論文 - チッテリオ，　ダニエル

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年04月

ISSN  09254005

　概要を見る

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年03月

ISSN  10431802

　概要を見る

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年02月

ISSN  14730197

　概要を見る

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年02月

ISSN  09254005

　概要を見る

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年02月

ISSN  00263672

　概要を見る

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年01月

ISSN  00222860

　概要を見る

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年01月

ISSN  16182642

　概要を見る

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年

　概要を見る

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年11月

ISSN  14394227

　概要を見る

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年09月

ISSN  09277765

　概要を見る

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年05月

ISSN  03667022

　概要を見る

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年01月

ISSN  14770520

　概要を見る

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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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年

ISSN  05251931

　概要を見る

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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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年

ISSN  05251931

　概要を見る

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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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年10月

ISSN  01437208

　概要を見る

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年09月

ISSN  00032654

　概要を見る

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年07月

　概要を見る

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年07月

ISSN  00032654

　概要を見る

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年06月

　概要を見る

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年05月

　概要を見る

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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