Papers - Citterio, Daniel

Ratiometric fluorometry on microfluidic paper-based analytical device for simultaneous glucose and cholesterol detection using MnFe-layered double hydroxides as peroxidase mimic

Kitchawengkul N., Prakobkij A., Saenmuangchin R., Citterio D., Nacapricha D., Jarujamrus P.

Sensors and Actuators B: Chemical 435 2025.07

ISSN  09254005

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A highly sensitive ratiometric fluorescence sensing system was developed for simultaneous glucose and total cholesterol (TC) detection in whole blood using MnFe-layered double hydroxides (MnFe-LDHs) as a peroxidase mimic, combined with an o-phenylenediamine (OPD) substrate and nitrogen-doped graphene quantum dots (N-GQDs). The detection platform, an X-shaped laminated microfluidic paper-based analytical device (XL-μPAD), was fabricated via laser printing and cutting. The MnFe-LDHs' large surface area and layered structure provide a high affinity for OPD, with a Michaelis–Menten constant (KM) of 0.0127 mmol L−1. Upon placing a drop of blood on the XL-μPAD sample pad, the enzymatic reactions of glucose and TC produce H2O2, which MnFe-LDHs convert to hydroxyl radicals (•OH). These radicals oxidize OPD into fluorescent 2,3-diamino phenazine (DAP) with emission at 560 nm. Meanwhile, the N-GQDs emit fluorescence at 415 nm, which is quenched by DAP through the inner filter effect (IFE) and dynamic quenching, enabling ratiometric sensing via the intensity ratio (I560/I415). As H2O2 levels increase, a visible green emission appears, correlating with glucose and TC levels. This XL-μPAD system demonstrates promising potential as a portable device for multiplex biomarker detection and diagnostic applications.

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Origami Paper-Based Immunoassay Device with CRISPR/Cas12a Signal Amplification

Suzuki H., Tong G., Nath P., Hiruta Y., Citterio D.

ACS Sensors 10 ( 3 ) 1811 - 1821 2025.03

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In clinical diagnosis, the determination of target proteins at low concentration levels is generally performed by immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), which is a time-consuming process. To date, paper-based ELISA platforms enabling faster and less expensive analysis have been developed, but their important issue for clinical applications is the limited sensitivity compared to conventional ELISA. To address this challenge, this paper introduces a simple, rapid, and highly sensitive detection method for non-nucleic acid targets achieved by integrating the CRISPR/Cas12a system into paper-based ELISA. An origami-type paper-based device enabling simple assay operation has been designed, and the detection of targets on the paper substrates is based on observing the fluorescence signal induced by the CRISPR/Cas12a enzyme cleaving a probe single-stranded DNA (ssDNA) labeled with fluorophore and quencher (FQ reporter). To enhance sensitivity, antibodies labeled with a network of multiple DNA activating the CRISPR/Cas12a enzyme have been utilized as detection antibodies. As a result, the developed device successfully boosted the detection sensitivity for both human IgG and the hepatitis B virus surface antigen (HBsAg). In particular, the limit of detection (LOD) for HBsAg was estimated to be 12 pg/mL, representing over 10-fold higher sensitivity compared with commercially available HBsAg ELISA kits (LOD: 200 pg/mL). In addition, the fluorescence response toward porcine whole blood samples containing different HBsAg concentrations was also confirmed by capturing images with a smartphone, followed by quantitative data analysis. These results demonstrate the potential applicability of the proposed platform for clinical tests at the point of care.

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Bioluminescence readout lateral flow immunoassay using nanobody targeting aflatoxin B1

Takahashi S., Hiruta Y., Citterio D.

Analyst 150 ( 8 ) 1563 - 1570 2025.03

ISSN  00032654

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Multiple signal detection methods are known for lateral flow immunoassays (LFIAs), with colorimetric approaches dominating the field. However, their limited sensitivity is a remaining challenge. Fluorescence-based signaling is regarded as a more sensitive method, but it comes at the cost of partial sacrifice of the user-friendliness of LFIAs due to the requirement of an excitation light source. In this context, bioluminescence providing an inherently high signal to noise ratio without the need of excitation light could be an attractive alternative. But only a few studies have demonstrated the application of bioluminescence signaling in LFIAs. This work aimed at the development of a simple bioluminescence-based LFIA for the detection of aflatoxin B1 (AFB1), used as a model target in a competitive LFIA format. Signal transduction was achieved by nanobody-nanoluciferase (Nluc) fusion proteins. These small-sized recombinant heavy-chain-only antibodies derived from the camelidae family directly linked with the Nluc enzyme produce high intensity glow-type bioluminescence in combination with the furimazine substrate. LFIA devices consisting of a sample pad, nitrocellulose membrane and absorbent pad with AFB1-BSA conjugate deposited at the test line on the nitrocellulose membrane, achieved an LOD of 0.26 ng mL−1 for aqueous AFB1 solutions pre-mixed with Nanobody-Nluc and bioluminescence emission observed on an imaging system. More user-friendly LFIA devices with integrated conjugate pad and pre-deposited Nanobody-Nluc provided clear AFB1 concentration-dependent bioluminescence signals with low background and enabled readout with a standard digital camera, resulting in an LOD of 1.12 ng mL−1. Finally, the LFIA strips have been applied in AFB1-spiked oat milk samples. The LOD of 4.09 ng mL−1 achieved in the real sample matrix is well below the maximum allowable residual concentration of AFB1 in the U.S. (20 ng mL−1).

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Ratiometric Imaging for Quantification of Elevated Ca²⁺ in Neurons Using Synthetic Low-Affinity Fluorescent Probe

Kuronuma Y., Shindo Y., Kumada R., Sakama A., Citterio D., Oka K., Hiruta Y.

ACS Chemical Neuroscience 16 ( 4 ) 649 - 658 2025.02

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The availability of various calcium ion (Ca2+) fluorescent probes has contributed to revealing physiological events related to intracellular Ca2+. However, conventional probes face challenges for quantitatively and selectively visualizing high Ca2+ concentrations in cells induced by any stimuli, including biomolecules or electrical signal that disrupt Ca2+ homeostasis. In this report, we designed and synthesized a low-affinity ratiometric Ca2+ probe, KLCA-Fura, utilizing o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate (APDAP) as a ligand, for which we recently demonstrated the suitability as a new low-affinity ligand for Ca2+. KLCA-Fura showed a blue shift in excitation wavelength with increasing Ca2+ concentration based on the intramolecular charge transfer (ICT). Its affinity for Ca2+ is lower than commercially available conventional Ca2+ probes. Furthermore, the selectivity for Ca2+ and the fluorescence intensity were considered sufficient to accurately detect Ca2+. The corresponding acetoxymethyl ester, KLCA-FuraAM, was synthesized for intracellular imaging and applied to Ca2+ quantification in neurons. KLCA-FuraAM enabled quantitative ratiometric monitoring of the two-step Ca2+ concentration increase induced by glutamate stimulation. While this two-step response was not clearly observed with a commercially available low-affinity ratiometric Ca2+ probe, Fura-FF, KLCA-FuraAM has demonstrated the potential to quantitatively visualize the behavior of high Ca2+ concentrations. The ratiometric low-affinity Ca2+ probe, KLCA-Fura, is expected to be a powerful tool for discovering new functions of Ca2+ in neurons.

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Present and future of smartphone-coupled chemiluminescence and electrochemiluminescence assays: a mini-review

Dutta C., Citterio D., Nath P.

Analyst 150 ( 6 ) 1033 - 1047 2025.02

ISSN  00032654

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The convergence of smartphones with chemiluminescence and electrochemiluminescence (CL/ECL) assays marks a transformative leap in the realm of sensing technologies. The traditional CL/ECL assays, known for their high sensitivity and versatility, find extensive applications in medical diagnostics, environmental monitoring, food safety, and forensic sciences. However, these techniques have long been constrained due to the requirement of expensive instrumentation and complex reagent handling and hence their accessibility within certain environments is limited. In an era where rapid, accurate, and routine analysis is critical, smartphone-enabled CL/ECL systems offer substantial advantages over conventional analytical methods. By leveraging the universal accessibility and technological sophistication of smartphones and combining them with CL/ECL-based sensing, the smartphone has evolved into a cost-efficient and accessible analytical platform. The ability of the combined platform to conduct on-site analysis in real-time with minimal effort has emerged as a game-changer, particularly in low-resource settings. This mini-review explores the rapid evolution of smartphone-coupled CL/ECL systems over the last five years. The article covers the areas where the combined platform has been implemented in recent years for various sensing applications. The review further identifies key challenges that are associated with such combined platforms and finally highlights the future perspectives of the present topic.

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Development of a Semiquantitative Barcode Readout Approach for Paper-Based Analytical Devices (PADs) for Enzymatic H<inf>2</inf>O<inf>2</inf> and Glucose Detection

Manmana Y., Kinugasa S., Hiruta Y., Citterio D.

Analytical Chemistry 97 ( 3 ) 1500 - 1506 2025.01

ISSN  00032700

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The integration of barcode technology with smartphones on paper-based analytical devices (PADs) presents a promising approach to bridging manual detection with digital interpretation and data storage. However, previous studies of 1D barcode approaches have been limited to providing only a “yes/no” response for analyte detection. Herein, a method of using barcode readout for semiquantitative signal detection on PADs has been achieved through the integration of barcode technology with a distance-based measurement concept on PADs. To demonstrate the feasibility of this concept, a PAD fabrication strategy incorporating barcodes was explored, using the enzymatic reaction between horseradish peroxidase (HRP), 3,3′-diaminobenzidine (DAB), and H2O2 as a model system. The enzyme-catalyzed polymerization of DAB to polyDAB in the presence of hydrogen peroxide results in the appearance of color observable by the naked eye inside a paperfluidic channel, with the color-changed length depending on the H2O2 concentration. At the same time, the barcode pattern displayed as a result of this distance-based color evolution overlaid with a paper-based barcode layer can be read using a smartphone application. Parameters affecting the signal readout performance were studied. The developed device can be used to detect H2O2 concentrations in the range of 0.25 to 10 mM within 90 min with 79.6% of barcode signals correctly readable. Additionally, results from different smartphone models showed a consistent reading performance (78.4-79.6%). Finally, the quantification of glucose levels in artificial urine samples was demonstrated. This developed PAD signaling strategy offers end-users more simplicity and can be used as a standalone device or in conjunction with other digital devices.

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Polymeric hydrogel integrated paper-based potentiometric ion-sensing device for the determination of sodium ions in human urine

Teekayupak K., Preechakasedkit P., Chuaypen N., Dissayabutra T., Lieberzeit P.A., Chailapakul O., Ruecha N., Citterio D.

Analyst 150 ( 5 ) 841 - 850 2025.01

ISSN  00032654

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A paper-based potentiometric sensor integrated with a polymeric hydrogel has been developed for sodium ion (Na+) determination in human urine. The construction of an all-solid-state ion selective electrode (s-ISE) and an all-solid-state reference electrode (s-RE) on a photo paper substrate was achieved using an inkjet printing method. For s-ISE fabrication, carbon nanotubes (CNTs) and gold nanoparticles (AuNPs) were printed on the substrate as a nanocomposite solid contact. A polymeric hydrogel containing lithium acetate (CH3COOLi) was then prepared and used as an intermediate layer to improve the adhesion between the ion selective membrane (ISM) and the AuNP/CNT solid contact, leading to enhanced detection sensitivity. The printed s-RE consisted of a pseudo silver/silver chloride electrode (p-Ag/AgCl) coated with a polymeric hydrogel containing KCl to improve the potential stability of the sensor. Under the optimal conditions, the hydrogel-integrated paper-based potentiometric sensor provided a response toward Na+ over a linear range of 10−7 M to 1 M with a near Nernstian slope of 56.42 ± 0.68 mV per decade. This sensor exhibited fast response, good sensitivity, and reasonable selectivity for Na+ measurement. Furthermore, the developed sensor was effectively applied for the detection of Na+ in urine samples with high accuracy. The presented work can be considered as a good addition to the growing field of potentiometric analytical platforms suitable for large-scale production using inkjet printing technology.

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3D-printed devices for multiplexed semiquantitative competitive lateral flow immunoassays

Tong G., Misawa K., Jarujamrus P., Hiruta Y., Citterio D.

Analytical Methods 17 ( 6 ) 1206 - 1213 2025.01

ISSN  17599660

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Lateral flow immunoassays (LFIAs) are widely used for the simple and rapid detection of various targets at the point of need. However, LFIAs enabling the simultaneous detection of multiple analytes and the possibility for naked-eye semi-quantitative analysis are facing various challenges, including the requirement of large sample volumes, low efficiency, and accuracy. This is particularly the case for the competitive immunoassay format targeting the detection of low molecular weight compounds, such as, for example, drugs. Due to limited space for multiple reaction zones on a single planar nitrocellulose membrane, conducting multiplexed tests requires the addition of more test strips, which consequently increases the size of the whole device. To overcome these spatial constraints, two 3D-printed devices fitting eight assay lanes of both backed and unbacked nitrocellulose membranes have been designed. For proof of concept, 8-OHdG, caffeine, and acetaminophen were used as model analytes. Inkjet printing was applied to deposit capture reagents in the form of text symbols while controlling the concentration thresholds for text readability to achieve an intuitive result expression. A comparably small sample volume of 350 mL was sufficient to simultaneously visually distinguish 4 concentration levels of caffeine (0, 4, 10, 175 ng mL-1) and acetaminophen (0, 4, 8, 12 ng mL-1) in mixed solutions without crosstalk. This study demonstrates the potential of 3D-printed LFIA devices for multiplex and semiquantitative analyte detection.

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Amplification-free CRISPR/Cas based dual-enzymatic colorimetric nucleic acid biosensing device

Tong G., Nath P., Hiruta Y., Citterio D.

Lab on a Chip 25 ( 4 ) 536 - 545 2025.01

ISSN  14730197

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Nucleic acid testing (NAT) is widely considered the gold standard in analytical fields, with applications spanning environmental monitoring, forensic science and clinical diagnostics, among others. However, its widespread use is often constrained by complicated assay procedures, the need for specialized equipment, and the complexity of reagent handling. In this study, we demonstrate a fully integrated 3D-printed biosensensing device employing a CRISPR/Cas12a-based dual-enzymatic mechanism for highly sensitive and user-friendly nucleic acid detection. A plastic probe stick was designed to host small-sized gold nanoparticles, enhancing enzyme labeling density. Alkaline phosphatase (ALP) was then conjugated via single-stranded DNA, requiring only a single enzyme substrate addition to generate a simple visual signal change. This approach eliminates the need for amplification or centrifugation steps, achieving a limit of detection (LOD) as low as 10 pM – among the highest sensitivities reported for amplification-free colorimetric nucleic acid detection. Furthermore, we developed a device that incorporates this probe stick, integrates all necessary reagents, and features a smartphone-compatible accessory for quantitative analysis. This allows end-users to perform visual or quantitative DNA analysis with simple operations, achieving a visual detection limit of approximately 100 pM, comparable to other CRISPR-based non-amplified nucleic acid detection methods. Additionally, the system successfully distinguished perfectly matched from mismatched nucleic acid sequences, demonstrating its specificity and versatility. Although certain design limitations affected the sensitivity of the integrated device compared to the probe stick alone, the simplicity and portability of this device make it a promising tool for rapid nucleic acid screening in clinical diagnostics, environmental monitoring, and food safety control. This study paves the way for the development of practical biosensors for point-of-care testing (POCT) applications.

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Lateral flow immunoassay device to detect staphylococcal enterotoxin B (SEB) in durian candy

Relox P.E., Israel K.A.C., Hiruta Y., Citterio D.

Analytical Sciences 41 ( 1 ) 3 - 11 2025.01

ISSN  09106340

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Staphylococcal enterotoxin B (SEB), a potent enterotoxin produced by Staphylococcus aureus, has been implicated in incidences of Staphylococcal food poisoning in the Philippines. The use of lateral flow immunoassay devices to detect this toxin in solid food samples, like durian candy, at the point of sampling is constrained by the requirement for sample purification (e.g. centrifugation). This problem is also true with the other applications of LFIA devices on food samples. To overcome this challenge, a lateral flow immunoassay (LFIA) device capable of detecting SEB in unpurified durian candy sample was developed in this study. A modified LFIA device was assembled with three layers of glass fiber pads functioning as sample pads instead of a conventional cellulose fiber pad. Unlike with the cellulose fiber pad, the glass fiber sample pads acted as filter and allowed the flow of a 1:5 dilution of durian candy. The LFIA device applied to spiked 1:5 diluted durian candy samples achieved a visual limit of detection of 5 ng/mL for SEB, which is twofold lower than reported for previous LFIA devices designed to detect SEB in food samples.

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IgG-Binding Peptidomimetic Mixed-Charge Polymer-Modified Resins for Chromatographic Purification of Antibodies

Deura K., Sakama A., Moriwaki Y., Citterio D., Hiruta Y.

ACS Applied Materials and Interfaces 16 ( 49 ) 67468 - 67476 2024.12

ISSN  19448244

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The process of antibody purification using Fc affinity ligands such as protein A, G, and L faces several challenges including high cost, low stability, and loss of antibody activity due to harsh elution conditions. Here, we describe a chromatographic purification of antibodies utilizing a pH-responsive mixed-charge polymer that mimics the IgG-binding peptide (Z34C) derived from the B domain of protein A. The protein A mimetic resins were prepared by modifying the surface of a TOYOPEARL, methacrylate resin with a polymer that mimics the amino acid sequence of Z34C and the functions of histidine and acidic and neutral amino acids using histamine methacrylamide (HisMA), methacrylic acid, and neutral monomers. The therapeutic monoclonal antibody (mAb), rituximab, was retained on the column at pH 7 and eluted under mildly acidic conditions at pH 5 using a protein A mimetic resin (HisMA20-EEMA) optimized for antibody interaction. The injected antibodies were selectively captured on the column by hydrophobic and electrostatic interactions with the protein A mimetic polymer under neutral conditions and eluted by electrostatic repulsion under acidic conditions. The HisMA20-EEMA column successfully purified mAbs from mixtures with BSA, mouse ascites fluid, and hybridoma cell culture supernatant. In addition, the HisMA20-EEMA column consistently achieved 90% antibody recovery in 100 consecutive purifications from cell culture supernatant. The antibody purification method presented in this study is low cost, highly durable, easy to synthesize, and allows for mild elution conditions. The results demonstrate that the approach of mimicking IgG-binding peptides with mixed-charge polymers is useful for the development of column packing materials for antibody purification.

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A selective dual-signal electrochemical paper-based device using imprinted sensors for voltammetric and impedance analysis of 4-NQO and carcinoembryonic antigen (CEA)

Ngaosri P., Karuwan C., Wanram S., Jarujamrus P., Citterio D., Amatatongchai M.

Analytica Chimica Acta 1330 2024.11

ISSN  00032670

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Background: Carcinoembryonic Antigen (CEA) and 4-nitroquinoline-N-oxide (4-NQO) are cancer markers that play a crucial role in tumor risk assessment and early cancer diagnosis. Therefore, it is in demand to develop a fast, accurate, simple, and cost-effective method to detect these cancer markers for quick and early stage-cancer diagnosis and treatment. Results: Herein, we report a dual signaling approach for direct and indirect signal transduction of cancer biomarker binding on molecularly imprinted-electrodes integrated on an ePAD, enabling sensitive and selective quantitative analysis of 4-NQO and CEA. Multiple test zones on a single device based on artificial recognition sites using core-shell Cu-MOF@MIP were developed to create a selective “Dual-signal-ePAD”. An effective and facile imprinting method on a Cu-MOF surface based on the synthesized of 4-VPBA/MAA/TRIM polymer. It resulted in a novel MIP useable as a high-performance sensing tool for the ultrasensitive and specific quantification of the target cancer biomarkers. Multiple tests consisted of voltammetric analysis of 4-NQO via oxidation of the analyte on a WE1, and impedance analysis of CEA via the molecular interaction with the imprinted WE2. Significance: The proposed device provides enhanced electrochemical signal amplification due to its high conductivity, electrocatalytic activity, and the target-specific recognition sites for accurate determination of 4-NQO and CEA targets. The Dual-signal-ePAD exhibits good voltammetric and EIS response, oxidation currents of 4-NQO at −0.06 V and RCT of CEA, resulting in high selectivity and sensitivity with a linear response range covering the concentration range of clinical interest for both analytes. Dual-signal ePAD is a good candidate for clinical screening and POCT.

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Peptide nucleic acid probe-assisted paper-based electrochemical biosensor for multiplexed detection of respiratory viruses

Lomae A., Teekayupak K., Preechakasedkit P., Pasomsub E., Ozer T., Henry C.S., Citterio D., Vilaivan T., Chailapakul O., Ruecha N.

Talanta 279 2024.11

ISSN  00399140

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The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43–25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases.

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Sensor Arrays for Electrochemical Detection of PCR-Amplified Genes Extracted from Cells Suspended in Environmental Waters

Aoki H., Kawaguchi M., Kumakura Y., Kamo H., Miura K., Hiruta Y., Simizu S., Citterio D.

