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.

Rapid DNA Sequencing Technology Based on the Sanger Method for Bacterial Identification.

Antimicrobial resistance, a global health concern, has been increasing due to inappropriate use of antibacterial agents. To facilitate early treatment of sepsis, rapid bacterial identification is imperative to determine appropriate antibacterial agent for better therapeutic outcomes. In this study, we developed a rapid PCR method, rapid cycle sequencing, and microchip electrophoresis, which are the three elemental technologies for DNA sequencing based on the Sanger sequencing method, for bacterial identification. We achieved PCR amplification within 13 min and cycle sequencing within 14 min using a rapid thermal cycle system applying microfluidic technology. Furthermore, DNA analysis was completed in 14 min by constructing an algorithm for analyzing and performing microchip electrophoresis. Thus, the three elemental Sanger-based DNA sequencing steps were accomplished within 41 min. Development of a rapid purification process subsequent to PCR and cycle sequence using a microchip would help realize the identification of causative bacterial agents within one hour, and facilitate early treatment of sepsis.

Early diagnosis of type 2 diabetes based on multiple biomarkers and non-invasive indices.

We previously reported that type 2 diabetes risk, early impaired glucose tolerance and insulin resistance can be predicted by measuring the fasting levels of certain biomarkers. Here we validated these findings in randomly recruited healthy volunteers (n = 101) based on biomarker expression as well as various non-invasive indices. Weight, body mass index, waist circumference and visceral fat differed between individuals with impaired fasting glucose and/or impaired glucose tolerance, and normal subjects. Fasting plasma levels of glycated hemoglobin, leptin, pro-insulin and retinol binding protein 4 differed between impaired fasting glucose/impaired glucose tolerance and normal subjects group and between newly detected diabetes and normal subjects group. Insulin resistance was correlated with fasting levels of insulin and leptin/adiponectin (r = 0.913); of insulin, retinol binding protein 4 and leptin/adiponectin (r = 0.903); and of insulin, glycated albumin, and leptin/adiponectin (r = 0.913). Type 2 diabetes risk, early impaired glucose tolerance and insulin resistance were predicted with >98% specificity and sensitivity by comparing fasting glucose levels to the estimated Matsuda Index based on fasting levels of insulin, adiponectin and leptin with or without oxidative lineolate metabolites. Non-invasive indices are slightly correlated with glucose tolerance and insulin resistance but do not increase the accuracy of predicting type 2 diabetes risk.

Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

Study of a liquid plug-flow thermal cycling technique using a temperature gradient-based actuator.

Easy-to-use thermal cycling for performing rapid and small-volume DNA amplification on a single chip has attracted great interest in the area of rapid field detection of biological agents. For this purpose, as a more practical alternative to conventional continuous flow thermal cycling, liquid plug-flow thermal cycling utilizes a thermal gradient generated in a serpentine rectangular flow microchannel as an actuator. The transit time and flow speed of the plug flow varied drastically in each temperature zone due to the difference in the tension at the interface between temperature gradients. According to thermal distribution analyses in microfluidics, the plug flow allowed for a slow heating process, but a fast cooling process. The thermal cycle of the microfluid was consistent with the recommended temperature gradient for PCR. Indeed, amplification efficiency of the plug flow was superior to continuous flow PCR, and provided an impressive improvement over previously-reported flow microchannel thermal cycling techniques.

Wireless electrochemiluminescent biosensors: Powering innovation with smartphone technology.

Energy supply and sensor response acquisition can be performed wirelessly, enabling biosensors as Internet of Thing (IoT) tools by linking wireless power supply and electrochemical sensors. Here, we used the electromagnetic induction method to clarify the conditions under which electrochemiluminescence is induced by a simple potential modulation circuit without an integrated circuit on the electrode chip that receives the power. Initially, the potential waveform obtained in a circuit with inductance and capacitance components that resonate with the transmission frequency and a diode for rectification was investigated to clarify the conditions inducing an electrochemiluminescence reaction at the printed electrode. A high-sensitivity complementary metal-oxide semiconductor camera built into the smartphone wirelessly detected the luminescence generated on the electrode chip. The images were quantitatively evaluated using open-source image analysis software which determine the sensitivity of detecting hydrogen peroxide. Glucose oxidase (GOD) encapsulated in a matrix of chitosan polymers and photocrosslinkable polymers was immobilized on a mass-producible and inexpensive printed electrode to maintain high activity. The immobilized membrane suppressed luminescence when immobilized on the working electrode; therefore, the enzyme was immobilized on the counter electrode for glucose measurement over a wide concentration. Thus, luminol electrochemiluminescence was induced on the electrode chip by wireless power supply from a smartphone. Human serum and artificial sweat samples were tested and indicated possibility for actual applications. In this way a fully wireless biosensor was developed with potential as an IoT biosensor.

