Yusuke Fuchiwaki

Ultra-rapid flow-through polymerase chain reaction microfluidics using vapor pressure.

A novel flow-through polymerase chain reaction (PCR) microfluidic system using vapor pressure was developed that can achieve ultra-rapid, small-volume DNA amplification on a chip. The 40-cycle amplification can be completed in as little as 120 s, making this device the fastest PCR system in the world. The chip device is made of a pressure-sensitive polyolefin (PSP) film and cyclo-olefin polymer (COP) substrate which was processed by cutting-work to fabricate the microchannel. The enclosed structure of the microchannel was fabricated solely by weighing the PSP film on the COP substrate, resulting in superior practical application. The vapor pressure in the denaturation zone of the destabilizing flow source was applied to the flow force, and ultra-rapid, efficient amplification was accomplished with a minimal amount of PCR reagents for detection. The flowing rhythm created by vapor pressure minimized the residual PCR products, leading to highly efficient amplification. For field test analysis, airborne dust was collected from a public place and tested for the presence of anthrax. The PCR chip had sufficient sensitivity for anthrax identification. The fastest time from aerosol sampling to detection was theoretically estimated as 8 min.

A fine pointed glucose oxidase immobilized electrode for low-invasive amperometric glucose monitoring

A low invasive type glucose sensor, which has a sensing region at the tip of a fine pointed electrode, was developed for continuous glucose monitoring. Platinum-iridium alloy electrode with a surface area of 0.045mm(2) was settled at the middle of pointed PEEK (Polyetheretherketone) tubing and was employed as sensing electrode. Electrodeposition of glucose oxidase in the presence of surfactant, Triton X-100, was performed for high-density enzyme immobilization followed by the electropolymerization of o-phenylenediamine for the formation of functional entrapping and permselective polymer membrane. Ag/AgCl film was coated on the surface of PEEK tubing as reference electrode. Amperometric responses of the prepared sensors to glucose were measured at a potential of 0.60V (vs. Ag/AgCl). The prepared electrode showed the sensitivity of 2.55μA/cm(2) mM with high linearity of 0.9986, within the glucose concentration range up to 21mM. The detection limit (S/N=3) was determined to be 0.11mM. The glucose sensor properties were evaluated in phosphate buffer solution and in vivo monitoring by the implantation of the sensors in rabbit, while conventional needle type sensors as a reference were used. The results showed that change in output current of the proposed sensor fluctuated similar with one in output current of the conventional needle type sensors, which was also in similar accordance with actual blood sugar level measured by commercially glucose meter. One-point calibration method was used to calibrate the sensor output current.

New Approach to a Practical Quartz Crystal Microbalance Sensor Utilizing an Inkjet Printing System

The present work demonstrates a valuable approach to developing quartz crystal microbalance (QCM) sensor units inexpensively for reliable determination of analytes. This QCM sensor unit is constructed by inkjet printing equipment utilizing background noise removal techniques. Inkjet printing equipment was chosen as an alternative to an injection pump in conventional flow-mode systems to facilitate the commercial applicability of these practical devices. The results demonstrate minimization of fluctuations from external influences, determination of antigen-antibody interactions in an inkjet deposition, and quantification of C-reactive protein in the range of 50–1000 ng(x000B7)mL−1. We thus demonstrate a marketable application of an inexpensive and easily available QCM sensor system.

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.

Characteristics of Molecularly Imprinted Polymer Thin Layer for Bisphenol A and Response of the MIP-Modified Sensor

We examine the characteristics of molecularly imprinted polymer (MIP) layers for bisphenol A (BPA) to investigate the effect of their thickness on the performance of the BPA sensor. MIP thin layers for bisphenol A were polymerized on a sputtered gold electrode by UV light irradiation for 2 to 30 min. Their thickness, as determined by a QCM analyzer, was 3.6 ±  0.3 nm after 5 min of irradiation and increased as the irradiation time increased to 30 min. AFM images of the MIP-modified surface suggested that the gold electrode was covered with a smooth MIP layer. The anodic peaks of BPA and ascorbic acid caused by gold electrode and the MIP-modified electrode were compared, and the electrode with MIP polymerized for 5 min showed more selectivity to BPA than that polymerized for 2 min. The MIP thin layer thus has potential as a sensing element of a chemical sensor.