Sensors 24 ( 22 )  2024.11

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Ecological surveys of living things based on DNAs from environmental samples are attractive. However, despite simplicity of water sampling from the target environment, it is still necessary to transport the samples to the laboratory for DNA analysis based on skillful next-generation sequencers. To perform DNA-oriented surveys based on a simple protocol without any special training, we demonstrated, in this study, the detection of genes from cell-containing environmental waters using gene sensor arrays that require no DNA labeling and no external indicators. Cell-suspended PBS or river water were used as models of environmental waters containing living things, and DNA samples were prepared by PCR amplification. Ferrocene-terminated probes were synthesized and immobilized on an electrode array to develop a sensor array. The sensor array showed a large response to a target DNA complementary to the probe and no response to a mismatched DNA, indicating sequence-specific detection. For DNA samples prepared from the cells in PBS, they showed good responses similar to those for the target DNA. They also significantly detected DNA samples from the cells in river water at a general environmental concentration (38 cells mL−1) with 28-fold larger responses than those for 0 cells mL−1.

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Paper-based analytical device for point-of-care nucleic acid quantification combining CRISPR/Cas12a and a personal glucose meter

Tanifuji Y., Tong G., Hiruta Y., Citterio D.

Analyst 149 ( 19 ) 4932 - 4939 2024.07

ISSN  00032654

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Although CRISPR-based nucleic acid detection has great potential in point-of-care testing due to its simplicity, it has been rarely integrated into paper-based analytical devices (PADs), which are attractive platforms to simplify assays. This work introduces a CRISPR-assisted nucleic acid quantification approach integrated into a PAD with signal readout by a personal glucose meter (PGM). Retention of magnetic beads by filter paper and pre-deposition of all required reagents by freeze-drying stabilized with trehalose enabled the indirect quantification of human papilloma virus (HPV) DNA through a PGM readout without complicated user intervention and complex reagent handling. The calculated limit of detection was 57 pM, which is comparable with other amplification-free CRISPR-based assays detecting nucleic acids. The fully integrated device exhibited good storage stability for up to 4 weeks, suggesting its applicability toward practical point-of-care nucleic acid quantification.

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Basic evaluation of the CRISPR/Cas system stability for application to paper-based analytical devices

Tanifuji Y., Suzuki H., Tong G., Hiruta Y., Citterio D.

Analytical Methods 16 ( 25 ) 4143 - 4149 2024.05

ISSN  17599660

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Despite the promising features of the CRISPR/Cas system for application to point-of-care nucleic acid tests, there are only a few reports on its integration into paper-based analytical devices (PADs) for the purpose of assay simplification. In most cases, paper platforms have only been used for the final signal readout in an assay otherwise performed in a test tube. Therefore, there is very limited information on the suitability of the CRISPR/Cas system for on-device reagent storage. To fill this gap, the current work primarily investigated the influence of various factors, including the type of paper, reagent drying method, effect of stabilizers, and storage condition on the storage stability of reagents necessary for CRISPR-based assays on paper substrates, by comparing the fluorescence signal emitted by the trans-cleavage of the dsDNA-activated Cas12a complex. The results obtained in the form of fluorescence signals emitted after trans-cleavage of a ssDNA probe through a dsDNA-activated Cas12a complex on paper substrates showed that CRISPR-related reagents spontaneously dried at room temperature on BSA blocked paper retained over 70% of their initial activity when stored at −20 °C for 28 days, independent of the type of paper substrates, which was improved by the addition of sucrose as a stabilizer. In addition, reagents dried on paper substrates under the optimized conditions exhibited stronger heat tolerance at temperatures above 65 °C compared to their corresponding solutions. This work is expected to contribute to the future development of fully integrated PADs relying on CRISPR/Cas systems for point-of-care applications requiring no additional reagent handling.

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Rational design of pH-responsive near-infrared spirocyclic cyanines: the effects of substituents and the external environment

Sakama A., Seo H., Hara J., Shindo Y., Ikeda Y., Oka K., Citterio D., Hiruta Y.

Chemical Communications 60 ( 46 ) 5984 - 5987 2024.05

ISSN  13597345

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pH-responsive spirocyclic cyanine dyes were designed and synthesized. The equilibrium constant for cyclization (pKcycl) could be rationally controlled by changing the nucleophilic moiety and the side chains. Encapsulation in polymeric micelles inhibited the H-aggregation of the dye, and the pKcycl could be shifted according to the amphiphilic polymer employed.

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Simplified capture, extraction, and amplification of cellular DNA from water samples

Kawaguchi M., Aoki H., Kamo H., Miura K., Hiruta Y., Simizu S., Citterio D.

Analytical Sciences (Analytical Sciences)  40 ( 3 ) 501 - 510 2024.03

ISSN  09106340

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DNA analysis in water samples is attracting attention in various fields. However, conventional methods for DNA analysis require a work-intensive and time-consuming sample pre-treatment. In this study, a simplified pre-treatment method for analyzing DNA in water samples was evaluated. The process consists of filtration, DNA extraction, and amplification, which can be achieved within a short time. In the filtration process, two types of filters, firstly a tissue paper (Kimwipe) and then a glass filter (GF/F), were used in sequence. The first large pore size filter enabled a reduction in filtration time by removing large particulate matter impurities present in river water matrix. Cells spiked into 1 L of river water were recovered at more than 90% within approximately 5 min filtration time. Also, DNA was extracted from the captured cells directly on the surface of the filter in only 5 min. Thus, DNA collection and extraction from a water sample can be completed within about 10 min. Furthermore, PCR amplification was performed directly from DNA-attached filter sections, which greatly reduced the number of required pre-treatment steps. Finally, we succeeded in establishing a simple and fast on-site pre-treatment system by using a hand-driven syringe filtration method. This pre-treatment system is expected to offer the possibility for the future establishment of a rapid and easy DNA analysis method applicable to various types of water samples. Graphical abstract: (Figure presented.)

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Peroxidase-like Activity of Aptamer-Gold Nanoparticles for Selective and Sensitive Fluorescence Detection of Low-Density Lipoproteins

Prakobkij A., Saenmuangchin R., Chunta S., Amatatongchai M., Citterio D., Jarujamrus P.

ACS Applied Nano Materials (ACS Applied Nano Materials)  7 ( 11 ) 12356 - 12365 2024

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Low-density lipoprotein cholesterol (LDL-C), commonly called “bad cholesterol”, is crucial to cardiovascular health. Increased LDL-C levels pose a substantial risk to human health. As a result, there is a demand for reliable, affordable, and highly sensitive analytical methods for LDL-C detection. Herein, a facile fluorometric aptasensor for LDL-C detection based on the aptamer-enhanced peroxidase-mimicking activity of gold nanoparticles (AuNPs) has been developed. AuNPs were functionalized with LDL-C-specific thiolated aptamer to enhance their intrinsic peroxidase-like activity, which could effectively catalyze the oxidation of o-phenylenediamine dihydrochloride (OPD) by H2O2 into the yellow-fluorescent product 2,3-diamino phenazine (DAP). The characterization studies confirmed that the presence of the aptamer enhances the affinity of AuNPs toward the OPD substrate, thereby leading to a marked increase in the peroxidase-mimicking activity. Moreover, after being functionalized with an aptamer, increased dispersibility and substrate affinity of AuNPs were achieved. Using LDL-C as a target analyte, under optimum conditions, a linear relationship between smartphone-recorded signal intensity and the logarithm of the analyte concentration was observed in the range of 0.05-1 mg dL-1 and the limit of detection was 0.0230 mg dL-1. The results agree with those obtained by a clinical laboratory method. This proposed method is readily deployable and a promising prototype for diverse diagnostic applications, particularly in biomarker detection within serum or plasma samples. It holds the potential to yield substantial advantages in point-of-care testing.

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Nonsequential and Sequential Dual Detection Probe Lateral Flow Immunoassay Device for Staphylococcal Enterotoxin B Detection

Relox P.E., Israel K.A.C., Hiruta Y., Citterio D.

Mindanao Journal of Science and Technology 22   235 - 259 2024

ISSN  22440410

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Staphylococcal enterotoxins (SEs) are among the leading causes of foodborne illnesses in the Philippines. Rapid and sensitive detection of these toxins in food, particularly staphylococcal enterotoxin B (SEB) - the most potent type of SE - is imperative. Rapid toxin detection can be achieved using a lateral flow immunoassay (LFIA) device. However, low sensitivity has been a persistent issue with LFIA devices. To develop an LFIA device with improved sensitivity, this study investigated the exclusive use of antibodies as biorecognition molecules in a dual-detection probe LFIA device format and the configuration of the LFIA device to release the probes nonsequentially and sequentially. The LFIA device was fitted with two layers of glass fiber sample pads, loaded separately with the two detection probes, and partially separated by a Scotch tape film. For the nonsequential LFIA device, the two sample pads were pretreated with blocking buffers containing the same sugar concentration, whereas for the sequential LFIA device, the two sample pads were pretreated with blocking buffers containing different sugar concentrations. When tested against a filtered, homogenized durian candy sample, the nonsequential LFIA device generated higher signal intensity than the sequential LFIA device. This result suggests that aggregation of the detection probes was superior to sequential binding in enhancing the signal of the LFIA device for SEB detection. The sensitivity of the nonsequential LFIA device at 5 ng/mL was two-fold higher compared to previous POCT LFIA devices for SEB detection in food samples.

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Label-Free Detection of Waterborne Pathogens Using an All-Solid-State Laser-Induced Graphene Potentiometric Ion Flux Immunosensor

Preechakasedkit P., Pulsrikarn C., Nuanualsuwan S., Rattanadilok Na Phuket N., Citterio D., Ruecha N.

Analytical Chemistry  2024

ISSN  00032700

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Waterborne pathogens are harmful microorganisms transmitted through water sources. Early and rapid pathogen detection is important for preventing illnesses and implementing stringent water safety measures to minimize the risk of contamination. This work introduces a miniaturized all-solid-state potentiometric ion flux immunosensor for the rapid and label-free detection of waterborne pathogens. A screen-printed silver/silver chloride electrode coated with a reference electrode membrane and polyurethane as an all-solid-state reference electrode was combined with a solid-state contact ion-selective electrode (ISE). An all-solid-state ISE was constructed on laser-induced graphene by coating it with a cationic marker and a carboxylated poly(vinyl chloride)-based membrane for immobilizing antibodies and controlling ion fluxes through the membrane. Proof-of-concept was achieved by detecting Escherichia coli and Salmonella enterica serovar Typhimurium using the assembled immunosensors within 10 min. The potentiometric response shift attributed to the blocking effect in the ion flux caused by pathogen-antibody interaction corresponded to pathogen concentration, indicating detection limits of 0.1 CFU/mL and working ranges of 0.1-105 CFU/mL. Furthermore, the developed sensors revealed high selectivity and were directly applied in groundwater and tap water without any sample preparation, demonstrating high recovery percentages. The simple operation and elimination of sample preparation are key benefits to further usability of the developed immunosensors for efficient pathogen detection.

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Development of Phosphinate Ligand-Based Low-Affinity Ca²⁺ Fluorescent Probes and Application to Intracellular Ca²⁺ Imaging

Kumada R., Sakama A., Shindo Y., Kuronuma Y., Iwasawa N., Citterio D., Oka K., Hiruta Y.

Analytical Chemistry (Analytical Chemistry)  95 ( 45 ) 16683 - 16691 2023.11

ISSN  00032700

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Divalent metal cations such as calcium ion (Ca2+) and magnesium ion (Mg2+) are indispensable to the regulation of various cellular activities. In this research, we developed the KLCA series utilizing o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate (APDAP) as a target binding site, which was reported recently as a highly free Mg2+-selective ligand. KLCA-301 with orange fluorescence based on a rhodamine fluorophore and KLCA-501 with near-infrared (NIR) fluorescence based on a Si-rhodamine fluorophore were synthesized, intended for application to multicolor imaging. The evaluation of the fluorescence response to Ca2+ and Mg2+ of the KLCA series indicated the applicability as low-affinity Ca2+ probes. While KLCA-301 mainly localized in the cytosol in cultured rat hippocampal neurons, KLCA-501 localized to the cytosol and granular organelles in neurons. Comparison of the fluorescence response of KLCA-301 and the high-affinity Ca2+ probe Fluo-4 upon stimulation by glutamate in stained neurons revealed that KLCA-301 could reflect the secondary large rise of intracellular Ca2+, which Fluo-4 could not detect. In addition, KLCA-501 showed a fluorescence response similar to the low-affinity Ca2+ probe Fluo-5N upon stimulation by glutamate in stained neurons, concluding that KLCA-301 and KLCA-501 could be used as low-affinity Ca2+ probes. The KLCA series offers new options for low-affinity Ca2+ probes. Moreover, KLCA-501 achieved simultaneous visualization of the change in Ca2+ and ATP concentrations and also in mitochondrial inner membrane potential in neurons. KLCA-501 is expected to be a strong tool that enables simultaneous multicolor imaging of multiple targets and elucidation of their relationship in cells.

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Cation/anion-exchange mode switching chromatography utilizing pH-responsive mixed charge polymer-modified silica beads

Kaku T., Deura K., Yoshii T., Citterio D., Hiruta Y.

Molecular Systems Design and Engineering (Molecular Systems Design and Engineering)  9 ( 1 ) 56 - 62 2023.11

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The separation capacity of a column typically remains constant. By applying stimuli-responsive materials to the stationary phase, the separation capacity in a single column can be tuned; however, the separation mode is not completely switched. In this study, we aimed to develop a cation/anion-exchange mode switching chromatography approach, in which the monomer ratio is adjusted, enabling the surface charge to become either negative or positive in response to mobile phase pH. Three types of beads were prepared, each modified with a pH-responsive mixed-charge polymer combining a cationic monomer, a pH-responsive carboxylic acid monomer, a neutral monomer, and a cross-linking monomer. The composition ratio of the cationic monomer to the pH-responsive carboxylic acid monomer was set at 1 : 2 so that the cation-exchange mode occurs at a pH above the pKa and the anion-exchange mode occurs below the pKa. At a pH below the pKa, the retention factor of the negatively charged compound increased. In contrast, at a pH above the pKa, the retention factor of the positively charged compound increased, confirming the charge switching on the bead surface. Switching to the cation- and anion-exchange mode enabled the separation of five basic antidepressants and acidic non-steroidal anti-inflammatory drugs, respectively. Utilizing a pH-responsive mixed-charge polymer, we attributed a cation/anion-exchange mode to a single column.

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Dual Colorimetric/Electrochemical Detection of Salmonella typhimurium Using a Laser-Induced Graphene Integrated Lateral Flow Immunoassay Strip

Preechakasedkit P., Panphut W., Lomae A., Wonsawat W., Citterio D., Ruecha N.

Analytical Chemistry (Analytical Chemistry)  95 ( 37 ) 13904 - 13912 2023.09

ISSN  00032700

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Foodborne illnesses caused by the ingestion of contaminated foods or beverages are a serious concern due to the millions of reported cases per year. It is essential to develop sensitive and rapid detection methods of foodborne pathogens to ensure food safety for producers and consumers. Unfortunately, current detection techniques still suffer from time-consuming operations and the need for highly skilled personnel. Here, we introduce a highly sensitive dual colorimetric/electrochemical detection approach for Salmonella enterica serovar typhimurium (S. typhimurium) based on a laser-induced graphene-integrated lateral flow immunoassay (LIG-LFIA) strip. The LIG electrode was fabricated by laser engraving on a polyimide tape containing a pseudo silver/silver chloride reference electrode from silver sintering and chlorination. Using double-sided tape inserted into the strip, automatic sequential reagent delivery was enabled for the dual-mode signal readout by single-sample loading. A gold-deposited gold nanoparticle strategy was first employed to simultaneously obtain a colorimetric signal for early screening and a signal turn-on electrochemical response for high-sensitivity and -quantitative analysis. A superior performance of the strip was established, characterized by a short analysis time (12 min assay +15 min sample preparation), a broad working concentration range (1 cfu/10 mL to 108 cfu/mL), and the lowest limit of detection (1 ± 0.5 cfu/10 mL; mean ± standard deviation, n = 3) among reported multimode S. typhimurium detection schemes. The strip was successfully applied in the analysis of various food products without any bacterial enrichment or amplification required, and the results were comparable to those of the standard culture method.

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Development of prediction model for cloud point of thermo-responsive polymers by experiment-oriented materials informatics

Hayakawa M., Sakano K., Kumada R., Tobita H., Igarashi Y., Citterio D., Oaki Y., Hiruta Y.

Polymer Chemistry (Polymer Chemistry)  14 ( 19 ) 2383 - 2389 2023.04

ISSN  17599954

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Thermo-responsive polymers having a lower critical solution temperature (LCST) have attracted attention for biological applications such as drug delivery, diagnosis, and coating materials. In recent years, research on predicting LCST by utilizing machine learning has been conducted. However, since these methods targeted only copolymers combining specific monomer structures, they are not versatile, and multiple trials are still required to obtain new thermo-responsive polymers with the desired LCST. In this study, a prediction model for cloud point temperature (TCP) was built by a combination of materials informatics and chemical insight, named sparse modeling for small data (SpM-S) using a small dataset of polymers collected from the literature as training data. This approach created a model that is interpretable, easy to calculate, and versatile. The prediction accuracy was validated using data from different literature sources and experimental test data. The model was able to predict the TCP of polymers containing monomers not included in the dataset as well as polymers containing monomers included in the dataset. The predictive model has the potential to guide the design of new thermo-responsive polymers, and to contribute to efficient development of thermo-responsive polymers.

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Evaluation of separation performance for eggshell-based reversed-phase HPLC columns by controlling particle size and application in quantitative therapeutic drug monitoring

Yoshii T., Nakano K., Okuda T., Citterio D., Hiruta Y.

Analytical Methods (Analytical Methods)  15 ( 14 ) 1790 - 1796 2023.03

ISSN  17599660

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Eggshell-based reversed-phase packing materials were applied to an analytical column for high-performance liquid chromatography. Commercially available eggshell powder was classified by a cyclone system to obtain three types of particles with different diameters (arithmetic mean ± standard deviation: 4.3 ± 3.8, 5.6 ± 3.3, and 9.5 ± 5.5 μm). Sedimentation separation removed tiny particles from each sample, resulting in particles with arithmetic means of 6.6 ± 5.5, 7.3 ± 4.5, and 10.2 ± 5.0 μm, respectively. The unclassified particles and three particle types treated with sedimentation separation were subsequently packed into analytical columns (150 mm × 4.6 mm I.D.), and their separation efficiencies were evaluated by comparing their height equivalent to a theoretical plate (HETP). The column without sedimentation separation exhibited the highest HETP, whereas the columns with sedimentation separation showed better separation efficiency and lower back pressure. The column with the best separation efficiency was applied for the separation of 10 alkylbenzenes and 5 steroids, and all peaks were observed with complete separation (peak resolution: RS > 1.5). Finally, the column was used for quantitative analysis of voriconazole, an azole antifungal agent, and imatinib, a first-generation molecularly targeted drug for cancer treatment, in spiked whole blood. Excellent accuracy (99.1-102.8%) and precision (0.6-1.9%) were observed for the spiked drugs and long-term stability (>3000 column volumes of mobile phase flow) indicated good applicability of the developed eggshell-based column as an analytical column for routine analyses of therapeutic drugs in blood.

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Evaluation of cellophane as platform for colorimetric assays on microfluidic analytical devices

Shigemori H., Maejima K., Shibata H., Hiruta Y., Citterio D.

Microchimica Acta (Microchimica Acta)  190 ( 2 )  2023.02

ISSN  00263672

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Due to their low cost, simplicity, and pump-free liquid transport properties, colorimetric assays on paper spots and microfluidic paper-based analytical devices (µPADs) are regarded as useful tools for point-of-care testing (POCT). However, for certain types of colorimetric assays, the “non-transparent” and “white” characters of paper can be a disadvantage. In this work, the possibilities of using cellophane as an alternative platform for colorimetric assays have been investigated. Cellophane is a low cost and easy-to-handle transparent film made of regenerated cellulose. Owing to its hydrophilic character, cellophane-based microfluidic channels fabricated through a print–cut–laminate approach enabled pump-free liquid transport into multiple detection areas, similar to µPADs. In addition, the water absorption characteristics of cellophane allowed the stable immobilization of water-soluble colorimetric indicators without any surface modification or additional reagents. The transparency of cellophane provides possibilities for simple background coloring of the substrates, increasing the dynamic signal range for hue-based colorimetric assays, as demonstrated for two model assays targeting H2O2 (46-fold increase) and creatinine (3.6-fold increase). Finally, a turbidity detection-based protein assay was realized on black background cellophane spots. The lowest limits of detection achieved with the cellophane-based devices were calculated as 7 µM for H2O2, 2.7 mg dL−1 for creatinine, and 3.5 mg dL−1 for protein (human serum albumin). Graphical Abstract: [Figure not available: see fulltext.]

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Fabrication process development and basic evaluation of eggshell-based column packing material for reversed-phase preparative separation

Yoshii T., Sakama A., Kanamori K., Nakanishi K., Imai H., Citterio D., Hiruta Y.