Surface Modification of Screen-Printed Carbon Electrode through Oxygen Plasma to Enhance Biosensor Sensitivity.

The screen-printed carbon electrode (SPCE) is a useful technology that has been widely used in the practical application of biosensors oriented to point-of-care testing (POCT) due to its characteristics of cost-effectiveness, disposability, miniaturization, wide potential window, and simple electrode design. Compared with gold or platinum electrodes, surface modification is difficult because the carbon surface is chemically or physically stable. Oxygen plasma (O2) can easily produce carboxyl groups on the carbon surface, which act as scaffolds for covalent bonds. However, the effect of O2-plasma treatment on electrode performance remains to be investigated from an electrochemical perspective, and sensor performance can be improved by clarifying the surface conditions of plasma-treated biosensors. In this research, we compared antibody modification by plasma treatment and physical adsorption, using our novel immunosensor based on gold nanoparticles (AuNPs). Consequently, the O2-plasma treatment produced carboxyl groups on the electrode surface that changed the electrochemical properties owing to electrostatic interactions. In this study, we compared the following four cases of SPCE modification: O2-plasma-treated electrode/covalent-bonded antibody (a); O2-plasma-treated electrode/physical adsorbed antibody (b); bare electrode/covalent-bonded antibody (c); and bare electrode/physical absorbed antibody (d). The limits of detection (LOD) were 0.50 ng/mL (a), 9.7 ng/mL (b), 0.54 ng/mL (c), and 1.2 ng/mL (d). The slopes of the linear response range were 0.039, 0.029, 0.014, and 0.022. The LOD of (a) was 2.4 times higher than the conventional condition (d), The slope of (a) showed higher sensitivity than other cases (b~d). This is because the plasma treatment generated many carboxyl groups and increased the number of antibody adsorption sites. In summary, the O2-plasma treatment was found to modify the electrode surface conditions and improve the amount of antibody modifications. In the future, O2-plasma treatment could be used as a simple method for modifying various molecular recognition elements on printed carbon electrodes.

Miniaturization of CRISPR/Cas12-Based DNA Sensor Array by Non-Contact Printing.

DNA microarrays have been applied for comprehensive genotyping, but remain a drawback in complicated operations. As a solution, we previously reported the solid-phase collateral cleavage (SPCC) system based on the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 12 (CRISPR/Cas12). Surface-immobilized Cas12-CRISPR RNA (crRNA) can directly hybridize target double-stranded DNA (dsDNA) and subsequently produce a signal via the cleavage of single-stranded DNA (ssDNA) reporter immobilized on the same spot. Therefore, SPCC-based multiplex dsDNA detection can be performed easily. This study reports the miniaturization of SPCC-based spots patterned by a non-contact printer and its performance in comprehensive genotyping on a massively accumulated array. Initially, printing, immobilization, and washing processes of Cas12-crRNA were established to fabricate the non-contact-patterned SPCC-based sensor array. A target dsDNA concentration response was obtained based on the developed sensor array, even with a spot diameter of 0.64 ± 0.05 mm. Also, the limit of detection was 572 pM, 531 pM, and 3.04 nM with 40, 20, and 10 nL-printing of Cas12-crRNA, respectively. Furthermore, the sensor array specifically detected three dsDNA sequences in one-pot multiplexing; therefore, the feasibility of comprehensive genotyping was confirmed. These results demonstrate that our technology can be miniaturized as a CRISPR/Cas12-based microarray by using non-contact printing. In the future, the non-contact-patterned SPCC-based sensor array can be applied as an alternative tool to DNA microarrays.

