Solid-Phase Collateral Cleavage System Based on CRISPR/Cas12 and Its Application toward Facile One-Pot Multiplex Double-Stranded DNA Detection.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12 (Cas12) system is attracting interest for its potential as a next-generation nucleic acid detection tool. The system can recognize double-stranded DNA (dsDNA) based on Cas12-CRISPR RNA (crRNA) and induce signal transduction by collateral cleavage. This property is expected to simplify comprehensive genotyping. Here, we report a solid-phase collateral cleavage (SPCC) reaction by CRISPR/Cas12 and its application toward one-pot multiplex dsDNA detection with minimal operational steps. In the sensor, Cas12-crRNA and single-stranded DNA (ssDNA) are immobilized on the sensing surface and act as enzyme and reporter substrates, respectively. We also report a dual-target dsDNA sensor prepared by immobilizing Cas12-crRNA and a fluorophore-labeled ssDNA reporter on separate spots. When a spot captures a target dsDNA sequence, it cleaves the ssDNA reporter on the same spot and reduces its fluorescence by 42.1-57.3%. Crucially, spots targeting different sequences do not show a reduction in fluorescence, thus confirming the one-pot multiplex dsDNA detection by SPCC. Furthermore, the sequence specificity has a two-base resolution, and the detectable concentration for the target dsDNA is at least 10-9 M. In the future, the SPCC-based sensor array could achieve one-pot comprehensive genotyping by using an array spotter as a reagent-immobilizing method.

Fully Transparent, Ultrathin Flexible Organic Electrochemical Transistors with Additive Integration for Bioelectronic Applications.

Optical transparency is highly desirable in bioelectronic sensors because it enables multimodal optical assessment during electronic sensing. Ultrathin (<5 µm) organic electrochemical transistors (OECTs) can be potentially used as a highly efficient bioelectronic transducer because they demonstrate high transconductance during low-voltage operation and close conformability to biological tissues. However, the fabrication of fully transparent ultrathin OECTs remains a challenge owing to the harsh etching processes of nanomaterials. In this study, fully transparent, ultrathin, and flexible OECTs are developed using additive integration processes of selective-wetting deposition and thermally bonded lamination. These processes are compatible with Ag nanowire electrodes and conducting polymer channels and realize unprecedented flexible OECTs with high visible transmittance (>90%) and high transconductance (≈1 mS) in low-voltage operations (<0.6 V). Further, electroencephalogram acquisition and nitrate ion sensing are demonstrated in addition to the compatibility of simultaneous assessments of optical blood flowmetry when the transparent OECTs are worn, owing to the transparency. These feasibility demonstrations show promise in contributing to human stress monitoring in bioelectronics.

Towards On-site Determination of Secretory IgA in Artificial Saliva with Gold-Linked Electrochemical Immunoassay (GLEIA) Using Portable Potentiostat and Disposable Printed Electrode.

Mental stress is closely connected with our physical and mental wellness. Therefore, stress measurement can contribute to assess our lifestyle and increase our quality of life. In this paper, we detect the secretory immunoglobulin A (sIgA), which is the candidate of salivary stress markers, with original electrochemical immunoassay: gold-linked electrochemical immunoassay (GLEIA). This biosensor is based on a sandwich-type immunosensor and adopts the electrochemical method to detect the reduction peak from Au nanoparticles linked to the secondary antibody. GLEIA is convenient and cost-effective that only requires a low sample volume (10 µL). In addition, the GLEIA show high sensitivity and selectivity. We obtained the linear response to relate the concentration of sIgA (10-300 ng/mL) in D-PBS buffer with the artificial saliva which includes salivary inorganic salt and typically glycoprotein (mucin). Furthermore, we obtained acceptable selectivity in the various solution with salivary proteins such as α-amylase, human serum albumin, immunoglobulin G (IgG), lysozyme, and mucin. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress markers in saliva.

Investigation of Polyurethane Matrix Membranes for Salivary Nitrate ISFETs to Prevent the Drift.