Journal of Chromatography A (Journal of Chromatography A)  1688 2023.01

ISSN  00219673

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Purification of basic drugs in reversed-phase mode is often difficult, mainly due to adsorption of positively charged compounds to the silica gel-based stationary phase. Since this adsorption can be suppressed under alkaline condition, columns with alkali-resistance are required. In addition, compounds with acid-sensitive structures are sometimes degraded during separation on silica gel-based columns which exhibit acidity due to their surface structure. We prepared an alkali-resistant reversed-phase packing material, Eggshell-PMAcO based on eggshells modified with an amphiphilic copolymer, poly(maleic acid-alt-1-octadecene) (PMAcO). The height equivalent to a theoretical plate (HETP) of the Eggshell-PMAcO column was improved by surface treatment with ammonium acetate buffer (900 mM, pH = 3.7), which is an inexpensive reagent, and the retention behavior for hydrophobic compounds was compared to a typical ODS column based on silica gel, resulting in sufficient selectivity of the eggshell-based column for hydrophobic compounds, as indicated by the ratio of retention factors of pentylbenzene and butylbenzene (Eggshell-PMAcO column: 1.55, ODS column: 1.65). Column temperature-dependent retention behavior of naphthalene was investigated in the temperature range from 25 °C to 45 °C, followed by the calculation of thermodynamic parameters. There was little difference in the standard molar enthalpy (Eggshell-PMAcO: −19.6 kJ/mol, ODS: −21.7 kJ/mol). The absolute value of the standard free Gibbs energy for the Eggshell-PMAcO column was much smaller than that of the ODS column (Eggshell-PMAcO: −0.284 kJ/mol, ODS: −13.0 kJ/mol), indicating that the Eggshell-PMAcO column had a weaker retention strength for naphthalene than the ODS column mainly due to the large difference in the standard molar entropy (Eggshell-PMAcO: −64.9 J/mol K, ODS column: −29.2 J/mol K). The retention capacities for imipramine under neutral (water/methanol) and alkaline (0.1% triethylamine water/methanol) conditions were 0.2 mg and 5 mg, respectively, based on injection mass-dependent HETP, retention factor and symmetry factor. Finally, the prepared column was applied to the purification of a building block for nucleic acid drugs. This study demonstrated that reversed-phase columns, which can be fabricated from eggshells and an amphiphilic copolymer in an inexpensive and eco-friendly way, have the ability to purify basic compounds and acid-sensitive compounds.

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Semi-quantitative microfluidic paper-based analytical device for ionic silica detection

Ogawa M., Katoh A., Matsubara R., Kondo H., Otsuka M., Sawatsubashi T., Hiruta Y., Citterio D.

Analytical Sciences (Analytical Sciences)  39 ( 8 ) 1361 - 1370 2023

ISSN  09106340

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Silicate ions (SiO32−), or ionic silica, are known to cause silica scaling in industrial water applications when excess amounts are present; hence, concentrations must be monitored and kept at a constant low level. Ionic silica is conventionally measured by spectrophotometry in the form of its silicomolybdic complex based on the molybdenum blue reaction, but the operation process is complicated and not suitable for on-site detection. To solve these issues, microfluidic paper-based analytical devices (µPADs) have been gaining attention as portable, low-cost analytical devices suitable for on-site detection. The foldable origami type device described in this work enabled silica detection based on the molybdenum blue reaction, in the range of 50–1000 mg/L, with a practically detectable lowest concentration of 50 mg/L. The device showed selectivity for silicate ions and stability over 21 days when stored at 4 °C. The semi-quantitative analytical performance makes the proposed paper-based device attractive for on-site industrial monitoring. Graphical abstract: [Figure not available: see fulltext.].

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Nitrogen-Doped Graphene Quantum Dots as “Off-On” Fluorescent Probes in Paper-Based Test Kits for Selective Monitoring of Cyanide in Food

Malahom N., Ma-In T., Naksen P., Anutrasakda W., Amatatongchai M., Citterio D., Jarujamrus P.

ACS Applied Nano Materials (ACS Applied Nano Materials)  6 ( 13 ) 11144 - 11153 2023

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We successfully developed a fluorometric paper-based test kit for the selective and sensitive determination of cyanide using nitrogen-doped graphene quantum dots (N-GQDs) as the fluorescent probe. Citric acid and tris(hydroxymethyl)aminomethane were precursors for the one-step synthesis of N-GQDs via in situ hydrothermal methods, providing a high quantum yield of 57.9%. The proposed mechanism uses a fluorescence turn-on approach. Specifically, the fluorescence of N-GQDs is quenched by the incorporation of Ag+ via a photoinduced electron transfer (PET). During the detection step, sulfuric acid converts cyanide (CN-) into hydrogen cyanide (HCN). The Ag+ species on the N-GQD surface then react with the evolved HCN via a coordination bond to form a silver cyanide complex, resulting in the fluorescence emission of the N-GQDs being turned back on. As a result, the fluorescence emission intensity of N-GQDs linearly increased with increasing CN- concentrations in the range of 0.5-25 mg L-1, with a limit of detection (LOD) of 0.08 mg L-1. Notably, the developed sensor has advantages in terms of simplicity, rapidity, low cost, and high selectivity toward CN-. The analytical performance of the test kit was also validated the performance of the test kit against a conventional precipitation titration method.

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Effect of the side chain composition of mixed-charge polymers on pH-selective cell–membrane interactions

Nakamura Y., Nasu M., Shindo Y., Oka K., Citterio D., Hiruta Y.

Polymer Journal (Polymer Journal)  55 ( 11 ) 1179 - 1188 2023

ISSN  00323896

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Zwitterionic polymers are characterized by their antifouling ability due to their net-neutral charge and affinity for cancer cell surfaces. We developed six pH-responsive mixed-charge polymers and investigated the effect of their side chain hydrophobicity on their interaction with cancer cell membranes. The polymers were synthesized using [2-(methacryloyloxy)ethyl]trimethylammonium chloride, carboxylic acid monomers, and butyl methacrylate (BMA) as the neutral spacer moiety, and differences in hydrophobicity were created by altering the type of carboxylic acid monomer and ratio of the spacer. The surface charge of the polymers was designed to switch from net-neutral to positive in response to the weakly acidic tumor environment. This tuning occurred in a more cancer-like environment with the usage of a more hydrophobic anionic monomer and a higher ratio of BMA due to the weakening of electrostatic interactions. This trend was further confirmed by evaluation of the interaction between the polymers and heparin, an anionic polysaccharide that is used as a model for cancer cell surfaces. The affinity of the mixed-charge polymers for cells was then evaluated, and both the hydrophobicity and pH-responsive nature of these polymers led to pH-selective toxicity, which was assumed to be caused by the disruption of cell membrane integrity.

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Microoptics Integrating smartphone applications reader with paper-based analyser for accurate estimation of nickel in water

Nakagawa M., Tong G., Nath P., Citterio D.

28th Microoptics Conference, MOC 2023  2023

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The estimation of nickel ions in water is critical from a human health perspective. Herein, we demonstrate the usability of a paper strip analyzer coupled with a smartphone application reader for accurate estimation of Ni2+ ions in water. The limit of detection of the proposed technique is found to be 0.764 μM.

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Improvement in sensitivity for lateral flow immunoassay of ferritin using novel device design based on gold-enhanced gold nanoparticles

Preechakasedkit P., Teekayupak K., Citterio D., Ruecha N.

Scientific Reports (Scientific Reports)  12 ( 1 )  2022.12

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This work introduces a low-cost adhesive tape combined with a hydroxylamine/polyvinyl alcohol/polyethylene oxide (HA/PVA/PEO) blend film to fabricate novel devices for improving sensitivity of gold nanoparticle (AuNP)-based lateral flow immunoassays (LFIAs) via two platforms: (1) LFIA device with integrated gold enhancement and (2) LFIA device with two independent sample inlets. The detection of ferritin has been used for proof-of-concept. The adhesive tape inserted in the devices assists to separate two solutions independently flowing from two different inlets toward a nitrocellulose membrane. On-device gold enhancement was achieved by the enlargement of AuNPs via the catalytic reaction of KAuCl4 and HA using the HA/PVA/PEO blend film easily prepared via a solution-casting technique, which could delay the flow of HA released from the film for 180s and improve storage stability of the device. Under optimal conditions evaluated by naked eyes, the gold enhancement (LOD = 0.5 ng/mL) and double-sample inlet (LOD = 2 ng/mL) devices exhibited 20-fold and fivefold higher sensitivity respectively than a conventional device, verifying the sensitivity improvement. Furthermore, the proposed device was successfully detected ferritin in human serum samples within 10 min via naked-eye observation, exhibiting rapidity and simplicity of use, and the capability to perform on-site assays.

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Amphiphilic Copolymer-Modified Eggshell-Based Column Packing Materials for the Preparative Separation of Basic Drugs

Yoshii T., Mochida M., Kaizu K., Soda Y., Kanamori K., Nakanishi K., Sato T., Imai H., Citterio D., Hiruta Y.

ACS Applied Polymer Materials (ACS Applied Polymer Materials)  4 ( 10 ) 6949 - 6957 2022.10

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A way to utilize eggshells is desired because most of the eggshell waste is disposed of in landfills. Despite eggshells having attractive features such as a thermodynamically stable porous crystalline structure, their effective utilization as a functional material has yet to be demonstrated. Herein, we developed an alkali-stable column packing material for reversed-phase preparative high-performance liquid chromatography (HPLC) based on eggshells. Nanocrystals and biopolymers present on eggshell powder were removed by surface treatment with buffered ethylenediaminetetraacetic acid (EDTA) and sodium hypochlorite solutions, resulting in bare calcium carbonate with a uniform porous surface. The obtained particles were modified with poly(maleic acid-alt-1-octadecene) (PMAcO) having hydrophobic alkyl chains and anionic carboxylate groups. The prepared PMAcO-modified eggshell-based material was applied to an HPLC column, and its performance is evaluated. The column succeeded in the separation of alkylbenzenes, steroids, and diastereomers. It was also used for the separation of basic tricyclic antidepressants in an alkaline mobile phase, showing excellent retention capacity and alkali stability. This study demonstrates the potential of eggshell utilization, providing not only a practical stationary phase for HPLC but also a solution for recycling calcium carbonate-based biomineral waste.

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Quantitative evaluation of reversed-phase packing material based on calcium carbonate microspheres modified with an alternating copolymer

Kaizu K., Mochida M., Imai H., Citterio D., Hiruta Y.

Journal of Chromatography A (Journal of Chromatography A)  1677 2022.08

ISSN  00219673

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Considering the vulnerability of silica gel to alkaline mobile phases, a highly alkaline stable stationary phase for HPLC is required to separate basic compounds with high separation efficiency. To address this issue, we have developed a high alkaline stable packing material (CaCO3-PMAcO) based on mesoporous calcium carbonate microspheres modified with poly(maleic acid-alt-1-octadecene). In this study, we report further investigation of the separation performance of CaCO3-PMAcO column by systematically evaluating the effects of particle size and chromatographic conditions. Based on the theory of the van Deemter equation, the separation efficiency was related to the size of CaCO3-PMAcO particles (2.9 – 5.7 µm). The evaluation of thermodynamics of retention by changing the column temperature from 20 °C to 45 °C implied that the retention mode was dominated by hydrophobic interaction associated with the exothermic enthalpy changes (-11.1 to -12.5 kJ/mol). The results of column selectivity tests revealed that the CaCO3-PMAcO column had hydrophobic selectivity comparable to C18 silica gel columns (αP/B; CaCO3-PMAcO column: 1.53, C18 column: 1.69), and higher shape/steric selectivity (αTri/Ter; CaCO3-PMAcO column: 1.56, C18 column: 0.955). In practice, the CaCO3-PMAcO column could be applied to the separation of not only alkylbenzenes and polycyclic aromatic hydrocarbons, but also to basic tricyclic antidepressants by using an alkaline mobile phase (pH 12).

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Organelle-selective near-infrared fluorescent probes for intracellular microenvironment labeling

Kurutos A., Shindo Y., Hiruta Y., Oka K., Citterio D.

Dyes and Pigments (Dyes and Pigments)  204 2022.08

ISSN  01437208

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The design, synthesis, and bio-applicability of a series of 8 near-infrared (NIR) molecular probes with tunable hydrophilic/hydrophobic properties are described in this paper. The title dyes comprise of symmetric heptamethine cyanines with various N-quaternary heterocyclic moieties including indole, benzo[e]indol, benzothiazole and naphtho[1,2-d]thiazole, along with an extra positive charge in close proximity to the polymethine backbone. Combined UV–Vis and spectrofluorimetric techniques in both organic and aqueous-buffered solutions were employed to investigate the optical properties of the NIR chromophores. Two of the fluorophores emerged remarkable fluorescence quantum yield (Φ = 0.083), while their brightness was found up to 3-fold that of the FDA approved Indocyanine green – ICG (Φ = 0.027). What is more, two of the dyes appear to outperform the later clinical benchmark fluorophore in terms of photostability. While the ICG was completely faded after 1 h of constant irradiation, nearly 70% of the original emission signal could be observed for one of the novel molecular probes. The log Pow values determined by a combination of HPLC and UV–Vis (partition between 1-octanol/water) cover a relatively large range, from −2.14 up to 7.92. Investigation on the cytotoxicity by means of the MTT assay revealed, that present dyes are suitable for bioanalytical purposes and are safe to use. This paper also includes fluorescence imaging and cellular applications, such as labeling of HeLa cervical cancer cells. The results from the confocal microscopy reveal the superior staining capability of current heptamethines since they show significantly brighter signal than the commercial ICG probe. An organelle-selective fluorescent staining was achieved depending on the dye chemical structure. Co-localization within the intracellular microenvironment (linear or granular - cytosol, mitochondria, lysosome, endoplasmatic reticulum or Golgi apparatus) was confirmed without interference, employing several conventional organelle dye trackers. To the best of our knowledge, heptamethine dyes represent the first example of a series NIR emitting probes, that exhibit rational structural modifications aiming to fine-tune the hydrophilic/hydrophobic properties furnishing organelle-targeted staining.

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Ag<inf>3</inf>PO<inf>4</inf>/Ag nanocomposite for selective and sensitive cyanide determination in food samples through catalytical colorimetry using a paper-based test kit

Malahom N., Jarujamrus P., Anutrasakda W., Siripinyanond A., Amatatongchai M., Citterio D., Nacapricha D.

Sensors and Actuators B: Chemical (Sensors and Actuators B: Chemical)  356 2022.04

ISSN  09254005

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A paper-based test kit for selective and sensitive determination of cyanide was successfully developed using a Ag3PO4/Ag nanocomposite for catalytic colorimetry. The Ag3PO4/Ag nanocomposite synthesized via a one-step chemical bath method could instantly oxidize the chromogenic substrate ABTS for the color development under acidic conditions without the requirement of H2O2. During the detection step, cyanide in the sample was converted into gaseous hydrogen cyanide (HCN) using sulfuric acid. The HCN species then reacted with the generated Ag+ species via coordinate covalent bonding to form silver cyanide complex ions [Ag (CN2)]-. The amount of Ag+ species used for oxidizing ABTS subsequently decreased and so did the color intensity on the paper-based test kit. Specifically, the color intensity decreased with increasing cyanide concentrations in the range of 2.50–20.0 mg L−1. The limit of detection (LOD) and limit of quantification (LOQ) were 0.48 mg L−1 and 1.59 mg L−1, respectively. The developed test kit showed relatively high selectivity towards cyanide and high tolerance towards other cations and anions present in real samples. When applied to real samples including fruit juices and food products, the performance of the rapid and low-cost test kit was well validated by a conventional precipitation titration method.

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A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level

Orioka M., Eguchi M., Mizui Y., Ikeda Y., Sakama A., Li Q., Yoshimura H., Ozawa T., Citterio D., Hiruta Y.

Bioconjugate Chemistry (Bioconjugate Chemistry)  33 ( 3 ) 496 - 504 2022.03

ISSN  10431802

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Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/NanoKAZ (NKAZ)-FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.

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Traffic light type paper-based analytical device for intuitive and semi-quantitative naked-eye signal readout

Ohta S., Hiraoka R., Hiruta Y., Citterio D.

Lab on a Chip (Lab on a Chip)  22 ( 4 ) 717 - 726 2022.02

ISSN  14730197

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Microfluidic paper-based analytical devices (μPADs) have attracted great attention as potential candidates for point-of-care testing (POCT). Nevertheless, only a limited number of μPADs expected to satisfy the standard of Clinical Laboratory Improvement Amendments (CLIA) waived tests as issued by the US Food and Drug Administration (FDA) have been reported. This work introduces a “traffic light type μPAD”, enabling highly intuitive semi-quantitative equipment-free naked-eye readout with no need for calibration, subjective interpretation or calculation. Assay results are displayed as traffic light colours reporting 5 analyte concentration levels (green/green & yellow/yellow/yellow & red/red). The device has been designed to never display all three colours simultaneously, eliminating any risk for misinterpretation. The mechanism relies on the modulation of sample flow through a network of paperfluidic channels modified with a hydrophobic to hydrophilic phase-switching substance responsive to H2O2. User operation is limited to sample application, followed by observing a clear and time-independent traffic light signal after approximately 10-30 min. Multiple factors influencing the H2O2 concentration-dependent appearance of a specific traffic light signal were studied. Making use of the possibilities for customising the concentration threshold levels for traffic light colour appearance, quantification of glucose at 5 levels in a clinically relevant concentration range was demonstrated in artificial urine as a model proof-of-concept. This platform is expected to offer the possibility for the future detection of other important metabolites.

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Microfluidic thread-based analytical devices for point-of-care detection of therapeutic antibody in blood

Shimazu R., Tomimuro K., Ni Y., Malegori C., Hamedpour V., Hiruta Y., Oliveri P., Merkx M., Citterio D.

Sensors and Actuators B: Chemical (Sensors and Actuators B: Chemical)  352 2022.02

ISSN  09254005

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In therapies involving therapeutic antibodies, therapeutic drug monitoring (TDM) has the potential to substantially increase clinical efficacy and to enable more economical drug use, because the clearance from the bloodstream after administration is patient dependent. Since TDM must be performed continuously and over extended time periods, it should ideally be achieved through an economical and easy to perform point-of-care (POC) assay. Here, we introduce a new analytical method for POC TDM of the clinically important therapeutic antibody cetuximab, using microfluidic thread-based analytical devices (µTADs) as a platform for a bioluminescence resonance energy-transfer (BRET) switching sensor protein for simple quantitative antibody detection. The µTAD is pre-treated with NaCl for blood plasma separation and a BRET switching sensor protein and its substrate for cetuximab detection, integrating all elements required for a reagent-free, single-step assay. A change in bioluminescence emission color depending on cetuximab concentrations occurs after whole blood application. The device design enables the quantification of cetuximab within the therapeutically relevant blood concentration range in 2.5 min using only 2 µL of whole blood. The results obtained are independent of the blood volume. This µTAD can be stored for about one month at − 20 °C, a temperature compatible with commonly available freezers. The application of mathematical morphology recognition (MMR)-integrated image processing techniques for the automatic selection of regions of interest (ROIs) yields results with minimal error. The simplicity of use, rapidity, and cost-effectiveness of this µTAD are expected to offer the possibility of achieving TDM of therapeutic antibodies at POC for individual patients.

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Nitrogen-doped carbon dots/Ni-MnFe-layered double hydroxides (N-CDs/Ni-MnFe-LDHs) hybrid nanomaterials as immunoassay label for low-density lipoprotein detection

Prakobkij A., Jarujamrus P., Chunta S., Chawengkirttikul R., Keawin T., Malahom N., Tamuang S., Amatathongchai M., Citterio D.

Microchimica Acta (Microchimica Acta)  189 ( 2 )  2022.02

ISSN  00263672

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Nitrogen-doped carbon dots/Ni-MnFe-layered double hydroxides (N-CDs/Ni-MnFe-LDHs) are demonstrated as superior peroxidase mimic antibody labels alternative to horseradish peroxidase (HRP) in an immunoassay, potentially overcoming some of the inherent disadvantages of HRP and other enzyme mimicking nanomaterials. They revealed efficient peroxidase-like activity and catalyzed the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to form the intense blue product (at 620 nm) in the presence of hydrogen peroxide (H2O2). Using low-density lipoprotein (LDL) as a model target, an ultra-low limit of detection (0.0051 mg/dL) and a linear range of 0.0625–0.750 mg/dL were achieved, exhibiting higher sensitivity than the HRP-based immunoassay. Thus, the proposed N-CDs/Ni-MnFe-LDHs can be used as HRP mimicking analogs for developing highly sensitive colorimetric immunosensors for detection of biomarkers, as well as trace chemical analysis. Graphical abstract: [Figure not available: see fulltext.]

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Synthesis and spectral properties of near-infrared cyanine dyes showing enhanced Stokes shift: A paradigm of ICT dipolar state polymethine chromophoric systems

Kurutos A., Citterio D.

Journal of Molecular Structure (Journal of Molecular Structure)  1247 2022.01

ISSN  00222860

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Three benzothiazole-derived heptamethine cyanine dyes were prepared via nucleophilic substitution at meso-position along the polymethine backbone. The synthetic route to the title polymethines includes a total of four steps starting from inexpensive commercially available materials. Structural elucidation of the newly synthesized chromophores was achieved using 1H-NMR, 13C-NMR and high-resolution mass spectrometry (HRMS) in ESI mode. Current functionalized amino-derived NIR dyes exhibit significant Stokes shift (Δν up to 1835 cm−1, 108 nm) compared to the parent unsubstituted chromophore (Δν = 303 cm−1, 23 nm). The probability of solvatochromism was determined from the correlation between the Stokes shift against the Dimroth-Reichardt ET (30) and the Lippert–Mataga function (ΔfLM) of orientation polarizability. Based on the experimental findings, this large Stokes shift presumably reflects a solvatochromic intramolecular charge transfer (ICT) across the molecule, which is induced by the introduced secondary amine.