Rotatable reagent cartridge for high-performance microvalve system on a centrifugal microfluidic device.

Recently, microfluidic lab-on-a-CD (LabCD) has attracted attentions of researchers for its potential for pumpless, compact, and chip-inclusive on-site bioassay. To control the fluids in the LabCD, microvalves such as capillary, hydrophobic, siphon, and sacrificial valves have been employed. However, no microvalve can regulate more than one channel. In a complicated bioassay with many sequential mixing, washing, and wasting steps, thus, an intricate fluidic network with many microchannels, microvalves, and reservoirs is required, which increases assay costs in terms of both system development and chip preparation. To address this issue, we developed a rotatable reagent cartridge (RRC), which was a column-shaped tank and has several rooms to store different reagents. By embedding and rotating the RRC in the LabCD with a simple mechanical force, only the reagent in the room connected to the following channel was injected. By regulating the angle of the RRC to the LabCD, conservation and ejection of each reagent could be switched. Our developed RRC had no air vent hole, which was achieved by the gas-permeable gap between the bottle and cap parts of the RRC. The RRC could inject 230 nL-10 µL of reagents with good recoveries more than 96%. Finally, an enzymatic assay of L-lactate was demonstrated, where the number of valves and reservoirs were well minimized, significantly simplifying the fluidic system and increasing the channel integratability. Well quantitative analyses of 0-100 µM L-lactate could easily be carried out with R(2) > 0.999, indicating the practical utility of the RRC for microfluidic bioanalysis.

Microarray of human P450 with an integrated oxygen sensing film for high-throughput detection of metabolic activities.

A microarray chip containing human P450 isoforms was constructed for the parallel assay of their metabolic activities. The chip had microwells that contained vertically integrated P450 and oxygen sensing layers. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). Human P450s (23 types) expressed in E. coli and purified as membrane fractions were immobilized in agarose matrixes on the oxygen sensing layer. The activities of P450s were determined by evaluating the fluorescence intensity enhancement of the oxygen sensor due to the oxygen consumption by the metabolic reaction. By normalizing the responses with the amounts of oxygen sensor and P450 enzymes in microwells, we could obtain fluorescence enhancement patterns that were characteristic to the combination of P450 isoforms and substrate material. The patterns obtained from two psoralen derivatives resembled each other, whereas a structurally different substrate (capsaicin) resulted in a distinct pattern. These results suggest the potential of the microarray to analyze the activities of diverse P450 isoforms in a high-throughput fashion. Furthermore, mechanism-based inactivation (MBI) of P450 could be detected by successively incubating a chip with different substrate solutions and measuring the residual activities.

Detection of expressed gene in isolated single cells in microchambers by a novel hot cell-direct RT-PCR method.

In order to be able to detect the expression of a gene in individual cells, the ability to isolate and lyse a single cell and to perform reverse transcription polymerase chain reaction (RT-PCR) in one device is important. As is common, when performing cell lysis and RT-PCR in the same reaction chamber, it is necessary to add the reagent for RT-PCR after cell lysis. In this study, we propose an original formula for cell lysis and RT-PCR in the same reaction chamber without the addition of reagent by only a heat process, which we termed hot cell-direct RT-PCR. Hot cell-direct RT-PCR was enabled by using Tth DNA polymerase, which is a thermostable polymerase and has high reverse transcription activity in the presence of manganese ions. Direct detection of RT-PCR products was performed by detecting fluorescence with the use of a double-dye fluorescent probe. We attempted to detect the mRNA of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in isolated Jurkat cells on a microfluidic device, which we had already developed for single cell isolation. After cell isolation and successive hot cell-direct RT-PCR on the device, fluorescent signals from RT-PCR products for a single cell were detected and differentiated from the chamber containing no cells. A highly positive linear relationship (r = 0.9933) was observed between the number of chambers containing cell(s) and those containing RT-PCR products from 10 to 400 cells µL(-1). Thus it was possible to use the novel hot cell-direct RT-PCR method to detect the expressed gene in isolated cells.

Rapid multiplex PCR assay for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary samples: A 30-minute assay.

