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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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Journal of Photochemistry and Photobiology A: Chemistry （Journal of Photochemistry and Photobiology A: Chemistry） 394 2020年05月

ISSN 10106030

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

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

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

Analytical and Bioanalytical Chemistry （Analytical and Bioanalytical Chemistry） 412 （ 14 ） 3489 - 3497 2020年05月

ISSN 16182642

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

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

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

ACS Sensors （ACS Sensors） 5 （ 4 ） 1110 - 1118 2020年04月

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

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Centrifugal Paperfluidic Platform for Accelerated Distance-Based Colorimetric Signal Readout

Maejima K., Hiruta Y., Citterio D.

Analytical Chemistry （Analytical Chemistry） 92 （ 7 ） 4749 - 4754 2020年04月

ISSN 00032700

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

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