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Colorimetric paper-based sarcosine assay with improved sensitivity

Masumoto M., Ohta S., Nakagawa M., Hiruta Y., Citterio D.

Analytical and Bioanalytical Chemistry (Analytical and Bioanalytical Chemistry)  414 ( 1 ) 691 - 701 2022.01

ISSN  16182642

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This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)–catalyzed conversion of the colorless indicator 3,3’,5,5’-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 μM and a significant lowering of the limit of detection (LOD) (0.6 μM) compared to the unmodified paper substrate (12.6 μM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 μM) is below the 5 μM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at − 20 °C for a period of 50 days were confirmed. Graphical abstract: [Figure not available: see fulltext.]

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Feasibility of Using Poly[oligo(ethylene glycol) Methyl Ether Methacrylate] as Tumor-Targeted Carriers of Diagnostic Drugs

Sano K., Umemoto K., Miura H., Ohno S., Iwata K., Kawakami R., Munekane M., Yamasaki T., Citterio D., Hiruta Y., Mukai T.

ACS Applied Polymer Materials (ACS Applied Polymer Materials)  4 ( 7 ) 4734 - 4740 2022

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Poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) is a water-soluble polymer in which multiple short ethylene glycol (EG) chains are grafted along the hydrophobic methyl methacrylate backbone. We investigated whether POEGMA could be used as a drug carrier for tumor diagnosis. For this purpose, we synthesized POEGMA derivatives with different molecular weights (11, 21, and 30 kDa) and EG side chain lengths, and labeled them with radioisotopes (indium-111; 111In) or fluorescence dyes (indocyanine green; ICG) as signal emitters. Fluorescence microscopy studies using colon26 tumor cells treated with ICG-labeled POEGMA derivatives revealed that the POEGMA derivatives were efficiently taken up by tumor cells compared to the control, poly(ethylene glycol) (PEG), suggesting the involvement of a POEGMA-specific cellular uptake pathway. The radiolabeled POEGMA derivatives were intravenously injected into colon26 tumor-bearing mice, and their biodistribution was evaluated. As the molecular weight of POEGMA derivatives increased, the circulating time was prolonged, and their accumulation in the liver and spleen increased. In particular, 21 kDa POEGMA showed excellent tumor uptake and tumor-to-normal tissue ratio. Therefore, we investigated the effect of EG chain length on the tumor uptake of POEGMA (21 kDa) and observed high tumor uptake for all POEGMA derivatives; however, relatively higher abdominal uptake was observed for POEGMA derivatives with longer EG chain lengths. Finally, in vivo fluorescence imaging using optimized POEGMA derivatives labeled with ICG clearly visualized the tumor tissues, suggesting that the POEGMA derivatives could be suitable drug carriers for sensitive tumor diagnosis.

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Differential Effect of Azetidine Substitution in Firefly Luciferin Analogues

Ikeda Y., Orioka M., Nomoto T., Hiruta Y., Nishiyama N., Citterio D.

ChemBioChem (ChemBioChem)  22 ( 21 ) 3067 - 3074 2021.11

ISSN  14394227

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Replacing an N,N-dimethylamino group in a classical fluorophore with a four membered azetidine ring provides an improved luminescence quantum yield. Herein, we extended this strategy to bioluminescent firefly luciferin analogues and evaluated its general validity. For this purpose, four types of luciferin cores were employed, and a total of eight analogues were evaluated. Among these analogues, unexpectedly, only the benzothiazole core analogue benefited from an azetidine substitution and showed enhanced bioluminescence. In addition, fluorescence measurements revealed that an azetidine substitution improved the fluorescence quantum yield by 2.3-times compared to a N,N-dimethylamino group. These findings clarify the differential effects of azetidine substituents in luciferins and present one possible strategy for enhancing photon output in benzothiazole type luciferins through a synthetic approach.

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Anion species-triggered antibody separation system utilizing a thermo-responsive polymer column under optimized constant temperature

Nomoto D., Nagase K., Nakamura Y., Kanazawa H., Citterio D., Hiruta Y.

Colloids and Surfaces B: Biointerfaces (Colloids and Surfaces B: Biointerfaces)  205 2021.09

ISSN  09277765

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Although the field of antibody drugs has grown larger, the antibody production still faces several challenges. Effective antibody purification is required, but the conventional purification method for antibodies is cost intensive and often causes aggregation problems, indicating the need for new alternative antibody purification methods. In the present study, a constant temperature antibody purification system for use with a thermo-responsive polymer column was developed based on switching of anion species in eluents. By adjusting the temperature for each antibody, the developed column enabled separation of the therapeutic monoclonal antibodies, rituximab and trastuzumab, from contaminants without changing salt concentration or pH of the eluents. The thermo-responsive hydrogel-modified column packing material was synthesized by introducing n-butyl methacrylate, acrylic acid, N,N′-methylenebisacrylamide and N-isopropylacrylamide to the surface of silica beads with an initiator by a graft-from approach. Elution behavior of antibodies with three types of anions, such as citrate, phosphate, and chloride were tested under three different temperature conditions. It was demonstrated that the thermo-responsive hydrogel grafted column showed a switchable antibody retention behavior at constant temperature and salt concentration, with antibody adsorption by NaCl eluent and desorption by citric acid buffer eluent.

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Small molecule-based alkaline-earth metal ion fluorescent probes for imaging intracellular and intercellular multiple signals

Hiruta Y., Shindo Y., Oka K., Citterio D.

Chemistry Letters (Chemistry Letters)  50 ( 5 ) 870 - 887 2021.05

ISSN  03667022

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This highlight review overviews recent progress in fluorescence imaging technology for the alkaline-earth metal ions calcium, and magnesium, by small molecule-based fluorescent probes. Fluorescent probes for Ca2+ using 1,2-bis-(o-aminophenoxy)- ethane-N,N,N′,N′-tetraacetic acid (BAPTA) as a recognition unit are introduced. Focus is set on the fluorescence characteristics and intracellular dynamics of Ca2+ fluorescent probes depending on the differences in fluorophore units. In addition, this review comprehensively covers Mg2+ fluorescent probes from Mg2+ selective ligands and fluorophore units to their application. In particular, the influence of the molecular structure of the fluorescent probes on organelle localization and fluorescence wavelength for imaging intracellular signals in organelles and intercellular multiple signal interactions are summarized, providing a strategy for probe design for chemists, and for selecting the appropriate imaging technique for biologists.

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Long-term single cell bioluminescence imaging with C-3 position protected coelenterazine analogues

Mizui Y., Eguchi M., Tanaka M., Ikeda Y., Yoshimura H., Ozawa T., Citterio D., Hiruta Y.

Organic and Biomolecular Chemistry (Organic and Biomolecular Chemistry)  19 ( 3 ) 579 - 586 2021.01

ISSN  14770520

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Bioluminescence is a powerful imaging modality for monitoring biological phenomena bothin vitroandin vivo. Bioluminescence imagin (BLI) is becoming a seamless imaging technology covering the range from cells to organs of small animals. Long-term imaging at the single cell level would lead to a true understanding of the dynamics of life phenomena. This work presents a long-term single cell bioluminescence imaging technology accomplished with C-3 position protected furimazines (FMZs), a CTZ analogues, which generate intense blue emission when paired with a highly stable engineered luciferase, Nanoluc. Four types of FMZs protected at the C-3 position have been synthesized. The type and steric bulkiness of the protection group strongly contributed to storage stability and the kinetics of the bioluminescence reactions of the analogues in human living cells. In particular, two developed FMZ analogues resulted in significantly longer bioluminescence emission with higher S/N ratio than FMZ at single cell level. Long-term bioluminescence single cell imaging technology with the developed FMZ analogues will lead to seamless imaging in the range from cells to organs of small animals.

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Fluorescent and Bioluminescent Probes based on Precise Molecular Design

Kumada R., Orioka M., Citterio D., Hiruta Y.

Bunseki Kagaku (Bunseki Kagaku)  70 ( 10.11 ) 601 - 616 2021

ISSN  05251931

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Fluorescence and bioluminescence have been actively applied to various chemical sensors and biosensors, including to cellular/in vivo imaging. On application to the detection of specific targets, it is necessary to develop fluorescent/bioluminescent probes that selectively respond to the target analytes such as molecules, ions or cells by changing their optical properties. The authors have focused on the development of small molecular probes by means of molecular design based on the desired function. In this paper, we describe the approaches to molecular design of fluorescent/bioluminescent probes and their applications, including the introduction of our recent research works. Finally, a future outlook for further functionalization of small molecular probes is provided.

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Ion-selective optodes integrated paper-based device for simultaneous colorimetric quantification of salivary Na⁺, K⁺ and Ca²⁺

Suenaga Y., Shibata H., Maejima K., Hiruta Y., Citterio D.

Bunseki Kagaku (Bunseki Kagaku)  70 ( 3 ) 165 - 173 2021

ISSN  05251931

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The quantification of electrolytes in biological fluids plays an important role for evaluating the human health condition. This work proposes a disposable and simple microfluidic paperbased analytical device (µPAD) for the quantitative colorimetric detection of salivary Na+, K+ and Ca2+, relying on ion-selective optodes (ISOs). Nanoscale ISOs composed of pluronic F-127 surfactant-based micelles were used as a colorimetric indicator system, reducing the assay time compared to the previously reported bulk membrane approach. Devices consist of three functionalized paper layers assembled by lamination, with the optical signal read out from the bottom layer. Saliva was chosen as the biological specimen because it is painlessly collected and easy-to-handle, which makes it suitable for POC assays. Simultaneous Na+, K+ and Ca2+ detection by a single µPAD was successfully confirmed by using aqueous metal ion samples with various concentrations of Na+, K+ and Ca2+ within the biologically relevant range. The simultaneous detection of the three cationic species enabled the correction of the three ion quantities by eliminating the interference from other ions using the calculated selectivity coefficients. For proof-of-concept, Na+ quantification in a human saliva sample was conducted by the developed µPADs. The calibration curve for this assay was prepared by applying artificial saliva to the µPADs. The concentrations of Na+ in spiked human saliva were also measured by the µPADs. The obtained recovery rates were 78-132 %, which indicates the quantification of the added Na+ in the real sample matrix. Finally, this µPAD was successfully integrated into a smartphone-based assay, which enables salivary electrolyte quantification in resource poor settings. The developed µPADs satisfy the criteria of low-cost (< $0.3/device), rapidity (< 5 min) and simple analytical procedure compared to the conventional ion-selective electrode (ISE) method.

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Near-infrared pH responsive heptamethine cyanine platforms: Modulating the proton acceptor

Kurutos A., Shindo Y., Hiruta Y., Oka K., Citterio D.

Dyes and Pigments (Dyes and Pigments)  181 2020.10

ISSN  01437208

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In this report, we wish to present the rational design, synthesis, and study of optical characteristics of near-infrared (NIR) molecular probes emitting over 800 nm. The title pH sensing platforms consist of a piperazine moiety serving as a hydronium ion receptor, coupled with a rigidified symmetric heptamethine backbone. The spectrally fine-tuned response of the fluorophores was studied in both organic solvents and aqueous buffer solutions, employing combined UV–Vis and steady state fluorimetric techniques. In parallel, computational approach and optical spectroscopy were employed aiming to ascribe the origin of spectral behavior of the amino-substituted heptamethines. Herein, we also investigated the halochromic effect of the differently substituted piperazines on the spectral properties of the NIR cyanine dyes. Under acidic conditions, the protonation of the piperazine moiety promoted a gradual bathochromic shift in the absorption spectra. The Δλ between the neutral and the protonated forms are indirectly related to the modifications applied to the proton acceptor. The calculated pKa values (5.13–7.06) were found to be dependent on the dye chemical structure. A good linear correlation was established between the pKa of the newly synthesized dyes and that of the reported piperazine moieties. This aspect allows the future expansion of the current facile strategy since sensing chromophores with predicted pH response could readily be designed based on pKa databases for substituted piperazine derivatives. A detailed literature study along with NMR and computational methods were found to be in agreement with our assumption, that both nitrogen atoms of the 1,4-disubstituted amine are involved in the sensing process. The covered pKa range allows us to recommend the title fluorophores as promising potential candidates for qualitative analysis of biological samples. A cytotoxicity study by the MTT assay showed that the NIR dyes are safe and applicable for bioanalytical purposes at dye concentrations up to 10 μM. Fluorescence imaging and cellular applications, including staining and visualization of HeLa cervical cancer cells was successfully demonstrated.

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All-printed semiquantitative paper-based analytical devices relying on QR code array readout

Katoh A., Maejima K., Hiruta Y., Citterio D.

Analyst (Analyst)  145 ( 18 ) 6071 - 6078 2020.09

ISSN  00032654

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The wide spread of smartphones and QR codes for various end-user applications has had an impact beyond traditional fields of use, recently also reaching point-of-care testing (POCT). This work presents the integration of QR code recognition into paper-based analytical devices (PADs) with "distance-based"colorimetric signalling, resulting in semiquantitative readout fully relying on straightforward barcode reader solutions. PADs consist of an array of QR codes arranged in series inside a paperfluidic channel. A mask dye concept has been developed, which enables utilisation of colour changing indicators by initially hiding QR codes. The colour change of the indicator induced by the presence of an analyte of interest results in gradual unmasking of QR codes, which become recognisable by the smartphone barcode reader app. To reproducibly fabricate devices, all fabrication steps were performed by commercial desktop solid ink and inkjet printing. The QR code masking function was optimised by controlling the amount of printed mask dye through adjusting the opacity of printing patterns during the inkjet deposition process. For proof-of-concept, a model assay in the form of colorimetric copper ion (Cu2+) detection in the concentration range of 0.4 mM to 3.2 mM was evaluated. Consistent results independent of the smartphone model and environmental light condition were achieved with a free barcode reader app. To the best of our knowledge, this work is the first demonstration of a semiquantitative assay approach fully relying on QR code readout without digital colour analysis, customised app or hardware modification.

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Text-Displaying Semiquantitative Competitive Lateral Flow Immunoassay Relying on Inkjet-Printed Patterns

Misawa K., Yamamoto T., Hiruta Y., Yamazaki H., Citterio D.

ACS Sensors (ACS Sensors)  5 ( 7 ) 2076 - 2085 2020.07

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This work describes a colorimetric signaling approach for competitive lateral flow immunoassays (LFIAs) enabling sensitive and semiquantitative direct visual result readout in the form of "text", demonstrated on the example of 8-hydroxy-2′-deoxyguanosine (8-OHdG) detection. The distinctive feature of the developed text-displaying LFIA (TD-LFIA) is the test zone system consisting of a combination of two types of inkjet-deposited capture molecules referred to as "mask antigen"and "text antibody", allowing for sensitive turn-on signaling as opposed to the inverse response of conventional competitive LFIAs. The user operation is limited to sample application, followed by direct reading of assay results written in text after approximately 10 min. TD-LFIAs enabled the visual detection of 8-OHdG at concentrations down to 3 ng/mL, which is a 2-3 orders of magnitude lower visual detection limit than that achieved with the corresponding conventional design and is comparable to the existing LFIAs relying on external signal readout equipment. Highly reproducible observer-independent assay performance was confirmed, and the result interpretation is not influenced by sample color and readout timing. Making use of customizable threshold settings for text appearance, a device for semiquantitative assays was developed and successfully applied to the detection of 8-OHdG at four concentration levels (trace, low, medium, and high) in 54 human urine samples within the clinically relevant concentration range. The sensitive and intuitive signaling method of the developed system offers great potential for an alternative competitive LFIA platform suitable for real-world point-of-care testing applications.

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Acid-base titration using a microfluidic thread-based analytical device (μTAD)

Jarujamrus P., Prakobkij A., Puchum S., Chaisamdaeng S., Meelapsom R., Anutrasakda W., Amatatongchai M., Chairam S., Citterio D.

Analyst (Analyst)  145 ( 13 ) 4457 - 4466 2020.07

ISSN  00032654

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This work presents the development and application of a novel analytical approach for the determination of acid and base concentrations by titration using a microfluidic thread-based analytical device (μTAD). This approach proved to be a simple to fabricate and to use, high precision, and cost-efficient means of acid-base quantification. The μTAD was fabricated by immobilizing the untreated cotton threads onto a wood frame, followed by pre-coating with an indicator (20 μL) and a primary standard solution (3 μL), and was tested using real samples including drug, food, and household products where 3 μL of each sample was dropped onto the center of a thread. Afterward, the distance of color change on the thread, easily observed and measured using the naked eye and a ruler, was used for analysis. The analysis using the μTAD, completed within 2 minutes and validated by the conventional titration, showed high accuracy and precision (RSD < 12.9%), good linearity ranges and low limit of quantification. The fabricated μTAD also remained stable for an extended period of time (>2 weeks under various storage conditions).

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Thread-Based Bioluminescent Sensor for Detecting Multiple Antibodies in a Single Drop of Whole Blood

Tomimuro K., Tenda K., Ni Y., Hiruta Y., Merkx M., Citterio D.

ACS Sensors (ACS Sensors)  5 ( 6 ) 1786 - 1794 2020.06

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Antibodies are important biomarkers in clinical diagnostics in addition to being increasingly used for therapeutic purposes. Although numerous methods for their detection and quantification exist, they predominantly require benchtop instruments operated by specialists. To enable the detection of antibodies at point-of-care (POC), the development of simple and rapid assay methods independent of laboratory equipment is of high relevance. In this study, we demonstrate microfluidic thread-based analytical devices (μTADs) as a new platform for antibody detection by means of bioluminescence resonance energy-transfer (BRET) switching sensor proteins. The devices consist of vertically assembled layers including a blood separation membrane and a plastic film with a sewn-in cotton thread, onto which the BRET sensor proteins together with the substrate furimazine have been predeposited. In contrast to intensity-based signaling, the BRET mechanism enables time-independent, ratiometric readout of bioluminescence signals with a digital camera in a darkroom or a smartphone camera with a 3D-printed lens adapter. The device design allows spatially separated deposition of multiple bioluminescent proteins on a single sewn thread, enabling quantification of multiple antibodies in 5 μL of whole blood within 5 min. The bioluminescence response is independent of the applied sample volume within the range of 5-15 μL. Therefore, μTADs in combination with BRET-based sensor proteins represent user-friendly analytical tools for POC quantification of antibodies without any laboratory equipment in a finger prick (5 μL) of whole blood.

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Metal-Free Colorimetric Detection of Pyrophosphate Ions by Inhibitive Nanozymatic Carbon Dots

Chen C.Y., Tan Y.Z., Hsieh P.H., Wang C.M., Shibata H., Maejima K., Wang T.Y., Hiruta Y., Citterio D., Liao W.S.

ACS Sensors (ACS Sensors)  5 ( 5 ) 1314 - 1324 2020.05

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The pyrophosphate ion (P2O74-, PPi) plays a critical role in various biological processes and acts as an essential indicator for physiological mechanism investigations and disease control monitoring. However, most of the currently available approaches for PPi species detection for practical usage still lack appropriate indicator generation, straightforward detection requirements, and operation convenience. In this study, a highly sensitive and selective PPi detection approach via the use of nanozymatic carbon dots (CDs) is introduced. This strategy eliminates the common need for metal ions in the detection process, where a direct indicator-PPi interaction is adopted to provide straightforward signal reports, and importantly, through a green indicator preparation. The preparation of this nanozymatic CDs' indicator utilizes an aqueous solution refluxing, employing galactose and histidine as the precursor materials. The mild conditions of the solution refluxing produce fluorescent CDs exhibiting peroxidase-mimic properties, which can catalyze the o-phenylenediamine oxidation under the presence of H2O2. The introduction of PPi species, interestingly, inhibits this process very efficiently, the extent of which can be colorimetrically monitored by the generated yellow product 2,3-diaminophenazine. Spectroscopic results point to CD surface functional groups' selective binding toward PPi species, which severely interferes with the electron transfer process in the enzymatic catalysis. Relying on this CD peroxidase-mimetic property inhibition, sensitive and selective recognition of PPi reaches a detection limit of 4.29 nM, enabling practical usage in complex matrixes. Owing to the superior compatibility and high stability of nanozymatic CDs, they can also be inkjet-printed on paper-based devices to create a portable and convenient platform for PPi detection. Both the solution and the paper-device-based selective recognitions confirm this unique and robust metal-free inhibitive PPi detection, which is supported by a convenient green preparation of nanozymatic CDs.

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Quantitative evaluation of luminescence intensity from enzymatic luminescence reaction of coelenterazine and analogues

Ikeda Y., Tanaka M., Nishihara R., Hiruta Y., Citterio D., Suzuki K., Niwa K.