We developed a rapid multiplex PCR assay available in bedside for the simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis, which enables diagnosis in less than 30 minutes. In this study, we validated the clinical utility of this assay including its sensitivity and specificity for NG and CT detection.

Compact disk (CD)-shaped device for single cell isolation and PCR of a specific gene in the isolated cell.

For immediate discrimination among isolated cells we propose a novel device and technique for isolation of cells and sequential detection of specific gene(s) within them by polymerase chain reaction (PCR). In this study, we isolated Salmonella enterica cells and detected the Salmonella-specific invA gene from isolated cells by PCR on a compact disk (CD)-shaped device. This device enabled liquid flow by centrifugal force without a micro pump, and was fabricated from silicon wafer and glass to avoid evaporation of a small amount of reagent. One device has 24 microchannels, and 313 microchambers integrated on each microchannel. One microliter of PCR mixture containing cells was separated into microchambers on the device at 5000 rpm for 30 s. Each microchamber contained approximately 1.5 nL PCR mixture. A Poisson distribution of S. enterica cells was observed for different densities of cell suspension. At 200 cells µL(-1) of S. enterica or less, isolated single cells could be determined on the device by amplification of DNA of the invA gene; at 400 cells µL(-1), chambers containing no, one, two, or three cells could be determined on the device. Selective detection of S. enterica was achieved by PCR from a mixture of S. enterica and Escherichia coli on the CD-shaped device.

Flow analysis on microcasting with degassed polydimethylsiloxane micro-channels for cell patterning with cross-linked albumin.

Patterned cell culturing is one of the most useful techniques for understanding the interaction between geometric conditions surrounding cells and their behaviors. The authors previously proposed a simple method for cell patterning with an agarose gel microstructure fabricated by microcasting with a degassed polydimethylsiloxane (PDMS) mold. Although the vacuum pressure produced from the degassed PDMS can drive a highly viscous agarose solution, the influence of solution viscosity on the casting process is unknown. This study investigated the influences of micro-channel dimensions or solution viscosity on the flow of the solution in a micro-channel of a PDMS mold by both experiments and numerical simulation. It was found experimentally that the degassed PDMS mold was able to drive a solution with a viscosity under 575 mPa·s. A simulation model was developed which can well estimate the flow rate in various dimensions of micro-channels. Cross-linked albumin has low viscosity (1 mPa·s) in aqueous solution and can undergo a one-way dehydration process from solution to solid that produces cellular repellency after dehydration. A microstructure of cross-linked albumin was fabricated on a cell culture dish by the microcasting method. After cells were seeded and cultivated on the cell culture dish with the microstructure for 7 days, the cellular pattern of mouse skeletal myoblast cell line C2C12 was observed. The microcasting with cross-linked albumin solution enables preparation of patterned cell culture systems more quickly in comparison with the previous agarose gel casting, which requires a gelation process before the dehydration process.

Heat denaturation of the antibody, a multi-domain protein.

The antibody is one of the most well-studied multi-domain proteins because of its abundance and physiological importance. In this article, we describe the effect of the complex, multi-domain structure of the antibody on its denaturation by heat. Natural antibodies are composed of 6 to 70 immunoglobulin fold domains, and are irreversibly denatured at high temperatures. Although the separated single immunoglobulin fold domain can be refolded after heat denaturation, denaturation of pairs of such domains is irreversible. Each antibody subclass exhibits a distinct heat tolerance, and IgE is especially known to be heat-labile. IgE starts unfolding at a lower temperature compared to other antibodies, because of the low stability of its CH3 domain. Each immunoglobulin domain starts unfolding at different temperatures. For instance, the CH3 domain of IgG unfolds at a higher temperature than its CH2 domain. Thus, the antibody has a mixture of folded and unfolded structures at a certain temperature. Co-existence of these folded and unfolded domains in a single polypeptide chain may increase the tendency to aggregate which causes the inactivation of the antibody.

Rapid Enzyme-linked Immunosorbent Assays for Diagnosis of Diabetes in a Compact Disc-shaped Microfluidic Device.

We have developed a compact disc (CD)-shaped microfluidic device for multiple, rapid enzyme-linked immunosorbent assays (ELISA). The device has a versatile design that can be adapted for the detection of various proteins by selecting the push-in-type reaction parts and appropriate reagents for each target. In this paper, we report the rapid quantification of insulin, adiponectin, and leptin, which can be used for the early diagnosis of diabetes, in human serum in only 16 min with our device.