Design and Construction of Automated Amperometric Immunosensing System

An automated amperometric immunosensors for the quantification of transferrin was fabricated using two monoclonal IgG class anti-rat transferrin antibodies, 15C2H3 and 22A06D2. The former was immobilized on a gold electrode, while the latter was conjugated with the positively charged polymers containing ferrocenyl groups. The quantitative analysis of transferrin was investigated by measuring the electrooxidation current of ferrocene, which is proportional to the transferrin concentration for the equivalently observed sandwich formation. The deposition of electroconductive polymers, polypyrrole (poly-P) and poly⌈ferrocenyl-methyl-3-(pyrrol-l-yl)propyldimethylammonium bromide⌉ (poly-FP), were performed on the surface of antibody-immobilized gold electrode in order to increase the efficiency of the electron transfer between the antibody-cojugated ferrocence and the gold electrode, and to eliminate the nonspecific binding of ferrocene-labeled antibody by a mutual repulsion.

UV-laser-assisted modification of poly(methyl methacrylate) and its application to capillary-driven-flow immunoassay

A concave microchannel surface was formed by nanosecond pulse laser ablation to allow antibody immobilization on a capillary flow immunoassay chip. Microscopic analysis showed that UV laser ablation of poly(methyl methacrylate) at 193 nm and 1.76 J cm−2 allowed excellent immobilization of Cy5 conjugated antibody. The concave structure was 10 µm deep and 260 µm wide and supported uniform antibody printing on the microchannel surface. The characteristics of immobilized antibodies on this surface and on a commercially available polymer coating were comparable. Quantitative analysis of procollagen type I C-peptide (PICP) at different concentrations provided a linear relationship in the range 0–600 ng ml−1 PICP, which is sufficient for clinical estimation of PICP in the blood. The results may provide a new benchmark for a mechatronic antibody immobilization-based capillary flow immunoassay chip.

The Establishment of Bisphenol A Sensing System Utilizing Molecularly Imprinted Polymer Receptor and Electrochemical Determination

A sensing system of bisphenol A (BPA) based on the electrochemical detection utilizing molecularly imprinted polymer (MIP) as a receptor of BPA was investigated. MIP for BPA was polymerized thermally from 4-vinylpyridine as a functional monomer and ethylene dimethacrylate (EDMA) as a cross-linker and served to prepare an MIP packed column. BPA in an aqueous solution was adsorbed to an MIP packed column and eluted by acetonitrile/phosphate buffer (60/40, v/v). From aqueous solution, BPA was adsorbed to the column and eluted completely in the eluent. The eluted BPA was electrochemically detected by cyclic voltammetry. Optimum pH and scan rate were 7.0 and 0.1 V/s in phosphate buffer. Electrochemical detection of BPA in acetonitrile/phosphate buffer was performed, and linear relationship between BPA and anodic peak current was observed at the range of 10–100 μM. In the eluent, anodic peak current of BPA was observed around 650 mV.

Electrochemical Sensor Based on Biomimetic Recognition Utilizing Molecularly Imprinted Polymer Receptor

Biological recognition elements such as antibodies, enzymes and aptamers have been utilized as specific receptors to a target molecule in a wide variety of assays and sensors. However, many difficulties for their practical use exist as they lack stability and reusability. Moreover, it is not easy to obtain and prepare sufficient natural bioreceptors. Since practically there are many extraneous inhibitors against biological receptors, scientists have attempted to develop specific recognition elements alternative to bioreceptors. One approach was the synthesis of hosts which possess a structure capable of binding complementary guests. The synthesis of specific recognition sites has been accomplished by coordinating functional monomers around the target molecule, and then cross-linking to position functional monomers around the target molecule (Fig. 1). Such receptors are

Electrochemical Sensing System Utilizing Simazine-Imprinted Polymer Receptor for the Detection of Simazine in Tap Water

A simazine sensing system, composed of column packed with a molecularly imprinted polymer (Sim-MIP) and an electrochemical analyzer, was scaled down in order to easily determine the concentration of simazine, an environmentally restricted chemical, in tap water. In order to enhance the detection limit, the ratio of the eluent (dilution rate) in the electrolyte was optimized to 10%. A new in-house built column size with  mm was prepared, and 3 mg of Sim-MIP particles was packed in the column. During the sensing process, 90% of the simazine loaded to the column was collected by elution. The reductive current of simazine was determined up to 1–10 M. Solid phase extraction through the Sim-MIP column enabled simazine to be selectively detected from a mixed aqueous solution containing structural analogues in the range of 10–40 nM. Whether the concentration of simazine in tap water had reached environmentally restricted levels (10–40 nM) was determined within 1 hour using this system.