論文

© 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.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. L-type amino acid transporter 1 (LAT1) is more highly expressed in cancer cells compared with normal cells. LAT1 targeting probes would therefore be a promising tool for cancer cell imaging. In this study, LAT1-targeting thermoresponsive fluorescent polymer probes based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)) were synthesized and their affinity for LAT1 was evaluated. The synthesized polymer probes interacted with LAT1 on HeLa cells, and inhibition of L-[3 H]-leucine, one of the substrates for LAT1 uptake, was investigated. L-Tyrosine-conjugated P(NIPAAm-co-DMAAm) inhibited the uptake of L-[3 H]-leucine, while P(NIPAAm-co-DMAAm) and L-phenylalanine-conjugated P(NIPAAm-co-DMAAm) did not. This result indicated that L-tyrosine-conjugated polymer has a high affinity for LAT1. The fluorescent polymer probes were prepared by modification of a terminal polymer group with fluorescein-5-maleimide (FL). Above the polymer transition temperature, cellular uptake of the polymer probes was observed because the polymers became hydrophobic, which enhanced the interaction with the cell membrane. Furthermore, quantitative analysis of the fluorescent probe using flow cytometry indicated that L-tyrosine-conjugated P(NIPAAm-co-DMAAm)-FL shows higher fluorescence intensity earlier than P(NIPAAm-co-DMAAm)-FL. The result suggested that cellular uptake was promoted by the LAT1 affinity site. The developed LAT1-targeting thermoresponsive fluorescent polymer probes are expected to be useful for cancer cell imaging.

© 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.

© 2019 Society for Laboratory Automation and Screening. Lactoferrin is an abundant glycoprotein in human body fluids and is known as a biomarker for various diseases. Therefore, point-of-care testing (POCT) for lactoferrin is of interest. Microfluidic paper-based analytical devices (µPADs) have gained a lot of attention as next-generation POCT device candidates, due to their inexpensiveness, operational simplicity, and being safely disposable. This work presents a colorimetric sensing approach for quantitative lactoferrin analysis. The detection mechanism takes advantage of the high affinity of lactoferrin to ferric ions (Fe3+). Lactoferrin is able to displace an indicator from a colorimetric 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP)-Fe3+ complex, resulting in a color change. A 5-Br-PADAP-Fe3+ complex was encapsulated into water-dispersible poly(styrene-block-vinylpyrrolidone) particles, whose physical entrapment in the cellulosic fiber network results in the immobilization of the complex to the paper matrix. The complex-encapsulating particles showed a color change response in accordance with lactoferrin concentration. Both color intensity-based paper well plates and distance readout-based µPADs are demonstrated. Color intensity-based devices allowed quantitative analysis of lactoferrin concentrations with a limit of detection of 110 µg/mL, using a smartphone and a color readout app. On the other hand, distance readout-based µPADs showed changes of the length of colored sections in accordance with lactoferrin concentration. In summary, we successfully developed both colorimetric intensity-based paper wells and distance-based µPADs for lactoferrin detection. This work demonstrates a user-friendly colorimetric analysis platform for lactoferrin without requiring lab equipment and expensive antibodies.

Copyright © 2019 American Chemical Society. The cell manipulation technique using thermoresponsive polymers is currently attracting much attention for applications in the medical field. To achieve arbitrary and accurate cell control, it is necessary to intensely research fibronectin behavior. A smart surface, which has thermoresponsive wettability and which can adsorb or desorb fibronectin repeatedly without the presence of cells, was fabricated by an electrospinning method. The fabricated coating changed its structure as the temperature was changed, and this transformation could substitute for the pulling force generated by the cytoskeletal contraction of cells. Moreover, a coated quartz crystal microbalance was able to detect the fibronectin behavior as frequency shifts, which could be used in the estimation of the mass shift with the aid of suitable equations. This coating and measurement system can contribute greatly not only to the development in the medical field centered on biomaterial manipulation technologies, but also to the improvement of medical instruments.

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

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

Copyright © 2020 American Chemical Society. 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.

© 2020 Elsevier B.V. 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.

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.

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.

Urinalysis is widely used in routine medical checkups. Conventionally, colorimetric paper strips currently available on the market are applied. Although they allow to rapidly obtain results, their use is often not straightforward, because a timed readout is required, followed by comparison with a color chart. D-μPADs (distance-based microfluidic paper-based analytical devices) have gained attention for enabling highly intuitive signal readout. During liquid flow through a paperfluidic channel, target analytes are sequentially depleted from the sample through reaction with colorimetric indicators and/or adsorption to the paper surface, so that the length of color-changed sections correspond to the analyte content. Therefore, D-μPADs are expected to solve the problems of existing urinalysis tests. The final goal is the development of D-μPADs for the simultaneous detection of urinary protein (albumin), glucose and hemoglobin. Colorimetric distance-based calibration curves were acquired for all analytes in artificial urine matrix containing electrolytes and organic compounds. For quantitative assays, evaluation of color-changed distances was conducted by computer software. The first cases of distance-based albumin, glucose and hemoglobin readouts in relatively complex artificial urine matrix are reported. For albumin detection, the color uniformity of the colorimetric indicator was improved by paper surface modification with poly(4-styrenesulfonic acid), an anionic polymer. For glucose detection, signal visibility was significantly improved by paper surface modification with chitosan, a biopolymer that works as immobilization support for enzymes. Finally, we evaluated the effect of urinary components on the assay results. It was confirmed that D-μPADs for the hemoglobin assay require a treatment to reduce the effect of albumin adsorption on the paper substrate.

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.

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