Flexible manipulation of microfluids using optically regulated adsorption/desorption of hydrophobic materials.

To realize highly integrated micro total analysis systems (microTAS), a simply controlled miniaturized valve should be utilized on microfluidic device. In this paper, we describe the application of photo-induced super-hydrophilicity of titanium dioxide (TiO2) to microfluidic manipulation. In addition, we found a new phenomenon for reversibly converting the surface wettability using a polydimethylsiloxane (PDMS) matrix and the photocatalytic properties of TiO2. While PDMS polymer was irradiated with UV, it was confirmed that hydrophobic material was released from the polymer to air. Several prepolymers were identified as the hydrophobic material with a gas chromatograph and mass spectrometer (GC/MS). Here, we successfully demonstrated the flexible manipulation of microfluid in a branched microchannel using the reversible wettability as micro opto-switching valve (MOS/V). The simultaneous control of MOS/Vs was also demonstrated on a 256-MOS/V integrated disk. The MOS/V promises to be one of the most effective flow switching valves for advanced applications in highly integrated micro/nano fluidics.

A single-bead analysis on a disk-shaped microfluidic device using an antigen-immobilized bead.

A disk-shaped microfluidic device (lab-on-a-Disk) was developed to allow the evaluation of mental stress. As a standard sample, secretory immunoglobulin A (sIgA), which is a candidate marker of mental stress, was measured by a heterogeneous enzyme immunoassay (EIA) on the lab-on-a-Disk. Centrifugal force provided a microfluidic control on the lab-on-a-Disk. We examined the relationship between the rotational speed, the channel profile, and the position of the microfluidic chambers from the center of rotation to manipulate sample solutions into each reaction reservoir through microchannels sequentially, i.e., retain in a reservoir or flow into a subsequent reservoir. A single glass bead with immobilized sIgA on its surface was injected into a reservoir for a competitive antigen-antibody reaction, and applied to a specific surface in a heterogeneous assay. It is expected that the lab-on-a-Disk would be suitable for miniaturization and automation of the processes in EIA compared with a conventional EIA using a titer plate.

On-site identification of meat species in processed foods by a rapid real-time polymerase chain reaction system.

Correct labeling of foods is critical for consumers who wish to avoid a specific meat species for religious or cultural reasons. Therefore, gene-based point-of-care food analysis by real-time Polymerase Chain Reaction (PCR) is expected to contribute to the quality control in the food industry. In this study, we perform rapid identification of meat species by our portable rapid real-time PCR system, following a very simple DNA extraction method. Applying these techniques, we correctly identified beef, pork, chicken, rabbit, horse, and mutton in processed foods in 20min. Our system was sensitive enough to detect the interfusion of about 0.1% chicken egg-derived DNA in a processed food sample. Our rapid real-time PCR system is expected to contribute to the quality control in food industries because it can be applied for the identification of meat species, and future applications can expand its functionality to the detection of genetically modified organisms or mutations.

On-chip micellar electrokinetic chromatographic separation of phenolic chemicals in waters.

This paper describes on-chip micellar electrokinetic chromatography (MEKC) separation of bisphenol A and 3 kinds of alkylphenols, which have been recently recognized as endocrine disrupting chemicals for fish by the Japanese government, using microchip capillary electrophoresis with UV detection. We successfully obtained high-speed separation of the phenolic chemicals within 15 s as optimizing in microfluidic controls and MEKC separation conditions. We obtained fairly good linearity with correlation coefficient of over 0.98 from 0 to 50 mg/l phenolic chemicals except for 4-nonylphenol, which sample is the mixture of many geometrical isomers (r = 0.86). The values of the relative standard deviation for peak height in 50 mg/l phenolic chemicals were less than 8% except for bisphenol A (11.0%). The limits of detection obtained at a signal-to-noise ratio of 3 were from 5.6 to 20.0 mg/l. To realize on-site monitoring, we described strategy for on-chip MEKC analysis of the phenolic chemicals in waters using a portable analyzer based on microfluidic devices.