Journal of Photochemistry and Photobiology A: Chemistry (Journal of Photochemistry and Photobiology A: Chemistry)  394 2020.05

ISSN  10106030

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Recent advances in bioluminescence technology rely on the discovery of luciferin-luciferase pairs having excellent luminescence activity. Here, we focus on coelenterazine (CTZ) and its analogues and quantitatively investigate reaction factors, including bioluminescence reaction quantum yield (φBL) and enzyme reaction kinetics (turnover rate, kcat), determining the luminescence intensity to design brighter reaction systems. Initially, it was confirmed that the CTZ was kept stable during the storage and the φBL value measuring experiment in terms of comparative study using benzyl-protected CTZ. Using a luminometer whose absolute responsivity is calibrated for each luminescence reaction spectrum, we absolutely measured φBL and other reaction factors of CTZ and 3 types of blue-shifted CTZ analogues, CTZ 400a, 6-pi-H-CTZ and 6-pi-Ph-CTZ. The results suggested that there was no significant difference in reaction kinetics, but a clear difference in φBL (up to 22-fold), which was presumably due to an improvement in fluorescence quantum yield (φFL) of the corresponding coelenteramide (CTMD), oxidized form of CTZ. Our evaluation suggests that the design of CTZ analogues considering φFL is important for developing a bright luminescent reaction system.

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Inkjet-printed pH-independent paper-based calcium sensor with fluorescence signal readout relying on a solvatochromic dye

Shibata H., Ikeda Y., Hiruta Y., Citterio D.

Analytical and Bioanalytical Chemistry (Analytical and Bioanalytical Chemistry)  412 ( 14 ) 3489 - 3497 2020.05

ISSN  16182642

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A challenge for paper-based cation sensors relying on classical carrier-based ion-selective optodes (ISOs) is their pH-cross response caused by the use of H+-sensitive chromoionophores as optical signal transducers. This work demonstrates fully pH-independent fluorescence-based calcium detection with a paper-based plasticizer-free ISO. To achieve a pH-independent assay, a solvatochromic dye (SD) instead of a traditional H+-sensitive chromoionophore has been applied to the paper-based ISO by means of inkjet printing technology. The detection principle depends on an ionophore-driven phase-transfer ion-exchange reaction between target cations and the positively charged SD, which no longer involves H+ in the optical signal transduction process. The developed paper-based ISOs with the SD resulted in Ca2+ concentration-dependent response curves not affected by the sample pH (pH 6.0, 7.0, and 8.0). The dynamic range obtained for Ca2+ detection was from 10−5 to 1 mol L−1 with a detection limit of 19.3 μmol L−1. Additionally, excellent selectivity derived from the used ionophore has been confirmed. As a simple practical application, the determination of Ca2+ in mineral water has been achieved without the pH-buffering process required for conventional cation-exchange ISOs.

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Paper-Based Device for Naked Eye Urinary Albumin/Creatinine Ratio Evaluation

Hiraoka R., Kuwahara K., Wen Y.C., Yen T.H., Hiruta Y., Cheng C.M., Citterio D.

ACS Sensors (ACS Sensors)  5 ( 4 ) 1110 - 1118 2020.04

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This paper introduces the concept of "Drawing-PADs" (Drawing paper-based microfluidic analytical devices) allowing to intuitively evaluate the urinary albumin (Alb) index, a clinically important parameter used for the early detection of renal deficiencies related to diabetes, among others. To enable regular monitoring of the Alb index, a simple examination method suitable for self-diagnosis is highly desirable. The Drawing-PADs rely on the simultaneous naked eye detection of Alb and creatinine (Cre) on a single device according to the distance-based microfluidic PAD (μPAD) approach. The Alb index is visualized by simply drawing a straight line connecting the top of two color-changed assay channel sections (Alb and Cre channels), followed by visually confirming the position of the intercept of the drawn straight line. The semiquantitative Alb index evaluation performed with Drawing-PADs does not require any equipment such as a camera, software, or a color reference chart. The obtained results are independent of the sample volume and are not influenced by changes in the absolute Alb and Cre concentrations caused by urine excretion variations, making spot urine assays possible. Classification of Alb index values according to clinically relevant criteria (normoalbuminuria, microalbuminuria, and macroalbuminuria) is readily achieved within 15 min and has been validated for 15 human urine samples including diabetic patients and healthy volunteers.

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Centrifugal Paperfluidic Platform for Accelerated Distance-Based Colorimetric Signal Readout

Maejima K., Hiruta Y., Citterio D.

Analytical Chemistry (Analytical Chemistry)  92 ( 7 ) 4749 - 4754 2020.04

ISSN  00032700

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Distance-based readout is one of the most user-friendly and simple colorimetric signaling methods widely applied for paper-based analytical devices (PADs). This work presents the integration of distance readout PADs into a centrifugal platform enabling affordable, rapid, and sample volume-independent colorimetric assays. Centrifugally assisted distance-based PADs (CD-PADs) eliminate the requirement to use micropipets for sample introduction and reduce the overall time for distance-based assays. All device fabrication steps were performed through computer-controlled print, cut, and laminate (PCL) techniques oriented toward mass production. The inexpensive centrifugal platform was built on a recycled DVD player combined with an open source microcomputer (Arduino). Assay protocols, including rotational velocity and rotation time, were optimized to obtain a maximum dynamic range and reproducible results for sample volume metering (coefficient of variation 3.62%). A colorimetric Ni2+ assay chosen to demonstrate measurements on CD-PADs allowed the detection of nickel ions (Ni2+) with naked-eye interpretation within 1.5 min and a limit of detection (LOD) of 44.1 ng of Ni2+, which to the best of our knowledge is the lowest value reported for a distance-based Ni2+ assay.

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Ring-Fused Firefly Luciferins: Expanded Palette of Near-Infrared Emitting Bioluminescent Substrates

Ikeda Y., Nomoto T., Hiruta Y., Nishiyama N., Citterio D.

Analytical Chemistry (Analytical Chemistry)  92 ( 6 ) 4235 - 4243 2020.03

ISSN  00032700

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Firefly bioluminescence is broadly applied as a noninvasive imaging modality in the biomedical research field. One limitation in firefly bioluminescence imaging is the limited variety of luciferins emitting in the near-infrared (NIR) region (650-900 nm), where tissue penetration is high. Herein, we describe a series of structure-inherent NIR emitting firefly luciferin analogues, NIRLucs, designed through a ring fusion strategy. This strategy resulted in pH-independent structure-inherent NIR emission with a native firefly luciferase, which was theoretically supported by quantum chemical calculations of the oxidized form of each luciferin. When applied to cells, NIRLucs displayed dose-independent improved NIR emission even at low concentrations where the native d-luciferin substrate does not emit. Additionally, excellent blood retention and brighter photon flux (7-fold overall, 16-fold in the NIR spectral range) than in the case of d-luciferin have been observed with one of the NIRLucs in mice bearing subcutaneous tumors. We believe that these synthetic luciferins provide a solution to the longstanding limitation in the variety of NIR emitting luciferins and pave the way to the further development of NIR bioluminescence imaging platforms.

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Chemometric challenges in development of paper-based analytical devices: Optimization and image processing

Hamedpour V., Oliveri P., Leardi R., Citterio D.

Analytica Chimica Acta (Analytica Chimica Acta)  1101   1 - 8 2020.03

ISSN  00032670

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Although microfluidic paper-based analytical devices (μPADs) get a lot of attention in the scientific literature, they rarely reach the level of commercialization. One possible reason for this is a lack of application of machine learning techniques supporting the design, optimization and fabrication of such devices. This work demonstrates the potential of two chemometric techniques including design of experiments (DoE) and digital image processing to support the production of μPADs. On the example of a simple colorimetric assay for isoniazid relying on the protonation equilibrium of methyl orange, the experimental conditions were optimized using a D-optimal design (DO) and the impact of multiple factors on the μPAD response was investigated. In addition, this work demonstrates the impact of automatic image processing on accelerating color value analysis and on minimizing errors caused by manual detection area selection. The employed algorithm is based on morphological recognition and allows the analysis of RGB (red, green, and blue) values in a repeatable way. In our belief, DoE and digital image processing methodologies are keys to overcome some of the remaining weaknesses in μPAD development to facilitate their future market entry.

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Microfluidic Paper-Based Analytical Devices for Colorimetric Detection of Lactoferrin

Kudo H., Maejima K., Hiruta Y., Citterio D.

SLAS Technology (SLAS Technology)  25 ( 1 ) 47 - 57 2020.02

ISSN  24726303

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Lactoferrin is an abundant glycoprotein in human body fluids and is known as a biomarker for various diseases. Therefore, point-of-care testing (POCT) for lactoferrin is of interest. Microfluidic paper-based analytical devices (µPADs) have gained a lot of attention as next-generation POCT device candidates, due to their inexpensiveness, operational simplicity, and being safely disposable. This work presents a colorimetric sensing approach for quantitative lactoferrin analysis. The detection mechanism takes advantage of the high affinity of lactoferrin to ferric ions (Fe3+). Lactoferrin is able to displace an indicator from a colorimetric 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP)-Fe3+ complex, resulting in a color change. A 5-Br-PADAP-Fe3+ complex was encapsulated into water-dispersible poly(styrene-block-vinylpyrrolidone) particles, whose physical entrapment in the cellulosic fiber network results in the immobilization of the complex to the paper matrix. The complex-encapsulating particles showed a color change response in accordance with lactoferrin concentration. Both color intensity-based paper well plates and distance readout-based µPADs are demonstrated. Color intensity-based devices allowed quantitative analysis of lactoferrin concentrations with a limit of detection of 110 µg/mL, using a smartphone and a color readout app. On the other hand, distance readout-based µPADs showed changes of the length of colored sections in accordance with lactoferrin concentration. In summary, we successfully developed both colorimetric intensity-based paper wells and distance-based µPADs for lactoferrin detection. This work demonstrates a user-friendly colorimetric analysis platform for lactoferrin without requiring lab equipment and expensive antibodies.

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Near-infrared fluorescent probes for imaging of intracellular mg²⁺ and application to multi-color imaging of mg²⁺, atp, and mitochondrial membrane potential

Murata O., Shindo Y., Ikeda Y., Iwasawa N., Citterio D., Oka K., Hiruta Y.

Analytical Chemistry (Analytical Chemistry)  92 ( 1 ) 966 - 974 2020.01

ISSN  00032700

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The magnesium ion (Mg2+) is an essential cation to maintain proper cellular activities. To visualize the dynamics and functions of Mg2+, there is a great need for the development of Mg2+-selective fluorescent probes. However, conventional Mg2+ fluorescent probes are falling behind in low selectivity and poor fluorescence color variation. In this report, to make available a distinct color window for multi-color imaging, we designed and synthesized highly Mg2+-selective and near-infrared (NIR) fluorescent probes, the KMG-500 series consisting of a charged β-diketone as a selective binding site for Mg2+ and a Si-rhodamine residue as the NIR fluorophore, which showed photoinduced electron transfer (PeT)-type OFF-ON response to the concentration of Mg2+. Two types of KMG-500 series probes, tetramethyl substituted Si-rhodamine KMG-501 and tetraethyl substituted Si-rhodamine KMG-502, were synthesized for the evaluation of cell permeability. For intracellular application, the membrane-permeable acetoxymethyl derivative KMG-501 (KMG-501AM) was synthesized and allowed to stably stain cultured rat hippocampal neurons during imaging of intracellular Mg2+. On the other hand, KMG-502 was cell membrane permeable without AM modification, preventing the probe from staying inside cells during imaging. KMG-501 distributed mainly in the cytoplasm and partially localized in lysosomes and mitochondria in cultured rat hippocampal neurons. Mg2+ increase in response to the FCCP uncoupler inducing depolarization of the mitochondrial inner membrane potential was detected in the KMG-501 stained neurons. For the first time, KMG-501 succeeded in imaging intracellular Mg2+ dynamics with NIR fluorescence. Moreover, it allows one to simultaneously visualize changes in Mg2+ and ATP concentration and also mitochondrial inner membrane potential and their interactions. This probe is expected to be a strong tool for multi-color imaging of intracellular Mg2+

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Traffic-light-type microfluidic paper-based analytical device for hydrogen peroxide detection

Ohta S., Hiraoka R., Hiruta Y., Citterio D.

MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences)     444 - 445 2020

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This work reports an intuitive signaling approach enabling naked-eye semi-quantitative result readout in the form of a “traffic-light” for microfluidic paper-based analytical devices (µPADs). The detection of hydrogen peroxide has been demonstrated with the aim of quantifying metabolites relevant as biomarkers of various lifestyle-related diseases. User operation is limited to simple sample application, followed by observing the traffic-light signal after approximately 10 min. Due to its intuitiveness with very low probability of result misinterpretation, this system is expected to offer great potential for home-based self-diagnostic point-of-care testing (POCT) applications.

pH-Responsive Tunable Mixed-Charge Polymers for pH-Selective Interaction with Anionic Biological Constituents

Hiruta Y., Sawada K., Mizui Y., Citterio D.

Bulletin of the Chemical Society of Japan (Bulletin of the Chemical Society of Japan)  93 ( 4 ) 547 - 552 2020

ISSN  00092673

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A pH-responsive mixed-charge polymer consists of uniformly distributed cationic and anionic units in the polymer chain. Carboxylic acid monomers are generally used as anionic monomers. The pH responsive range is confined to a relatively narrow pH of Â5, which is limiting the biological application of pH-responsive polymers. Therefore, we investigated the effect of a series of carboxylic acid and neutral monomers on the pKa value of pH-responsive mixed-charge polymers. We synthesized the polymers using [2-(methacryloyloxy)ethyl]-trimethylammonium chloride, carboxylic acid monomers, and neutral monomers as the spacer moiety. As the monomer hydrophobicity and the composition ratio of neutral monomers increased, the pKa shifted to higher values. Finally, the polymer pKa values were tuned from 4.6 to 6.1. We confirmed that the pH-dependent interaction between anionic tissue constituent and pH-responsive mixed-charge polymers depends on their pKa.

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Cellophane-based analytical devices for pump-free liquid transport and background-tinted colorimetric assays

Shigemori H., Maejima K., Shibata H., Hiruta Y., Citterio D.

MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences)     422 - 423 2020

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This report suggests utilizing “cellophane” as a new substrate material for point-of-care testing (POCT) devices. Cellophane-based microfluidic devices were fabricated by a print, cut and lamination (PCL) methodology and sample liquid was transported into four assay areas without external pumping. Furthermore, making use of the transparency of cellophane, background-tinted devices were fabricated. Based on a blue background-tinted cellophane device, the dynamic range of a hue-based H2O2 assay was 46-fold increased compared to performing the same assay on a white background device. Finally, turbidity testing for albumin was implemented on black background-tinted devices with a visual detection limit of 15 mg/dL.

Fabrication and application of microfluidic paper-based analytical devices (μPAD)

Shigemori H., Hiruta Y., Citterio D.

Kinoshi Kenkyu Kaishi/Annals of the High Performance Paper Society, Japan (Kinoshi Kenkyu Kaishi/Annals of the High Performance Paper Society, Japan)  59   17 - 23 2020

ISSN  02885867

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Bio)chemical sensors based on paper substrates show attractive features such as being low cost, portable and disposable. Therefore, the technology is currently used in urinary dipsticks and immunochromatographic test strip. In recent years, many research groups report studies on microfluidic paper-based analytical devices (μPADs). In spite of the simplicity of paper-based platforms, μPADs enable performing complicated analytical procedures, multiplex target analysis and equipment-free detection. Due to these useful functions, μPADs are paid attention for point-of-care testing (POCT) and for field-deployable analysis. Herein, we introduce the fabrication methodology and clinical applications of μPADs developed by our research group.

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Ink-jet printed microfluidic paper-based analytical devices relying on QR code detection

Katoh A., Maejima K., Hiruta Y., Citterio D.

MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences)     456 - 457 2020

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Microfluidic paper-based analytical devices (µPADs) that display semi-quantitative analysis results by reading a QR code have been developed. The only user operation required is to apply the sample and to read the displayed QR code with a mobile device to obtain the result. Copper ions were detected as a model target and the copper ion concentration-dependent display of QR codes was achieved. This simple assay method can be applied to other targets and has great potential for point-of-care testing (POCT) applications.

Biothiol-activatable bioluminescent coelenterazine derivative for molecular imaging in vitro and in vivo

Nomura N., Nishihara R., Nakajima T., Kim S., Iwasawa N., Hiruta Y., Nishiyama S., Sato M., Citterio D., Suzuki K.

Analytical Chemistry (Analytical Chemistry)  91 ( 15 ) 9546 - 9553 2019.08

ISSN  00032700

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© 2019 American Chemical Society. There is a high demand for sensitive biothiol probes targeting cysteine, glutathione, and homocysteine. These biothiols are known as playing essential roles to maintain homeostasis and work as indicators of many diseases. This work presents a bioluminescent probe (named AMCM) to detect biothiols in live mammalian cells and in vivo with a limit of detection of 0.11 μM for cysteine in solution and high selectivity for biothiols, making it suitable for real-time biothiol detection in biological systems. Upon application to live cells, AMCM showed low cytotoxicity and sensitively reported bioluminescence in response to changes of biothiol levels. Furthermore, a bioluminescence resonance energy transfer system consisting of AMCM combined with the near-infrared fluorescent protein iRFP713 was applied to in vivo imaging, with emitted tissue-permeable luminescence in living mice. In summary, this work demonstrates that AMCM is of high practical value for the detection of biothiols in living cells and for deep tissue imaging in living animals.

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Disposable electrochemical biosensor based on surface-modified screen-printed electrodes for organophosphorus pesticide analysis

Hua Q., Ruecha N., Hiruta Y., Citterio D.

Analytical Methods (Analytical Methods)  11 ( 27 ) 3439 - 3445 2019.07

ISSN  17599660

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© 2019 The Royal Society of Chemistry. A disposable enzymatic electrochemical biosensor for amperometric organophosphorus pesticide determination based on a screen-printed carbon electrode (SPCE) platform was successfully developed. The working electrode consists of acetylcholine esterase (AChE) immobilized onto the surface of multi-walled carbon nanotube (MWCNT), chitosan (CS) and gold nanoparticle (AuNP)-modified SPCE. Ferricyanide was used as a redox mediator in solution. Two linear dynamic response ranges from 0.01 to 10 μg L-1 and from 10 to 100 μg L-1 were found for paraoxon-ethyl, with a detection limit of 0.03 μg L-1 (calculated as the amount of pesticide resulting in 10% of enzyme inhibition). High sensitivity was achieved due to a synergistic effect of AuNPs and MWCNTs deposited on the SPCE surface. Moreover, 83% of enzyme activity was retained after a dry storage period of 49 days (4 °C). Finally, the results of the electrochemical biosensor for paraoxon-ethyl-spiked spinach sample analysis showed a good agreement with those obtained from conventional HPLC.

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Molecular imaging of retinoic acids in live cells using single-chain bioluminescence probes

Kim S., Fujii R., Nishihara R., Bose R., Citterio D., Suzuki K., Massoud T., Paulmurugan R.

ACS Combinatorial Science (ACS Combinatorial Science)  21 ( 6 ) 473 - 481 2019.06

ISSN  21568952

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© 2019 American Chemical Society. Retinoic acid (RA) is a key metabolite necessary for embryonic development and differentiation in vertebrates. We demonstrate the utility of genetically encoded, ligand-activatable single-chain bioluminescence probes for detecting RAs from different biological sources. We examined 13 different molecular designs to identify an efficient single-chain probe that can quantify RA with significant sensitivity. The optimal probe consisted of four components: the N- and C-terminal fragments of artificial luciferase variant-16 (ALuc16), the ligand binding domain of retinoic acid receptor α (RARα LBD), and an LXXLL interaction motif. This probe showed a 5.2-fold greater bioluminescence intensity in response to RA when compared to the vehicle control in live mammalian cells. The probe was highly selective to all-trans-RA (at-RA), and highly sensitive in determining at-RA levels in cells derived from tumor xenografts created using MDA-MB-231 cells engineered to stably express the probe. We also detected RA levels in serum and cerebrospinal fluid. Using this probe, the detection limit for at-RA was 10-9.5 M, with a linear range of two orders. We present a highly useful technique to quantitatively image endogenous at-RA levels in live mammalian cells expressing novel single-chain bioluminescence probes.

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Printed low-cost microfluidic analytical devices based on a transparent substrate

Fujisaki S., Shibata H., Yamada K., Suzuki K., Citterio D.

Analyst (Analyst)  144 ( 8 ) 2746 - 2754 2019.04

ISSN  00032654

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© 2019 The Royal Society of Chemistry. This work describes the development of a microfluidic analytical device prepared on a transparent OHP film substrate, named the microfluidic transparent film-based analytical device (μTFAD). Printing technologies including wax printing for microchannel patterning and inkjet printing for chemical assay component deposition have been employed for the μTFAD fabrication. The fully printed μTFAD allowed gravity-assisted pump-free transportation of the sample liquid (50 μL) and an absorbance measurement-based iron ion (Fe 2+ ) assay using nitroso-PSAP as the colorimetric reagent within a wax-patterned microfluidic structure. By measuring absorbance values at the Fe 2+ -nitroso-PSAP complex-specific wavelength (756 nm), a response curve with a linear range of 0-200 μM was obtained. The limit of detection (1.18 μM) obtained with the proposed μTFADs was comparable to the results achieved with a conventional 96-well microplate assay (0.92 μM) and lower than that in the case of digital colour analysis-assisted filter paper spot tests (7.71 μM) or the absorbance analysis of refractive index-matched translucent filter paper spots (37.2 μM). In addition, highly selective Fe 2+ detection has been achieved in the presence of potentially interfering metal ions (Cu 2+ , Co 2+ , Ni 2+ ) without the use of any masking reagents, owing to the selection of the target complex-specific wavelength in the absorbance measurement on μTFADs.