We have investigated human-stress monitoring by making use of salivary nitrate, which can be a candidate for stress markers, with ion-selective field-effect transistors (ISFETs). ISFETs are suitable for on-site single-drop analysis of salivary nitrate within 10 s. However, when ISFETs are used for salivary nitrate, ISFETs have a problem that is called the initial drift. The initial drift makes accurate nitrate monitoring difficult. Thus, the purpose of this study is to prevent the initial drift and to search for a new, simple polymer to possess a better performance of sensor responses than conventional matrix membranes, such as PVC. In this research, we investigated ISFETs using specific matrix membranes, for example KP-13, Pellethane®--, and P7281-PU. The initial drift was evaluated from the fluctuations of the response values generated by the ISFETs when immersed in saliva or aqueous solution. As a result, P7281-PU showed a prevention effect on the initial drift, both in the whole saliva and in various solutions. Furthermore, the cause of drift may be H+ diffusion, and the drift prevention effect of P7281-PU may be affected by urethane bond capturing H+ in the ion-selective membrane. This result suggests that a continuous nitrate monitoring is feasible and may be applied to wearable sensors.

Label-Free Detection of Human Glycoprotein (CgA) Using an Extended-Gated Organic Transistor-Based Immunosensor.

Herein, we report on the fabrication of an extended-gated organic field-effect transistor (OFET)-based immunosensor and its application in the detection of human chromogranin A (hCgA). The fabricated OFET device possesses an extended-gate electrode immobilized with an anti-CgA antibody. The titration results of hCgA showed that the electrical changes in the OFET characteristics corresponded to the glycoprotein recognition ability of the monoclonal antibody (anti-CgA). The observed sensitivity (detection limit: 0.11 µg/mL) and selectivity indicate that the OFET-based immunosensor can be potentially applied to the rapid detection of the glycoprotein concentration without any labeling.

Selective nitrate detection by an enzymatic sensor based on an extended-gate type organic field-effect transistor.

First selective nitrate biosensor device based on an extended-gate type organic field-effect transistor (OFET) is reported. The fabricated sensor device consists of the extended-gate electrode functionalized by a nitrate reductase with a mediator (=a bipyridinium derivative) and an OFET-based transducer. The mechanism of the nitrate detection can be explained by an electron-relay on the extended-gate electrode, resulting in changes of the electric properties of the OFET. The detection limit of nitrate in water is estimated to be 45 ppb, which suggests that the sensitivity of our fabricated sensor is comparable to those of some conventional detection methods. As a practical application of the OFET sensor, the nitrate detection in diluted human saliva has been successfully demonstrated; the results agreed well with those by conventional colorimetric measurement. The advantages of OFETs are printability, mechanical flexibility, stretchability and disposability, meaning that the fabricated OFET could open up a new approach for low-cost electronic devices toward on-site detection of nitrate in aqueous media.

Time-shared optical tweezers with a microlens array for dynamic microbead arrays.

Dynamic arrays of microbeads and cells offer great flexibility and potential as platforms for sensing and manipulation applications in various scientific fields, especially biology and medicine. Here, we present a simple method for assembling and manipulating dense dynamic arrays based on time-shared scanning optical tweezers with a microlens array. Three typical examples, including the dynamic and simultaneous bonding of microbeads in real-time, are demonstrated. The optical design and the hardware setup for our approach are also described.

An Organic Field-effect Transistor with an Extended-gate Electrode Capable of Detecting Human Immunoglobulin A.

We herein report on the development of an extended-gate type organic field-effect transistor (OFET)-based immunosensor for the detection of human immunoglobulin A (IgA). The titration results of IgA exhibited shifts in the transfer characteristics of the OFET sensor device with increasing IgA concentration. A linear detection range from 0 to 10 µg/mL was realized with a detection limit of 2.1 µg/mL, indicating that the OFET-based immunosensor can be potentially applied to the monitoring of infectious diseases and psychological stress in daily life.

Nanoparticles speckled by ready-to-conjugate lanthanide complexes for multimodal imaging.

