研究者詳細 - チッテリオ，　ダニエル

論文 - チッテリオ，　ダニエル

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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年

© 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年

ISSN 20507518

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年

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.

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

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

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.

Distance readout-based paper devices for multiplexed urinalysis

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

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 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年11月

ISSN 14337851

© 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年06月

ISSN 10431802

© 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年05月

ISSN 00032654

© 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<sup>+</sup> 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年04月

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

チッテリオ　ダニエル

Analytical and Bioanalytical Chemistry （Analytical and Bioanalytical Chemistry） 410 （ 9 ） 2305 - 2313 2018年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1618-2642

© 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

チッテリオ　ダニエル

Chemistry - An Asian Journal （Chemistry - An Asian Journal） 13 （ 6 ） 648 - 655 2018年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1861-4728

© 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

チッテリオ　ダニエル

Analyst （Analyst） 143 （ 5 ） 1234 - 1241 2018年03月

研究論文（学術雑誌）, 査読有り, ISSN 0003-2654

© 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

チッテリオ　ダニエル

Analyst （Analyst） 143 （ 3 ） 678 - 686 2018年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0003-2654

© 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

チッテリオ　ダニエル

Chemical Communications （Chemical Communications） 54 （ 14 ） 1774 - 1777 2018年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1359-7345

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Quantitative evaluation of analyte transport on microfluidic paper-based analytical devices (μPADs)

チッテリオ　ダニエル

Analyst （Analyst） 143 （ 3 ） 643 - 653 2018年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0003-2654

© 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

チッテリオ　ダニエル

Analyst （Analyst） 143 （ 2 ） 564 - 570 2018年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0003-2654

© 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

チッテリオ　ダニエル

Analytical Sciences （Japan Society for Analytical Chemistry） 34 （ 1 ） 5 - 6 2018年01月

研究論文（学術雑誌）, 共著, ISSN 0910-6340

Wax-assisted one-step enzyme-linked immunosorbent assay on lateral flow test devices

チッテリオ　ダニエル

Analytical Sciences （Analytical Sciences） 34 （ 1 ） 51 - 56 2018年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0910-6340

© 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年

ISSN 14730197

© 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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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年

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).

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