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Equipment-Free Detection of K ⁺ on Microfluidic Paper-Based Analytical Devices Based on Exhaustive Replacement with Ionic Dye in Ion-selective Capillary Sensors

Soda Y., Citterio D., Bakker E.

ACS Sensors (ACS Sensors)  4 ( 3 ) 670 - 677 2019.03

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© 2019 American Chemical Society. A distance-based analysis of potassium ion (K + ) is introduced that is performed on a microfluidic paper-based analytical device (μPAD) coupled to an ion-selective capillary sensor. The concept is based on two sequential steps, the selective replacement of analyte ion with an ionic dye, and the detection of this dye in a distance-based readout on paper. To achieve the first step, the capillary sensor holds a poly(vinyl chloride) (PVC) membrane film layer plasticized by dioctyl sebacate (DOS) that contains the potassium ionophore valinomycin, a lipophilic cation-exchanger and the ionic indicator Thioflavin T (ThT) on its inner wall. Upon introduction of the sample, K + in the aqueous sample solution is quantitatively extracted into the film membrane and replaced with ThT. To convert the ion exchange signal into a distance-based analysis, this solution was dropped onto the inlet area of a μPAD to flow the ThT along a channel defined by wax printing, resulting in the electrostatic binding of ThT to the cellulose carboxylic groups. The initial amount of K + determines the amount of ThT in the aqueous solution after ion-exchange, and consequently the distance of ThT-colored area reflects the sample K + concentration. The ion exchange reaction was operated in a so-called "exhaustive sensing mode" and gave a distinct response in a narrow range of K + concentration (1-6 mM) that cannot be achieved by the classical optode sensing mode. The absence of hydrogen ions from the equilibrium competition of the capillary sensor contributed to a complete pH-independence, unlike conventional optodes that contain a pH sensitive indicator. A very high selectivity for K + over Na + and Ca 2+ has been confirmed in separate solutions and mixed solutions tests. K + measurements in pooled serum samples at concentrations between 2 and 6 mM are successfully demonstrated on a temperature controlled support.

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Fully inkjet-printed distance-based paper microfluidic devices for colorimetric calcium determination using ion-selective optodes

Shibata H., Hiruta Y., Citterio D.

Analyst (Analyst)  144 ( 4 ) 1178 - 1186 2019.02

ISSN  00032654

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© 2019 The Royal Society of Chemistry. Although the determination of calcium ions (Ca 2+ ) is of high importance to monitor water hardness, currently available devices for on-site analysis suffer from a lack of user-friendliness and sensitivity. This work demonstrates fully inkjet-printed and low-cost microfluidic paper-based analytical devices (μPADs) for the simple naked-eye colorimetric determination of calcium ions (Ca 2+ ) in drinking and tap water samples. The quantification of Ca 2+ relies on visual readout of the length of a colour-changed detection channel modified with ionophore-doped ion-selective optode nanospheres (nano-optodes), eliminating the requirement of a scanner or a camera. All fabrication steps for deposition of assay reagents have been performed by means of a simple desktop thermal inkjet printer, which is expected to contribute to highly batch-to-batch reproducible device preparation. The detectable Ca 2+ concentrations between 0.05 mmol L -1 and 5 mmol L -1 cover the range recommended by the International Organization for Standardization (0.05-2.5 mmol L -1 ) and the World Health Organization (WHO) guideline for Ca 2+ quantification in drinking water (less than 5 mmol L -1 ). The lowest concentration of Ca 2+ detectable by the naked eye was found to be 0.05 mmol L -1 , which is below the value achieved with previously reported paper-based devices. μPAD quantified Ca 2+ concentrations in tap or drinking waters were within 15% error of the results obtained with a classical complexometric titration. Hence, distance-based μPADs relying on nano-optodes are sensitive and reproducible tools for equipment-free on-site assaying of Ca 2+ in real samples.

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A novel and innovative paper-based analytical device for assessing tear lactoferrin of dry eye patients

Sonobe H., Ogawa Y., Yamada K., Shimizu E., Uchino Y., Kamoi M., Saijo Y., Yamane M., Citterio D., Suzuki K., Tsubota K.

Ocular Surface (Ocular Surface)  17 ( 1 ) 160 - 166 2019.01

ISSN  15420124

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© 2018 The Authors Purpose: To elucidate the correlation between lactoferrin concentration in the tear film and signs and symptoms of severe dry eye disease (DED) using a novel microfluidic paper-based analytical device (μPAD) and enzyme-linked immunosorbent assay (ELISA). Methods: Twenty-four patients were recruited at the Keio University Hospital. Using a novel μPAD, lactoferrin concentrations were measured in 4 patients with GVHD-related DED, 3 patients with other types of DED and 2 controls (Group A). For validation by ELISA, 22 patients (7 patients from Group A) comprising 9 patients with GVHD-related DED, 6 patients with other types of DED and 7 controls were examined (Group B). The link between lactoferrin concentration and clinical data about the severity of aqueous tear deficient DED was also investigated by both μPAD and ELISA. Results: The lactoferrin concentration in tear fluid of the DED patients was positively correlated between μPAD and ELISA (p = 0.006, r = 0.886). The tear fluid of the GVHD patients showed low or undetectable lactoferrin concentration. Analysis by ELISA demonstrated that lactoferrin concentrations in the tear film from the GVHD patients were significantly lower than those from the non-GVHD patients (p = 0.010576). ELISA revealed lactoferrin concentration correlated with the value of Schirmer test and tear film breakup time, whereas it was inversely correlated with OSDI, fluorescein and rose bengal scores. Conclusions: The novel μPAD may pave the way for measuring lactoferrin concentration in tear fluid from DED patients. Our results suggested that lactoferrin concentration in tear fluid reflect the severity of DED.

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In vitro determination of Rapamycin-triggered FKBP-FRB interactions using a molecular tension probe

Kim S., Nishihara R., Fujii R., Paulmurugan R., Citterio D., Suzuki K.

Analytical Sciences (Analytical Sciences)  35 ( 1 ) 71 - 78 2019

ISSN  09106340

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© The Japan Society for Analytical Chemistry. As protein-protein interactions (PPI) have been mostly investigated in cellulo or in vivo, it is unclear whether the PPI-based imaging schemes are practically valid in a bioanalytical means in vitro. The present study exemplifies the PPI in vitro inside a unique single-chain probe, named TP2.4, which carries a full-length artificial luciferase (ALuc) sandwiched in between two model proteins of interest, e.g., FKBP and FRB, expressed in E. coli, and purified. We found that the TP2.4 efficiently recognizes its ligand in vitro and varies its molecular kinetics: i.e., rapamycin boosts the enzymatic affinities (K m ) of TP2.4 to its substrates, but does not or only weakly influences the turnover rates (K cat ) and the maximal velocity (V max ). The corresponding circular dichroism (CD) study shows that rapamycin weakly contributes to the enhancement of the α-helical contents in TP2.4. Kinetic constants according to the substrates revealed that a coelenterazine derivative, 6-N 3 -CTZ, exerted the best catalytic efficiency and the greatest variance in the total photon counts. The present study is the first in vitro example that demonstrates how intramolecular PPI works in a purified single-chain bioluminescent probe and what factors practically influence the biochemistry.

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Flow control-based 3D μPADs for organophosphate pesticide detection

Hua Q., Shibata H., Hiruta Y., Citterio D.

Analytical Sciences (Analytical Sciences)  35 ( 4 ) 393 - 399 2019

ISSN  09106340

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© 2019 The Japan Society for Analytical Chemistry. Flow control-based paper devices have recently shown great potential for point-of-need analysis, since they allow for the easy operation of multi-step assays by minimizing user operation. In this work, a wax printing method was evaluated as a means to control liquid flow in 3D microfluidic paper-based analytical devices (μPADs). The resulting flow controlbased 3D μPADs were applied to determine paraoxon-ethyl as a typical organophosphate pesticide model system. The analytical procedure is as simple as applying a 200-μL sample solution, resulting in reproducible (relative standard deviation of colorimetric signals from 6 independently fabricated devices, 2.63%) colorimetric signals within 1 h of the assay time with the limit of detection (LOD) reaching 25.0 μg/L. Finally, results obtained for pesticide-spiked water samples analyzed by flow control-based 3D μPADs showed good agreement with those from a conventional HPLC analysis with UV detection.

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Near-Infrared Bioluminescence Imaging with a through-Bond Energy Transfer Cassette

Abe M., Nishihara R., Ikeda Y., Nakajima T., Sato M., Iwasawa N., Nishiyama S., Paulmurugan R., Citterio D., Kim S., Suzuki K.

ChemBioChem (ChemBioChem)  20 ( 15 ) 1919 - 1923 2019

ISSN  14394227

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© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim A coelenterazine (CTZ) analogue emitting near-infrared (NIR) bioluminescence was synthesized for through-bond energy transfer (TBET)-based imaging modalities. The analogue, named Cy5-CTZ, was prepared by conjugating cyanine-5 (Cy5) dye to CTZ through an acetylene linker. This novel derivative is intrinsically fluorescent and emits NIR-shifted luminescence upon reacting with an appropriate luciferase, the Renilla luciferase. This Cy5-CTZ substrate is optically stable in physiological samples and rapidly permeabilize through the plasma membrane into the cytosolic compartment of live cells.

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Inkjet-Printed Colorimetric Paper-Based Gas Sensor Arrays for the Discrimination of Volatile Primary Amines with Amine-Responsive Dye-Encapsulating Polymer Nanoparticles

Shibata H., Ikeda Y., Citterio D.

Methods in Molecular Biology (Methods in Molecular Biology)  2027   101 - 114 2019

ISSN  10643745

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© Springer Science+Business Media, LLC, part of Springer Nature 2019. Arrays of gas sensors are of high interest for “electronic nose” applications. Here, we describe the fabrication of a colorimetric single-use gas sensor array made of paper allowing the discrimination of volatile primary amines based on their polarity. For this purpose, polymeric nanoparticles with different polarities containing an amine-sensitive chromogenic dye are deposited onto paper substrates by means of inkjet printing. Data processing is conducted by red-green-blue (RGB) color extraction, followed by principal component analysis (PCA) or agglomerative hierarchical clustering (AHC) analysis. The application to the discrimination of two cheese samples is demonstrated.

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Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo

Nishihara R., Paulmurugan R., Nakajima T., Yamamoto E., Natarajan A., Afjei R., Hiruta Y., Iwasawa N., Nishiyama S., Citterio D., Sato M., Kim S., Suzuki K.

Theranostics (Theranostics)  9 ( 9 ) 2646 - 2661 2019

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© Ivyspring International Publisher. Background: Bioluminescence imaging (BLI) is one of the most widely used optical platforms in molecular imaging, but it suffers from severe tissue attenuation and autoluminescence in vivo. Methods: Here, we developed a novel BLI platform on the basis of bioluminescence resonance energy transfer (BRET) for achieving a ∼300 nm blue-to-near infrared shift of the emission (NIR-BRET) by synthesizing an array of 18 novel coelenterazine (CTZ) derivatives, named “Bottle Blue (BBlue)” and a unique iRFP-linked RLuc8.6-535SG fusion protein as a probe. Results: The best NIR-BRET was achieved by tuning the emission peaks of the CTZ derivatives to a Soret band of the iRFP. In mammalian cells, BBlue2.3, one of the CTZ derivatives, emits light that is ∼50-fold brighter than DBlueC when combined with RLuc8.6-535SG, which shows stable BL kinetics. When we used a caged version of BBLue2.3, it showed a BL half decay time of over 60 minutes while maintaining the higher signal sensitivity. This NIR BL is sufficiently brighter to be used for imaging live mammalian cells at single cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. A single-chain probe developed based on this BLI platform allowed us to sensitively image ligand antagonist-specific activation of estrogen receptor in the NIR region. Conclusion: This unique optical platform provides the brightest NIR BLI template that can be used for imaging a diverse group of cellular events in living subjects including protein‒protein interactions and cancer metastasis.

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A biomimetic hybrid material consisting of CaCO<inf>3</inf> mesoporous microspheres and an alternating copolymer for reversed-phase HPLC

Mochida M., Nagai Y., Kumagai H., Imai H., Citterio D., Hiruta Y.

Journal of Materials Chemistry B (Journal of Materials Chemistry B)  7 ( 31 ) 4771 - 4777 2019

ISSN  20507518

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This journal is © The Royal Society of Chemistry. We developed a biomineral-inspired hybrid material composed of CaCO3 and an organic polymer as a column packing material for HPLC. This material combines a hierarchical mesoporous structure and the functionality of the polymer. The surface of monodispersed mesoporous CaCO3 microspheres was modified with poly(maleic acid-alt-1-octadecene) (PMAcO) comprising hydrophobic alkyl chains and anionic carboxylate groups. PMAcO adsorbed onto the surface of CaCO3 through electrostatic interaction between Ca2+ sites and carboxylate groups, resulting in an octadecene coated microsphere interface. These microspheres were applied as a HPLC column and exhibited reversed-phase retention behavior in the separation of alkylbenzenes. This column showed high alkaline mobile phase resistance compared with the conventionally applied ODS column packing material. Quantitative analysis of the basic antidepressants clomipramine and imipramine spiked into whole blood was achieved with an alkaline mobile phase, demonstrating the potential of the biomineral-inspired material as a HPLC stationary phase for practical applications in routine analyses of basic drugs requiring alkaline mobile phases.

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Single-step bioluminescence lateral flow immunoassays for diagnostics

Shimazu R., Kawahara J., Tomimuro K., Misawa K., Ni Y., Hiruta Y., Merkx M., Citterio D.

23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019 (23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019)     703 - 704 2019

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We present two diagnostic platforms, a microfluidic thread-based and a conventional immunochromatographic strip approach, for single-step lateral flow immunoassays (LFIAs) with bioluminescence (BL) signaling requiring only sample application and detection of emission color changes by a digital camera. Both rely on bioluminescence resonance energy transfer (BRET)-switching proteins. The detection of three clinically relevant targets is demonstrated: Cetuximab as example of an antibody-based anticancer drug requiring monitoring of blood drug levels in patients, and HIV1-p17 antibodies and antigens as examples of infectious disease markers.

Distance readout-based paper devices for multiplexed urinalysis

Sawano R., Hiroyuki S., Maejima K., Hiruta Y., Citterio D.

23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019 (23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019)     716 - 717 2019

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This work reports a basic study to simplify urinalysis, using distance-based microfluidic paper-based analytical devices (D-µPADs) for multiplexed detection. Intuitive detection of albumin (Alb), glucose (Glu) and hemoglobin (Hb) in artificial urine samples is demonstrated by a colorimetric distance-based readout method. The response ranges for the three analytes (15-1000 mg/dL, 25-2000 mg/dL, 0.25-1.0 mg/dL), fully cover the physiologically relevant domain. User operation is limited to sample application and readout of the color-changed length corresponding to the analyte concentration.

Paper-based device with integrated ion-selective optodes for colorimetric quantification of salivary metal ions

Suenaga Y., Shibata H., Hiruta Y., Citterio D.

23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019 (23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019)     697 - 698 2019

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A disposable and simple PPAD (microfluidic paper-based analytical device) for the quantitative colorimetric detection of salivary cation species, relying on ISOs (ion-selective optodes) is introduced. This work demonstrates the applicability of ISO-integrated PPADs to real saliva samples. Calibration enabling accurate salivary cation quantification was achieved by using artificial saliva. Multiple sensing areas on a single device can detect three different types of ions within 3 min by simple application of 20 PL sample. Three kinds of ISOs were optimized for salivary Na+, K+ and Ca2+ quantification. For proof-of-concept, salivary Na+ concentration was accurately quantified by the developed PPAD.

Paper-Based Antibody Detection Devices Using Bioluminescent BRET-Switching Sensor Proteins

Tenda K., van Gerven B., Arts R., Hiruta Y., Merkx M., Citterio D.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  57 ( 47 ) 15369 - 15373 2018.11

ISSN  14337851

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© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This work reports on fully integrated “sample-in-signal-out” microfluidic paper-based analytical devices (μPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET-based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume-independent and fully reagent-free operation, including on-device blood plasma separation. User operation is limited to the application of a single drop (20–30 μL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti-HIV1, anti-HA, and anti-DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user-friendly point-of-care testing in low-resource environments.

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Azide- and Dye-Conjugated Coelenterazine Analogues for a Multiplex Molecular Imaging Platform

Nishihara R., Hoshino E., Kakudate Y., Kishigami S., Iwasawa N., Sasaki S., Nakajima T., Sato M., Nishiyama S., Citterio D., Suzuki K., Kim S.

Bioconjugate Chemistry (Bioconjugate Chemistry)  29 ( 6 ) 1922 - 1931 2018.06

ISSN  10431802

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© 2018 American Chemical Society. Native coelenterazine (nCTZ) is a common substrate to most marine luciferases and photoproteins. In this study, nine novel dye- and azide-conjugated CTZ analogues were synthesized by conjugating a series of fluorescent dyes or an azide group to the C-2 or C-6 position of the nCTZ backbone to obtain bulkiness-driven substrate specificity and potential chemiluminescence/bioluminescence resonance energy transfer (C/BRET). The investigation on the optical properties revealed that azide-conjugated CTZs emit greatly biased bioluminescence to ALucs and ca. 130 nm blue-shifted bioluminescence with RLuc8.6 in living animal cells or lysates. The corresponding kinetic study explains that azide-conjugated CTZ exerts higher catalytic efficiency than nCTZ. Nile red-conjugated CTZ completely showed red-shifted CRET spectra and characteristic BRET spectra with artificial luciferase 16 (ALuc16). No or less spectral overlap occurs among [Furimazine-NanoLuc], [6-N 3 -CTZ-ALuc26], [6-pi-OH-CTZ-RLuc8.6], and [6-N 3 -CTZ-RLuc8.6] pairs, because of the substrate-driven luciferase specificity and color shifts, providing a crosstalk-free multiplex bioassay platform. The unique bioluminescence system appends a new toolbox to bioassays and multiplex molecular imaging platforms. This study is the first example that systematically synthesized fluorescent dye-conjugated CTZs and applied them for a bioluminescence assay system.

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Fabrication of paper-based analytical devices optimized by central composite design

Hamedpour V., Leardi R., Suzuki K., Citterio D.

Analyst (Analyst)  143 ( 9 ) 2102 - 2108 2018.05

ISSN  00032654

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© 2018 The Royal Society of Chemistry. In this work, an application of a design of experiments approach for the optimization of an isoniazid assay on a single-area inkjet-printed paper-based analytical device (PAD) is described. For this purpose, a central composite design was used for evaluation of the effect of device geometry and amount of assay reagents on the efficiency of the proposed device. The factors of interest were printed length, width, and sampling volume as factors related to device geometry, and amounts of the assay reagents polyvinyl alcohol (PVA), NH 4 OH, and AgNO 3 . Deposition of the assay reagents was performed by a thermal inkjet printer. The colorimetric assay mechanism of this device is based on the chemical interaction of isoniazid, ammonium hydroxide, and PVA with silver ions to induce the formation of yellow silver nanoparticles (AgNPs). The in situ-formed AgNPs can be easily detected by the naked eye or with a simple flat-bed scanner. Under optimal conditions, the calibration curve was linear in the isoniazid concentration range 0.03-10 mmol L -1 with a relative standard deviation of 3.4% (n = 5 for determination of 1.0 mmol L -1 ). Finally, the application of the proposed device for isoniazid determination in pharmaceutical preparations produced satisfactory results.

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Selective Detection of K⁺ by Ion-Selective Optode Nanoparticles on Cellulosic Filter Paper Substrates

Soda Y., Shibata H., Yamada K., Suzuki K., Citterio D.

ACS Applied Nano Materials (ACS Applied Nano Materials)  1 ( 4 ) 1792 - 1800 2018.04

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This work describes the generation of plasticized poly(vinyl chloride)-based ion-selective optode (ISO) nanoparticles by a piezoelectric inkjet dispensing device. The monodisperse inkjet-generated particles (polydispersity index: 0.177) with an average hydrodynamic diameter of 202 nm are suitable for the highly reproducible deposition onto cellulosic filter paper substrates, resulting in paper-based analytical devices (PADs) for colorimetric cation detection, as demonstrated on the example of potassium-responsive ISOs. In contrast to the deposition of ISO bulk membranes from organic solutions requiring specialized printing equipment, the ISO nanoparticles can be inkjet-deposited from aqueous dispersions by conventional office inkjet printing. The obtained ISO PADs showed highly reproducible (relative standard deviations of the hue mean values in the range of 0.06-0.83% for three independently fabricated batches) and immediate (t95% ≤ 1 min) response to aqueous K+ solutions buffered at pH 7.4, with high selectivity over Li+, Na+, Ca2+, and Mg2+ (log KK+,Mz+opt ≤ -5.2 (Li+); -3.6 (Na+); < -5.0 (Ca2+); < -5.2 (Mg2+)). In addition, causes for the distinctively different response observed for ISO nanoparticles in the liquid phase and on paper substrates were considered. For this purpose, several parameters related to the cation exchange reaction mechanism of ISOs were experimentally evaluated, allowing to semiquantitatively discuss the paper substrate-specific response of ISOs.