Multimodal and multifunctional contrast agents receive enormous attention in the biomedical imaging field. Such contrast agents are routinely prepared by the incorporation of organic molecules and inorganic nanoparticles (NPs) into host materials such as gold NPs, silica NPs, polymer NPs, and liposomes. Despite their non-cytotoxic nature, the large size of these NPs limits the in vivo distribution and clearance and inflames complex pharmacokinetics, which hinder the regulatory approval for clinical applications. Herein, we report a unique method that combines magnetic resonance imaging (MRI) and fluorescence imaging modalities together in nanoscale entities by the simple, direct and stable conjugation of novel biotinylated coordination complexes of gadolinium(III) to CdSe/ZnS quantum dots (QD) and terbium(III) to super paramagnetic iron oxide NPs (SPION) but without any host material. Subsequently, we evaluate the potentials of such lanthanide-speckled fluorescent-magnetic NPs for bioimaging at single-molecule, cell and in vivo levels. The simple preparation and small size make such fluorescent-magnetic NPs promising contrast agents for biomedical imaging.

Auger ionization beats photo-oxidation of semiconductor quantum dots: extended stability of single-molecule photoluminescence.

Despite the bright and tuneable photoluminescence (PL) of semiconductor quantum dots (QDs), the PL instability induced by Auger recombination and oxidation poses a major challenge in single-molecule applications of QDs. The incomplete information about Auger recombination and oxidation is an obstacle in the resolution of this challenge. Here, we report for the first time that Auger-ionized QDs beat self-sensitized oxidation and the non-digitized PL intensity loss. Although high-intensity photoactivation insistently induces PL blinking, the transient escape of QDs into the ultrafast Auger recombination cycle prevents generation of singlet oxygen ((1) O2 ) and preserves the PL intensity. By the detection of the NIR phosphorescence of (1) O2 and evaluation of the photostability of single QDs in aerobic, anaerobic, and (1) O2 scavenger-enriched environments, we disclose relations of Auger ionization and (1) O2 -mediated oxidation to the PL stability of single QDs, which will be useful during the formulation of QD-based single-molecule imaging tools and single-photon devices.

A Label-Free Immunosensor for IgG Based on an Extended-Gate Type Organic Field Effect Transistor.

A novel biosensor for immunoglobulin G (IgG) detection based on an extended-gate type organic field effect transistor (OFET) has been developed that possesses an anti-IgG antibody on its extended-gate electrode and can be operated below 3 V. The titration results from the target IgG in the presence of a bovine serum albumin interferent, clearly exhibiting a negative shift in the OFET transfer curve with increasing IgG concentration. This is presumed to be due an interaction between target IgG and the immobilized anti-IgG antibody on the extended-gate electrode. As a result, a linear range from 0 to 10 µg/mL was achieved with a relatively low detection limit of 0.62 µg/mL (=4 nM). We believe that these results open up opportunities for applying extended-gate-type OFETs to immunosensing.

Photouncaging nanoparticles for MRI and fluorescence imaging in vitro and in vivo.

Multimodal and multifunctional nanomaterials are promising candidates for bioimaging and therapeutic applications in the nanomedicine settings. Here we report the preparation of photouncaging nanoparticles with fluorescence and magnetic modalities and evaluation of their potentials for in vitro and in vivo bioimaging. Photoactivation of such bimodal nanoparticles prepared using photouncaging ligands, CdSe/ZnS quantum dots, and super paramagnetic iron oxide nanoparticles results in the systematic uncaging of the particles, which is correlated with continuous changes in the absorption, mass and NMR spectra of the ligands. Fluorescence and magnetic components of the bimodal nanoparticles are characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and elemental analyses using energy dispersive X-ray (EDX) spectroscopy and X-ray photoelectron spectroscopy (XPS). Bioconjugation of the nanoparticles with peptide hormones renders them with biocompatibility and efficient intracellular transport as seen in the fluorescence and MRI images of mouse melanoma cells (B16) or human lung epithelial adenocarcinoma cells (H1650). Biocompatibility of the nanoparticles is evaluated using MTT cytotoxicity assays, which show cell viability over 90%. Further, we combine MRI and NIR fluorescence imaging in C57BL/6 (B6) mice subcutaneously or intravenously injected with the photouncaging nanoparticles and follow the in vivo fate of the nanoparticles. Interestingly, the intravenously injected nanoparticles initially accumulate in the liver within 30 min post injection and subsequently clear by the renal excretion within 48 h as seen in the time-dependent MRI and fluorescence images of the liver, urinary bladder, and urine samples. Photouncaging ligands such as the ones reported in this article are promising candidates for not only the site-specific delivery of nanomaterials-based contrast agents and drugs but also the systematic uncaging and renal clearance of nanomaterials after the desired in vivo application.

