Details of a Researcher - Citterio, Daniel

Papers - Citterio, Daniel

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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

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

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

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 Ca<sup>2+</sup> 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

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

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

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

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

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

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

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

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

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

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

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

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

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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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

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

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

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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