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Chemometrics-Assisted Microfluidic Paper-Based Analytical Device for the Determination of Uric Acid by Silver Nanoparticle Plasmon Resonance

Hamedpour, Vahid, Postma, Geert J., van den Heuvel, Edwin, Jansen, Jeroen J., Suzuki, Koji, Citterio, Daniel

Analytical and Bioanalytical Chemistry (Springer)  410 ( 9 ) 2305 - 2313 2018.03

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1618-2642

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© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. This manuscript reports on the application of chemometric methods for the development of an optimized microfluidic paper-based analytical device (μPAD). As an example, we applied chemometric methods for both device optimization and data processing of results of a colorimetric uric acid assay. Box–Behnken designs (BBD) were utilized for the optimization of the device geometry and the amount of thermal inkjet-deposited assay reagents, which affect the assay performance. Measurement outliers were detected in real time by partial least squares discriminant analysis (PLS-DA) of scanned images. The colorimetric assay mechanism is based on the on-device formation of silver nanoparticles (AgNPs) through the interaction of uric acid, ammonia, and poly(vinyl alcohol) with silver ions under mild basic conditions. The yellow color originating from visible light absorption by localized surface plasmon resonance of AgNPs can be detected by the naked eye or, more quantitatively, with a simple flat-bed scanner. Under optimized conditions, the linearity of the calibration curve ranges from 0.1–5 × 10−3 mol L−1 of uric acid with a limit of detection of 33.9 × 10−6 mol L−1 and a relative standard of deviation 4.5% (n = 3 for determination of 5.0 × 10−3 mol L−1 uric acid). [Figure not available: see fulltext.].

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Highly Sensitive Bioluminescent Probe for Thiol Detection in Living Cells

Hemmi, Mayu, Ikeda, Yuma, Shindo, Yutaka, Nakajima, Takahiro, Nishiyama, Shigeru, Oka, Kotaro, Sato, Moritoshi, Hiruta, Yuki, Citterio, Daniel, Suzuki, Koji

Chemistry - An Asian Journal (Wiley-VCH)  13 ( 6 ) 648 - 655 2018.03

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1861-4728

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© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim The sensitive detection of thiols including glutathione and cysteine is desirable owing to their roles as indispensable biomolecules in maintaining intracellular biological redox homeostasis. Herein, we report the design and synthesis of SEluc-1 (sulfinate ester luciferin), a chemoselective probe exhibiting a ratiometric and turn-on response towards thiols selectively in fluorescence and bioluminescence, respectively. The probe, which was designed based on the “caged” luciferin strategy, displays excellent selectivity, high signal/noise ratio (>240 in the case of bioluminescence), and a biologically relevant limit of detection (LOD, 80 nm for cysteine), which are all desirable traits for a sensitive bioluminescent sensor. SEluc-1 was further applied to fluorescence imaging of thiol activity in living human cervical cancer HeLa cell cultures, and was successfully able to detect fluctuations in thiol concentrations induced by oxidative stress in a bioluminescent assay utilizing African green monkey fibroblast COS-7 cells and human breast adenocarcinoma MCF-7 cells.

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Simplified Determination of Complex Stoichiometry for Colorimetric Metal Indicators by Inkjet Printing

Kuwahara, Kento, Yamada, Kentaro, Suzuki, Koji, Citterio, Daniel

Analyst (The Royal Society of Chemistry)  143 ( 5 ) 1234 - 1241 2018.03

Research paper (scientific journal), Accepted,  ISSN  0003-2654

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© 2018 The Royal Society of Chemistry. This paper presents the determination of complex binding stoichiometry for colorimetric metal indicators according to the Job plot method by means of an office inkjet printer in combination with digital colour analysis. In contrast to the conventional solution-based analysis involving manual preparation of many mixed solutions, the elaborated inkjet-based approach allows convenient automated mixing of metal salt and indicator solutions in various ratios on a paper substrate. Inkjet-deposited liquid volumes and mixing ratios were controlled by simple colour value setting on a PC. For this purpose, a custom printing colour profile has been elaborated to achieve a linear relationship between the software set CMYK (Cyan, Magenta, Yellow and Key plate (Black)) colour values and the amount of deposited ink. The inkjet-based Job plot analysis was validated against the solution-based conventional approach for binding stoichiometry determination of five known metal ion-indicator systems. Simple colour scanning and colour analysis of printed spots on filter paper gave accurate results for five different stoichiometries and colorimetric response patterns. This inkjet printing approach using a customized printing colour profile allows rapid, flexible and precise deposition and mixing of reagent solutions, and thus, is expected to be useful for wide application purposes, which require the controlled deposition of reagents on a substrate.

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Implementation of a Plasticized PVC-Based Cation-Selective Optode System into a Paper-Based Analytical Device for Colorimetric Sodium Detection

Shibata, Hiroyuki, Henares, Terence G., Yamada, Kentaro, Suzuki, Koji, Citterio, Daniel

Analyst (The Royal Society of Chemistry)  143 ( 3 ) 678 - 686 2018.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0003-2654

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© 2018 The Royal Society of Chemistry. On the example of a colorimetric sodium assay, this work demonstrates the implementation of a classical cation-exchange optode relying on an ionophore-doped plasticized PVC membrane into a paper-based analytical device (PAD). An ion-selective optode (ISO) system has been arranged into a vertically-assembled PAD (vPAD) integrating a pH-buffering function. Capillary force-driven sample liquid transportation through the paper matrix enabled pH-adjustment prior to the optical detection of the analyte cation. Functionalized paper layers with inkjet-deposited ISO membranes were combined with whole device lamination to attain a stable ion-exchange equilibrium required for the theoretical behavior of ISOs. Whole device lamination limited rapid evaporation of sample liquid on vPADs to avoid an increase of target concentration. Sigmoidal response curves between 10-5 and 1 M of Na+ at pH 5.0-7.0 have been confirmed on vPADs, following the theory defined by the cation-exchange equilibrium reaction. Finally, the influence of the cellulosic paper substrate matrix acting as a cation-exchanger on the optode response behavior has been evaluated and compared with conventional plastic film optodes.

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An Allylated Firefly Luciferin Analogue with Luciferase Specific Response in Living Cells

Ikeda, Yuma, Saitoh, Tsuyoshi, Niwa, Kazuki, Nakajima, Takahiro, Kitada, Nobuo, Maki, Shojiro A., Sato, Moritoshi, Citterio, Daniel, Nishiyama, Shigeru, Suzuki, Koji

Chemical Communications (The Royal Society of Chemistry)  54 ( 14 ) 1774 - 1777 2018.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1359-7345

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© The Royal Society of Chemistry 2018. An allylated firefly luciferin was successfully synthesized and its bioluminescence properties were evaluated. When applied to cellular imaging in combination with Eluc, which is one of the commercially available luciferases, this analogue displayed a luciferase-specific bioluminescence signal with prolonged emission (>100 min).

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Quantitative Evaluation of Analyte Transport on Microfluidic Paper-Based Analytical Devices (µPADs)

Ota, Riki, Yamada, Kentaro, Suzuki, Koji, Citterio, Daniel

Analyst (The Royal Society of Chemistry)  143 ( 3 ) 643 - 653 2018.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0003-2654

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© 2018 The Royal Society of Chemistry. The transport efficiency during capillary flow-driven sample transport on microfluidic paper-based analytical devices (μPADs) made from filter paper has been investigated for a selection of model analytes (Ni2+, Zn2+, Cu2+, PO43-, bovine serum albumin, sulforhodamine B, amaranth) representing metal cations, complex anions, proteins and anionic molecules. For the first time, the transport of the analytical target compounds rather than the sample liquid, has been quantitatively evaluated by means of colorimetry and absorption spectrometry-based methods. The experiments have revealed that small paperfluidic channel dimensions, additional user operation steps (e.g. control of sample volume, sample dilution, washing step) as well as the introduction of sample liquid wicking areas allow to increase analyte transport efficiency. It is also shown that the interaction of analytes with the negatively charged cellulosic paper substrate surface is strongly influenced by the physico-chemical properties of the model analyte and can in some cases (Cu2+) result in nearly complete analyte depletion during sample transport. The quantitative information gained through these experiments is expected to contribute to the development of more sensitive μPADs.

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Gold Nanoparticle Core-Europium(III) Chelate Fluorophore-Doped Silica Shell Hybrid Nanocomposites for the Lateral Flow Immunoassay of Human Thyroid Stimulating Hormone with a Dual Signal Readout

Preechakasedkit, Pattarachaya, Osada, Kota, Katayama, Yuta, Ruecha, Nipapan, Suzuki, Koji, Chailapakul, Orawon, Citterio, Daniel

Analyst (The Royal Society of Chemistry)  143 ( 2 ) 564 - 570 2018.01

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0003-2654

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© 2018 The Royal Society of Chemistry. Hybrid nanocomposite particles composed of a gold core coated with a europium(iii)-chelate fluorophore-doped silica shell (AuNPs@SiO2-Eu3+) have been synthesized and applied as antibody labels in lateral flow immunoassay (LFIA) devices for the determination of human thyroid stimulating hormone (hTSH). Labeling of monoclonal anti-hTSH antibodies with AuNPs@SiO2-Eu3+ nanocomposites allows for both colorimetric and fluorometric observation of assay results on LFIA devices, relying on visible light absorption due to the localized surface plasmon resonance of the Au-core and the fluorescence emission of the Eu(iii)-chelate-modified shell under UV hand lamp irradiation (365 nm), respectively. The possibility for a dual signal readout provides an attractive alternative for LFIAs: instantaneous naked eye observation of the AuNP colorimetric signal as in conventional LFIAs for hypothyroidism detection, and more sensitive fluorescence detection to assess hyperthyroidism. The limits of detection (LOD) for naked eye observation of LFIA devices are 5 μIU mL-1 and 0.1 μIU mL-1 for the colorimetric and fluorimetric detection, respectively. Using the fluorescence detection scheme in combination with a smartphone and digital color analysis, a quantitative linear relationship between the red intensity and the logarithmic concentration of hTSH was observed (R2 = 0.988) with an LOD of 0.02 μIU mL-1. Finally, LFIA devices were effectively applied for detecting hTSH in spiked diluted human serum with recovery values between 100-116%.

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Guest Editorial

Citterio, Daniel, Kaneta, Takashi

Analytical Sciences (Japan Society for Analytical Chemistry)  34 ( 1 ) 5 - 6 2018.01

Research paper (scientific journal), Joint Work,  ISSN  0910-6340

Wax-Assisted One-Step Enzyme-Linked Immunosorbent Assay on Lateral Flow Test Devices

Ishii, Masanori, Preechakasedkit, Pattarachaya, Yamada, Kentaro, Chailapakul, Orawon, Suzuki, Koji, Citterio, Daniel

Analytical Sciences (Japan Society for Analytical Chemistry)  34 ( 1 ) 51 - 56 2018.01

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0910-6340

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© The Japan Society for Analytical Chemistry, 2018. Lateral flow tests (LFTs) are widely used analytical tools characterized by portability, operator simplicity and short analysis times. A remaining challenge is their limited analytical sensitivity, which in classical immunoassay formats is overcome by enzyme-linked immunosorbent assay (ELISA) formats. The implementation of ELISA to an LFT format however, is hampered by the complexity of the procedure requiring the enzyme substrate addition after sample addition. In this work, a simple method for automation of this procedure without user interference is presented. Originally used sample pads of LFTs have been replaced by hydrophobic wax-modified filter paper-based sample pads to realize a delayed flow a pre-deposited colorimetric ELISA substrate without other alterations to the classical lateral-flow immunoassay format. The performance of the system has been characterized by visualizing flow behavior and final proof-of-concept is provided by a model mouse IgG assay, achieving a limit of detection of 15.8 ng mL -1 from just a single application of the sample solution.

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"dip-and-read" paper-based analytical devices using distance-based detection with color screening

Yamada K., Citterio D., Henry C.

Lab on a Chip (Lab on a Chip)  18 ( 10 ) 1485 - 1493 2018

ISSN  14730197

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© 2018 The Royal Society of Chemistry. An improved paper-based analytical device (PAD) using color screening to enhance device performance is described. Current detection methods for PADs relying on the distance-based signalling motif can be slow due to the assay time being limited by capillary flow rates that wick fluid through the detection zone. For traditional distance-based detection motifs, analysis can take up to 45 min for a channel length of 5 cm. By using a color screening method, quantification with a distance-based PAD can be achieved in minutes through a "dip-and-read" approach. A colorimetric indicator line deposited onto a paper substrate using inkjet-printing undergoes a concentration-dependent colorimetric response for a given analyte. This color intensity-based response has been converted to a distance-based signal by overlaying a color filter with a continuous color intensity gradient matching the color of the developed indicator line. As a proof-of-concept, Ni quantification in welding fume was performed as a model assay. The results of multiple independent user testing gave mean absolute percentage error and average relative standard deviations of 10.5% and 11.2% respectively, which were an improvement over analysis based on simple visual color comparison with a read guide (12.2%, 14.9%). In addition to the analytical performance comparison, an interference study and a shelf life investigation were performed to further demonstrate practical utility. The developed system demonstrates an alternative detection approach for distance-based PADs enabling fast (∼10 min), quantitative, and straightforward assays.

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Text-displaying competitive immunochromatographic strips enabling naked-eye semi-quantitative analysis

Misawa K., Yamamoto T., Watanabe D., Hiruta Y., Yamazaki H., Citterio D.

22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018 (22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018)  2   1022 - 1025 2018

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Competitive lateral flow immunochromatographic assays (LFIAs) enabling naked-eye semi-quantitative result readout in the form of “text” have been developed. User operation is limited to sample application, followed by direct reading of assay results written in text representing the target concentration. LFIA strips enabled the detection of 8-hydroxy-2’-deoxyguanosine (8-OHdG) at concentrations down to 5 ng/mL, which is comparable to existing immunochromatography using external readout equipment. The system is readily adaptable to other analytical targets and allows for high flexibility in text design and detectable analyte concentrations. The simple assay handling and intuitive data interpretation offer great potential for point-of-care testing applications.

Thread-based analytical device for antibody detection in whole blood by using bioluminescent sensor proteins

Tomimuro K., Tenda K., Hiruta Y., Merkx M., Citterio D.

22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018 (22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018)  2   1018 - 1021 2018

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Bioluminescent protein integrating microfluidic thread-based analytical devices (µTADs) for point-of-care testing (POCT) of antibodies in whole blood have been developed. Devices consist of a blood separation membrane and a plastic film layer with sewn-in cotton thread, in which proteins and luciferin for signal generation are pre-deposited. Colorimetry-based response curves for three model antibodies spiked into porcine whole blood have been obtained. Stable signals from 6-18 min after sample application and no cross-talk when assaying multiple different antibodies are observed. The µTADs offer quantitative simultaneous detection of multiple antibodies in a small volume of whole blood (8 µL).

Fully Inkjet-Printed Paper-Based Potentiometric Ion-Sensing Devices

Ruecha, Nipapan, Chailapakul, Orawon, Suzuki, Koji, Citterio, Daniel

Analytical Chemistry (The American Chemical Society)  89 ( 19 ) 10608 - 10616 2017.10

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0003-2700

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Fabrication of a New Lineage of Artificial Luciferases from Natural Luciferase Pools

Kim, Sung Bae, Nishihara, Ryo, Citterio, Daniel, Suzuki, Koji

ACS Combinatorial Science (The American Chemical Society)  19 ( 9 ) 594 - 599 2017.09

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2156-8952

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Text-Displaying Colorimetric Paper-Based Analytical Device

Yamada, Kentaro, Suzuki, Koji, Citterio, Daniel

ACS Sensors (The American Chemical Society)  2 ( 8 ) 1247 - 1254 2017.08

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2379-3694

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"Drop-Slip" Bulk Sample Flow on Fully Inkjet-Printed Microfluidic Paper-Based Analytical Device

Henares Terence G., Yamada Kentaro, Takaki Shunsuke, Suzuki Koji, Citterio Daniel

Sensors and Actuators B - Chemical (Elsevier)  244   1129 - 1137 2017.06

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0925-4005

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Click Reaction Based on the Biosynthesis of Firefly Luciferin

Toyama, Tomohisa, Saitoh, Tsuyoshi, Takahashi, Yuka, Oka, Kotaro, Citterio, Daniel, Suzuki, Koji, Nishiyama, Shigeru

Chemistry Letters (The Chemical Society of Japan)  46 ( 5 ) 753 - 755 2017.05

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0366-7022

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Luciferase-Specific Coelenterazine Analogues for Optical Contamination-Free Bioassays

Nishihara Ryo, Abe Masahiro, Nishiyama Shigeru, Citterio Daniel, Suzuki Koji, Kim Sung Bae

Scientific Reports (Springer Nature)  7   908 2017.04

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2045-2322

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Paper-Based Analytical Device for Zinc Ion Quantification in Water Samples with Power-Free Analyte Concentration

Kudo Hiroko, Yamada Kentaro, Watanabe Daiki, Suzuki Koji, Citterio Daniel

Micromachines (MDPI)  8 ( 4 ) 127 2017.04

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2072-666X

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Spirolactam Capped Cyanine Dyes for Designing NIR Probes to Target Multiple Metal Ions

Kar, Chirantan, Shindo, Yutaka, Oka, Kotaro, Nishiyama, Shigeru, Suzuki, Koji, Citterio, Daniel

RSC Advances (The Royal Society of Chemistry)  7 ( 40 ) 24970 - 24980 2017

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2046-2069

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High-Resolution Microfluidic Paper-Based Analytical Devices for Sub-Microliter Sample Analysis

Tenda Keisuke, Ota Riki, Yamada Kentaro, Henares Terence G., Suzuki Koji, Citterio Daniel

Micromachines (MDPI)  7 ( 5 ) 80 2016.05

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2072-666X

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Real-time Monitoring and Detection of Primer Generation-Rolling Circle Amplification of DNA Using an Ethidium Ion-selective Electrode

Seichi Ayaka, Kozuka Nanami, Kashima Yuko, Tabata Miyuki, Goda Tatsuro, Matsumoto Akira, Iwasawa Naoko, Citterio Daniel, Miyahara Yuji, Suzuki Koji

Analytical Sciences (Japan Society for Analytical Chemistry)  32 ( 5 ) 505 - 510 2016.05

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0910-6340

Genetically Encoded Molecular Tension Probe for Tracing Protein - Protein Interactions in Mammalian Cells

Kim, Sung Bae, Nishihara, Ryo, Citterio, Daniel, Suzuki, Koji

BIOCONJUGATE CHEMISTRY 27 ( 2 ) 354 - 362 2016.02

Research paper (scientific journal), Joint Work,  ISSN  1043-1802

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Distance-Based Tear Lactoferrin Assay on Microfluidic Paper Device Using Interfacial Interactions on Surface-Modified Cellulose

Yamada, Kentaro, Henares, Terence G., Suzuki, Koji, Citterio, Daniel

ACS APPLIED MATERIALS & INTERFACES 7 ( 44 ) 24864 - 24875 2015.11

Research paper (scientific journal), Joint Work,  ISSN  1944-8244

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A Fluorous Biphasic Solvent Extraction System for Lanthanides with a Fluorophilic beta-Diketone Type Extractant

Nakamura, Etsuko, Hiruta, Yuki, Watanabe, Takafumi, Iwasawa, Naoko, Citterio, Daniel, Suzuki, Koji

ANALYTICAL SCIENCES 31 ( 9 ) 923 - 928 2015.09

Research paper (scientific journal), Joint Work,  ISSN  0910-6340

Near IR Emitting Red-Shifting Ratiometric Fluorophores Based on Borondipyrromethene

Hiruta, Yuki, Koiso, Hikaru, Ozawa, Hitoshi, Sato, Hiroyasu, Hamada, Kensaku, Yabushita, Satoshi, Citterio, Daniel, Suzuki, Koji

ORGANIC LETTERS 17 ( 12 ) 3022 - 3025 2015.06

Research paper (scientific journal), Joint Work,  ISSN  1523-7060

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Papierbasierte tintenstrahlgedruckte Mikrofluidiksysteme für die Analytik

Yamada, Kentaro, Henares, Terence G., Suzuki, Koji, Citterio, Daniel

Angewandte Chemie (Wiley)  127 ( 18 ) 5384 - 5401 2015.04

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1521-3757

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Bioluminescent coelenterazine derivatives with imidazopyrazinone C-6 extended substitution

Nishihara, Ryo, Suzuki, Hideyuki, Hoshino, Emi, Suganuma, Sakura, Sato, Moritoshi, Saitoh, Tsuyoshi, Nishiyama, Shigeru, Iwasawa, Naoko, Citterio, Daniel, Suzuki, Koji

Chemical Communications (Royal Society of Chemistry)  51 ( 2 ) 391 - 394 2015.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1359-7345

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New Trends in Near-Infrared Fluorophores for Bioimaging