A practical liquid plug flow-through polymerase chain-reaction system based on a heat-resistant resin chip.

Flow-through polymerase chain reaction (PCR) microfluidic systems for fast, small-volume DNA amplification on a single chip are significantly impacting medical and bioanalytical research. We have fabricated an improved, practical flow-through PCR chip by weighting a pressure-sensitive polyolefin (PSP) film onto a cyclo-olefin polymer (COP) substrate. The substrate was cut so as to produce microchannels, and was used to amplify DNA using a small moving liquid plug, in contrast to conventional continuous-flow-through PCR. Infrared (IR) imaging-based thermal analysis of the PSP film enabled the gradient and uniformity of the rapidly flowing microfluid to be accurately visualized, because the PSP film is thin, transparent and heat resistant. The liquid plug flow-through PCR, operating at 150 µl min(-1), showed approximately 55% amplification compared to a commercial PCR instrument, and required only 250 s for 40 cycles. To our knowledge, this is the fastest cycling time reported to date. In a real test sample, the liquid plug flow-through PCR could detect the presence or absence of anthrax in a sample solution in approximately 250 s. Thus, this liquid plug flow-through PCR system, based on our novel PCR chip, excelled in a practical applications compared to other devices.

Microfluidic polymer chip with an embedded ion-selective electrode detector for nitrate-ion assay in environmental samples.

A nitrate ion-selective electrode (NO(3)(-)-ISE) has been developed based on tetradodecylammonium bromide as an anion exchanger and 2-nitrophenyl octyl ether as a plasticizer. The NO(3)(-)-ISE shows an almost Nernstian response to nitrate ion over a concentration range between 1.0 x 10(-6) and 1.0 x 10(-1) M, with an anionic slope of -57.7 +/- 0.7 mV/decade. The selectivity coefficients of the NO(3)(-)-ISE for nitrate ion against chloride and sulfate (log k(NO(3)(-)Cl(-))(pot) = -2.42 and log k(NO(3)(-)SO(4)(2-))(pot) = -4.33) were obtained. A microfluidic polymer chip was fabricated using polystyrene plates and stainless-steel wires as a template for the channel. The microfluidic polymer chip is composed of a mixing chip and a NO(3)(-)-ISE detector chip. The microfluidic polymer chip, integrated with a NO(3)(-)-ISE detector consisting of the NO(3)(-)-ISE and a Na(+)-ISE as a reference electrode, showed an almost Nernstian response to nitrate ion over a concentration range between 1.0 x 10(-5) and 1.0 x 10(-1) M, with an anionic slope of -54.3 +/- 1.3 mV/decade. The microfluidic polymer chip was then applied to the determination of nitrate ion in environmental water samples, such as a tap water, a well-water sample and water for agricultural use.

Characterization of dissolved organic carbon at low levels in environmental waters by microfluidic-chip-based capillary gel electrophoresis with a laser-induced fluorescence detector.

A microfluidic analytical system for characterization of dissolved organic carbon (DOC) in environmental waters, based on a capillary gel electrophoresis (CGE) device with a laser-induced fluorescence (LEF) detector, was developed. The applied voltage and the running buffer were investigated to control the simple floating injection and CGE separation for convenient cross-type microchips made from polymethyl-methacylate. We obtained reproducible peaks for standard organic solutions and the determination time is less than 70 s. The values of the relative standard deviation (RSD) were 0.17-2.01% for repetitive injection (n = 12). We demonstrated high-throughput characterization of DOC in environmental water from the Biwa Lake and the Hino River using microfluidic chip and determined that the content of DOC in the Biwa Lake changed with the seasons.

Determination of adrenal steroids by microfluidic chip using micellar electrokinetic chromatography.

This paper describes a sensitive and convenient method to separate progesterone, 17alpha-hydroxy progesterone, cortexolone, hydrocortisone and cortisone, all of which are steroids and have similar structures, using microfluidic chip-based technology with UV detection at 252 nm. We successfully obtained high-speed separation of the five steroids within 70 s in optimized microfluidic controls and micellar electrokinetic chromatography (MEKC) separation conditions. Fairly good linearity with correlation coefficient of over 0.98 from 10 or 20 to 100 mg/l steroid chemicals was obtained. The limits of detection obtained at a signal to noise ratio of 3 were from 3.89 to 7.80 mg/l. The values of the relative standard deviation (RSD) were 0.98-1.34% for repetitive injection (n = 12) and the intraday and interday RSDs were below 6%. The highly stable response reflected the feasibility of this method.

Advanced sensing technology in environmental field.

Before the introduction of advanced sensing technology in environmental fields, environmental issues were discussed as several categories, such as local environmental issues in the 1970s, global environmental issues in the 1980s, living environmental issues in the 2000s and environmental stress issues in near future, which are of increasing interest in Japan. Using advanced sensing technologies, such as electrochemical sensors, chemically-sensitive field-effect transistors (ChemFETs) based on micro-electro mechanical system (MEMS) micromachining technology and subsequently electrophoretic separation and microfluidic Lab-on-a-Chip using MEMS technology, we have steered several kinds of environmental monitoring projects timely in response to the environmental issues for over the last 25 years. Among the local environmental issues, the global environmental issues and the living environmental issues, some fruits of R&D project will be introduced. Finally, our latest concern of the environmental stress monitoring was discussed and preliminary results were also introduced.

High-throughput assay of nitric oxide metabolites in human plasma without deproteinization by lab-on-a-chip electrophoresis using a zwitterionic additive.

In order to develop a high-throughput assay for nitric oxide metabolites, nitrite (NO2-) and nitrate (NO3-), in biological fluids, we have investigated the simultaneous determination of them using an electrophoretic lab-on-a-chip (microchip capillary electrophoresis, MCE) technique. In this study, in order to establish an MCE assay process without deproteinization, the addition of a zwitterionic additive into the running buffer to reduce the adsorption of protein onto the surface of channel was investigated. Initially, some zwitterionic additives were investigated by making a comparison of relative standard deviations (RSDs) of the migration times for NO2(-) and NO3(-) on capillary electrophoresis. From the results of our comparison of the RSD values, 2% (w/w) N-cyclohexyl-2-aminoethanesulfonic acid (CHES) was selected. As a result of the application of the running buffer with CHES to the MCE process, the complete separation of NO2(-) and NO3(-) in human plasma without deproteinization was achieved within 1 min. Since the RSD values of the positions of the peaks were less than 2.3%, beneficial reduction effects on MCE were suggested. When we used an internal standard method in order to correct the injection volume, the RSDs of the peak heights and areas were less than 10%, and the correlation coefficients of spiked calibration curves ranging from 0 to 350 microM were 0.999 and 0.997 for NO2(-) and NO3(-), respectively. The limits of detection (S/N=3) were 53 microM for NO2(-) and 41 microM for NO3(-). Moreover, the correlation coefficients in excess of 0.99 between the MCE method and a conventional Griess method were achieved for both NO2(-) and NO3(-). Consequently, the possibility of establishing a high-throughput assay process was obtained by utilizing 2% (w/w) CHES to reduce protein adsorption.