Umezawa, Keitaro, Citterio, Daniel, Suzuki, Koji

Analytical Sciences 30 ( 3 ) 327 - 349 2014.03

Research paper (scientific journal), Joint Work,  ISSN  0910-6340

Design and Synthesis of a FlAsH-Type Mg2+ Fluorescent Probe for Specific Protein Labeling

Fujii, Tomohiko, Shindo, Yutaka, Hotta, Kohji, Citterio, Daniel, Nishiyama, Shigeru, Suzuki, Koji, Oka, Kotaro

Journal of the American Chemical Society 136 ( 6 ) 2374 - 2381 2014.02

Research paper (scientific journal), Joint Work,  ISSN  0002-7863

• Access to Document (DOI)

An antibody-free microfluidic paper-based analytical device for the determination of tear fluid lactoferrin by fluorescence sensitization of Tb3+

Yamada, Kentaro, Takaki, Shunsuke, Komuro, Nobutoshi, Suzuki, Koji, Citterio, Daniel

Analyst 139 ( 7 ) 1637 - 1643 2014.01

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0003-2654

• Access to Document (DOI)

Steric hindrance effects in tripodal ligands for extraction and back-extraction of Ag+

Hiruta, Yuki, Watanabe, Takafumi, Nakamura, Etsuko, Iwasawa, Naoko, Sato, Hiroyasu, Hamada, Kensaku, Citterio, Daniel, Suzuki, Koji

RSC Advances 4 ( 19 ) 9791 - 9798 2014

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2046-2069

• Access to Document (DOI)

Inkjet-Printed Paper-Based Colorimetric Sensor Array for the Discrimination of Volatile Primary Amines

Soga, Tamaki, Jimbo, Yusuke, Suzuki, Koji, Citterio, Daniel

Analytical Chemistry 85 ( 19 ) 8973 - 8978 2013.10

Research paper (scientific journal), Joint Work,  ISSN  0003-2700

• Access to Document (DOI)

Development of UV-Excitable Red and Near-Infrared Fluorescent Labels and Their Application for Simultaneous Multicolor Bioimaging by Single-Wavelength Excitation

Mizuno, Tetsuya, Umezawa, Keitaro, Shindo, Yutaka, Citterio, Daniel, Oka, Kotaro, Suzuki, Koji

Journal of Fluorescence 23 ( 5 ) 1007 - 1018 2013.09

Research paper (scientific journal), Joint Work,  ISSN  1053-0509

• Access to Document (DOI)

Inkjet printed (bio)chemical sensing devices

Komuro, Nobutoshi, Takaki, Shunsuke, Suzuki, Koji, Citterio, Daniel

Analytical and Bioanalytical Chemistry 405 ( 17 ) 5785 - 5805 2013.07

Research paper (scientific journal), Joint Work,  ISSN  1618-2642

• Access to Document (DOI)

Li⁺-selective optodes – effect of fluoroionophore distribution in mesoporous silica thin films on Li⁺ response

Citterio Daniel

RSC Advances (The Royal Society of Chemistry)  3   6499-6506 2013.02

Research paper (scientific journal), Joint Work, Accepted

Inkjet printing: an integrated and green chemical approach to microfluidic paper-based analytical devices

Maejima, Kento, Tomikawa, Satoshi, Suzuki, Koji, Citterio, Daniel

RSC Advances 3 ( 24 ) 9258 - 9263 2013

Research paper (scientific journal), Joint Work,  ISSN  2046-2069

• Access to Document (DOI)

Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF)

Henares, Terence G., Uenoyama, Yuta, Nogawa, Yuto, Ikegami, Ken, Citterio, Daniel, Suzuki, Koji, Funano, Shun-ichi, Sueyoshi, Kenji, Endo, Tatsuro, Hisamoto, Hideaki

Analyst 138 ( 11 ) 3139 - 3141 2013

Research paper (scientific journal), Joint Work,  ISSN  0003-2654

• Access to Document (DOI)

Highly Durable Double Sol-Gel Layer Ratiometric Fluorescent pH Optode Based on the Combination of Two Types of Quantum Dots and Absorbing pH Indicators

Citterio Daniel

Analytical Chemistry (American Chemical Society)  84 ( 24 ) 10650−10656 2012.12

Research paper (scientific journal), Joint Work, Accepted

Gadolinium Containing Photochromic Micelles as Potential Magnetic Resonance Imaging Traceable Drug Carriers

Citterio Daniel

Photochemistry and Photobiology (Wiley-Blackwell)  88 ( 4 ) 876-883 2012.07

Research paper (scientific journal), Joint Work, Accepted

Quartz Crystal Microbalance Sensor Using Ionophore for Ammonium Ion Detection

Citterio Daniel

Journal of Nanoscience and Nanotechnology (American Scientific Publishers)  12 ( 1 ) 563-567 2012.01

Research paper (scientific journal), Joint Work, Accepted

Newly Developed Mg(2+)-Selective Fluorescent Probe Enables Visualization of Mg(2+) Dynamics in Mitochondria

Citterio Daniel

PLOS One (Public Library Science)  6 ( 8 ) e23684 2011.08

Research paper (scientific journal), Joint Work, Accepted

A Near-Infrared Fluorescent Calcium Probe: a New Tool for Intracellular Multicolour Ca²⁺ Imaging

Citterio Daniel

Chemical Communications (The Royal Society of Chemistry)  47 ( 37 ) 10407-10409 2011.08

Research paper (scientific journal), Joint Work, Accepted

Liquid-Phase Formation of Silver Nanoparticles on a Glass Substrate Regulated by Gold Nanoparticles

Citterio Daniel

Sensors and Materials (MYU)  23 ( 5 ) 251-261 2011

Research paper (scientific journal), Joint Work, Accepted

An Oxo-Bacteriochlorin Derivative for Long-Wavelength Fluorescence Ratiometric Alcohol Sensing

Citterio Daniel

Analyst (The Royal Society of Chemistry)  135   2334-2339 2010

Research paper (scientific journal), Joint Work, Accepted

A Fast-Response pH Optode Based on a Fluoroionophore Immobilized to a Mesoporous Silica Thin Film

Citterio Daniel

Analytical Sciences (The Japan Society for Analytical Chemistry)  26 ( 3 ) 297-301 2010

Research paper (scientific journal), Joint Work, Accepted

Inkjet-Printed Paperfluidic Immuno-Chemical Sensing Device

Citterio Daniel

Analytical and Bioanalytical Chemistry (Springer)  398 ( 2 ) 885-893 2010

Research paper (scientific journal), Joint Work, Accepted

A Highly Li⁺-Selective Glass Optode Based on Fluorescence Ratiometry

Citterio Daniel

Analyst (The Royal Society of Chemistry)  134 ( 11 ) 2314-2319 2009

Research paper (scientific journal), Joint Work, Accepted

Bright, Color-Tunable Fluorescent Dyes in the Vis/NIR Region: Establishment of New Tailor-Made Multicolor Fluorophores Based on Borondipyrromethene

Citterio Daniel

Chemistry - A European Journal (Wiley-VCH)  15 ( 5 ) 1096-1106 2009

Research paper (scientific journal), Joint Work, Accepted

A Novel Luciferin-Based Bright Chemiluminescent Probe for the Detection of Reactive Oxygen Species

Citterio Daniel

Chemical Communications (The Royal Society of Chemistry)  21   3047-3049 2009

Research paper (scientific journal), Joint Work, Accepted

Dextran Coated Gadolinium Phosphate Nanoparticles for Magnetic Resonance Tumor Imaging

Citterio Daniel

Journal of Materials Chemistry (The Royal Society of Chemistry)  19 ( 35 ) 6393-6399 2009

Research paper (scientific journal), Joint Work, Accepted

Cell-Based in Vivo Dual Imaging Probes Using Genetically Expressed Tags and Chemical Contrast Agents

Citterio Daniel

Chemical Communications (The Royal Society of Chemistry)  27   4040-4042 2009

Research paper (scientific journal), Joint Work, Accepted

Structural Characteristics and Optical Properties of a Series of Solvatochromic Fluorescent Dyes Displaying Long-Wavelength Emission

Citterio Daniel

Dyes and Pigments (Elsevier)  83 ( 2 ) 198-206 2009

Research paper (scientific journal), Joint Work, Accepted

Inkjet-Printed Microfluidic Multianalyte Chemical Sensing Paper

Citterio Daniel

Analytical Chemistry (American Chemical Society)  80 ( 18 ) 6928-6934 2008

Research paper (scientific journal), Joint Work, Accepted

Bright, Color-Tunable Fluorescent Dyes in the Visible-Near-Infrared Region

Citterio Daniel

Journal of the American Chemical Society (American Chemical Society)  130 ( 5 ) 1550-1551 2008

Research paper (scientific journal), Joint Work, Accepted

Water-Soluble NIR Fluorescent Probes Based on Squaraine and their Application for Protein Labeling

Citterio Daniel

Analytical Sciences (The Japan Society for Analytical Chemistry)  24 ( 2 ) 213-217 2008

Research paper (scientific journal), Joint Work, Accepted

Smart Taste Sensors

Citterio Daniel

Analytical Chemistry (American Chemical Society)  80   3965-3972 2008

Research paper (scientific journal), Joint Work, Accepted

A Ratiometric Fluorescent pH Glass Optode Based on a Boron-Dipyrromethene Derivative

Citterio Daniel

Sensors and Actuators B: Chemical (Elsevier)  121 ( 1 ) 74-82 2007

Research paper (scientific journal), Joint Work, Accepted

A Squaraine-Based Near-infrared Dye with Bright Fluorescence and Solvatochromic Property

Citterio Daniel

Chemistry Letters (The Chemical Society of Japan)  36 ( 12 ) 1424-1425 2007

Research paper (scientific journal), Joint Work, Accepted

Basic Design and Synthesis of Sulfonated Contrast Agents for Magnetic Resonance Imaging Based on Gadolinium Complexes

Citterio Daniel

Analytical Sciences (The Japan Society for Analytical Chemistry)  23 ( 10 ) 1159-1165 2007

Research paper (scientific journal), Joint Work, Accepted

Novel 15-Crown-5 Ether or -Diketone Incorporated Gadolinium Complexes for the Detection of Potassium Ions or Magnesium and Calcium Ions

Citterio Daniel

Analyst (The Royal Society of Chemistry)  132 ( 11 ) 1153-1160 2007

Research paper (scientific journal), Joint Work, Accepted

pH-Independent Fluorescent Chemosensor for Highly Selective Lithium Ion Sensing

Citterio Daniel

Analytical Chemistry (American Chemical Society)  79 ( 3 ) 1237-1242 2007

Research paper (scientific journal), Joint Work, Accepted

Gadolinium-Based Hybrid Nanoparticles as a Positive MR Contrast Agent

Citterio Daniel

Journal of the American Chemical Society (American Chemical Society)  128 ( 47 ) 15090-15091 2006.11

Research paper (scientific journal), Joint Work, Accepted

SPR Sensor Signal Amplification Based on Dye-Doped Polymer Particles

Citterio Daniel

Science and Technology of Advanced Materials (Elsevier)  7 ( 2 ) 150-155 2006

Research paper (scientific journal), Joint Work, Accepted

L-Glutamate Biosensor for Estimation of the Taste of Tomato Specimens

Citterio Daniel

Analytical and Bioanalytical Chemistry (Springer)  386 ( 2 ) 220-227 2006

Research paper (scientific journal), Joint Work, Accepted

Smart Chemical Taste Sensor for Determination and Prediction of Taste Qualities Based on a Two-Phase Optimized Radial Basis Function Network

Citterio Daniel

Analytical Chemistry (American Chemical Society)  77 ( 24 ) 7908-7915 2005.12

Research paper (scientific journal), Joint Work, Accepted

Single Molecular Multianalyte (Ca²⁺, Mg²⁺) Fluorescent Probe and Applications to Bioimaging

Citterio Daniel

Journal of the American Chemical Society (American Chemical Society)  127 ( 31 ) 10798-10799 2005.08

Research paper (scientific journal), Joint Work, Accepted

Single Molecular Multianalyte Sensor: Jewel Pendant Ligand

Citterio Daniel

Organic Letters (American Chemical Society)  7 ( 14 ) 2857-2859 2005.07

Research paper (scientific journal), Joint Work, Accepted

Fluorescence Enhancement Detection of Underivatized Amino Acids Using a Trifluoroacetophenone-Based Tripodal Fluoroionophore

Citterio Daniel

Chimia (Swiss Chemical Society)  59 ( 5 ) 204-208 2005

Research paper (scientific journal), Joint Work, Accepted

Planar Integrated Optical Waveguide Used as a Transducer to Yield Chemical Information: Detection of the Activity of Proteolytic Enzymes e.g. Serine-Proteases

Citterio Daniel

Optics and Lasers in Engineering (Elsevier)  43 ( 3-5 ) 603-617 2005

Research paper (scientific journal), Joint Work, Accepted

Highly Sodium-Selective Fluoroionophore Based on Conformational Restriction of Oligoethyleneglycol-Bridged Biaryl Boron-Dipyrromethene

Citterio Daniel

Journal of the Americal Chemical Society (American Chemical Society)  127 ( 19 ) 6956-6957 2005

Research paper (scientific journal), Joint Work, Accepted

Design and Synthesis of Highly Sensitive and Selective Fluorescein-Derived Magnesium Fluorescent Probes and Application to Intracellular 3D Mg²⁺ Imaging

Citterio Daniel

Journal of the American Chemical Society (American Chemical Society)  126 ( 50 ) 16353-16360 2004.12

Research paper (scientific journal), Joint Work, Accepted

Quantification of Ternary Mixtures of Heavy Metal Cations from Metallochromic Absorbance Spectra Using Neural Network Inversion

Citterio Daniel

Analytical Chemistry (American Chemical Society)  76 ( 19 ) 5726-5733 2004.10

Research paper (scientific journal), Joint Work, Accepted

Organic Tin Compounds Combined with Anionic Additives - an Ionophore System Leading to a Phosphate Ion-Selective Electrode?

Citterio Daniel

Talanta (Elsevier)  63 ( 1 ) 131-134 2004.05

Research paper (scientific journal), Joint Work, Accepted

Design and Synthesis of Labeling Reagents (MS Probes) for Highly Sensitive Electrospray Ionization Mass Spectrometry and their Application to the Detection of Carbonyl, Alcohol, Carboxylic Acid and Primary Amine Samples

Citterio Daniel

Analytical Sciences (The Japan Society for Analytical Chemistry)  20 ( 3 ) 475-482 2004.03

Research paper (scientific journal), Joint Work, Accepted

Chromogenic Betaine Lariates for Highly Selective Calcium Ion Sensing in Aqueous Environment

Citterio Daniel

Analytical Chimica Acta (Elsevier)  504 ( 2 ) 227-234 2004.02

Research paper (scientific journal), Joint Work, Accepted

Determination of Glycosylated Albumin Using Surface Plasmon Resonance Sensor

Citterio Daniel

Bunseki Kagaku (The Japan Society for Analytical Chemistry)  52 ( 5 ) 311-317 2003.05

Research paper (scientific journal), Joint Work

Molecular Design, Characterization, and Application of Multiinformation Dyes for Optical Chemical Sensing IV. Multiinformation Dyes with Extended Spectral Sensitivity in the Near-Infrared Spectral Range

Citterio Daniel

Analytica Chimica Acta (Elsevier)  482 ( 1 ) 19-28 2003.04

Research paper (scientific journal), Joint Work, Accepted

Application of the Absorption-Based Surface Plasmon Resonance Principle to the Determination of Glucose Using an Enzyme Reaction

Citterio Daniel

Instrumentation Science & Technology (Marcel Dekker)  31 ( 4 ) 343-356 2003

Research paper (scientific journal), Joint Work, Accepted

Application of an Absorption-Based Surface Plasmon Resonance Principle to the Development of SPR Ammonium Ion and Enzyme Sensors

Citterio Daniel

Analytical Chemistry (American Chemical Society)  74 ( 23 ) 6106-6110 2002.12

Research paper (scientific journal), Joint Work, Accepted

Ionophore-Based Lithium Ion Film Optode Realizing Multiple Color Variations Utilizing Digital Color Analysis

Citterio Daniel

Analytical Chemistry (American Chemical Society)  74 ( 22 ) 5766-5773 2002.11

Research paper (scientific journal), Joint Work, Accepted

Fluororeceptor for Zwitterionic Form Amino Acids in Aqueous Methanol Solution

Citterio Daniel

Tetrahedron Letters (Pergamon)  43 ( 40 ) 7243-7245 2002.09

Research paper (scientific journal), Joint Work, Accepted

Comparison of Two Molecular Design Strategies for the Development of an Ammonium Ionophore More Highly Selective than Nonactin

Citterio Daniel

Analytical Chemistry (American Chemical Society)  74 ( 18 ) 4845-4848 2002.09

Research paper (scientific journal), Joint Work, Accepted

Design and Synthesis of Mg²⁺-Selective Fluoroionophores Based on a Coumarin Derivative and Application for Mg²⁺ Measurement in a Living Cell

Citterio Daniel

Analytical Chemistry (American Chemical Society)  74 ( 6 ) 1423-1428 2002.03

Research paper (scientific journal), Joint Work, Accepted

Trifluoroacetophenone Derivatives as Amino Acid Selective Ionophores for the Potentiometric Determination of Phenylalanine

Citterio Daniel

Angewandte Chemie-International Edition (Wiley-VCH)  41 ( 16 ) 3005-3007 2002

Research paper (scientific journal), Joint Work, Accepted

Design and Synthesis of Preorganized Tripodal Fluororeceptors Based on Hydrogen Bonding of Thiourea Groups for Optical Phosphate Ion Sensing

Citterio Daniel

Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry (The Royal Society of Chemistry)     2309-2313 2001.12

Research paper (scientific journal), Joint Work, Accepted

Optical Determination of Low-Level Water Concentrations in Organic Solvents Using Fluorescent Acridinyl Dyes and Dye-Immobilized Polymer Membranes

Citterio Daniel

Analytical Chemistry (American Chemical Society)  73 ( 21 ) 5339-5345 2001.11

Research paper (scientific journal), Joint Work, Accepted

A new Type of Cation Responsive Chromoionophore with Spectral Sensitivity in the Near-Infrared Spectral Range

Citterio Daniel

Chemistry Letters (The Chemical Society of Japan)     552-553 2001.06

Research paper (scientific journal), Joint Work, Accepted

Application of Thiouronium Derivatives as Anion Ionophores for Ion Selective Electrodes

Citterio Daniel

Chemistry Letters (The Chemical Society of Japan)     382-383 2001.05

Research paper (scientific journal), Joint Work, Accepted

Synthesis of Novel Hg²⁺ Receptors Based on N-Benzyloxyamide Derivatives and their Application to Anion-Selective Electrodes

Citterio Daniel

Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (The Royal Society of Chemistry)     1366-1371 2001

Research paper (scientific journal), Joint Work, Accepted

Reversible Chemical Reactions as the Basis for Optical Sensors Used to Detect Amines, Alcohols and Humidity

Citterio Daniel

Journal of Materials Chemistry (The Royal Society of Chemistry)  9 ( 9 ) 2259-2264 1999.09

Research paper (scientific journal), Joint Work, Accepted

Observation of Silver and Hydrogen Ion Binding to Self-Assembled Monolayers Using Chemically Modified AFM Tips

Citterio Daniel

Langmuir (American Chemical Society)  15 ( 8 ) 2788-2793 1999.04

Research paper (scientific journal), Joint Work, Accepted

Reduced Dicyanovinyl Dyes: A Chromophore to Be Used for Optical Sensing in the Red and Near-Infrared Spectral Range

Citterio Daniel

Analytical Chemistry (American Chemical Society)  70 ( 16 ) 3452-3457 1998.08

Research paper (scientific journal), Joint Work, Accepted

Development of Chromogenic Reactands for Optical Sensing of Alcohols

Citterio Daniel

Sensors and Actuators B: Chemical (Elsevier)  49 ( 3 ) 226-234 1998.07

Research paper (scientific journal), Joint Work, Accepted

Dyes for Use in Integrated Optical Sensors

Citterio Daniel

Sensors and Actuators B: Chemical (Elsevier)  39 ( 1-3 ) 202-206 1997.03

Research paper (scientific journal), Joint Work, Accepted

Continuous Monitoring of Ethanol for Bioprocess Control by a Chemical Sensor

Citterio Daniel

Journal of Biotechnology (Elsevier)  50 ( 1 ) 37-46 1996.09

Research paper (scientific journal), Joint Work, Accepted

Development of New Dyes for Use in Integrated Optical Sensors

Citterio Daniel

Fresenius Journal of Analytical Chemistry (Springer)  354 ( 7-8 ) 836-840 1996.04

Research paper (scientific journal), Joint Work, Accepted

Integrated Optical Sensors Based on Refractometry of Ion-Selective Membranes

Citterio Daniel

Sensors and Actuators B: Chemical (Elsevier)  29 ( 1-3 ) 277-285 1995.10

Research paper (scientific journal), Joint Work, Accepted

From Molecular Recognition to Analytical Information Using Chemical Sensors - Development of a Combined Catheter Gastric pH-Probe

Citterio Daniel

Electroanalysis (VCH Publishers)  7 ( 9 ) 859-863 1995.09

Research paper (scientific journal), Joint Work, Accepted