Hiroyuki Kudo

Soft contact lens biosensor for in situ monitoring of tear glucose as non-invasive blood sugar assessment

A contact lens (CL) biosensor for in situ monitoring of tear glucose was fabricated and tested. Biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer and polydimethyl siloxane (PDMS) were employed as the biosensor material. The biosensor consists of a flexible Pt working electrode and a Ag/AgCl reference/counter electrode, which were formed by micro-electro-mechanical systems (MEMS) technique. The electrode at the sensing region was modified with glucose oxidase (GOD). The CL biosensor showed a good relationship between the output current and glucose concentration in a range of 0.03-5.0mM, with a correlation coefficient of 0.999. The calibration range covered the reported tear glucose concentrations in normal and diabetic patients. Also, the CL biosensor was applied to a rabbit for the purpose of tear glucose monitoring. The basal tear glucose was estimated to 0.11 mM. Also, the change of tear glucose induced by the change of blood sugar level was assessed by the oral glucose tolerance test. As a result, tear glucose level increased with a delay of 10 min from blood sugar level. The result showed that the CL biosensor is expected to provide further detailed information about the relationship between dynamics of blood glucose and tear glucose.

Biochemical gas sensor (bio-sniffer) for ultrahigh-sensitive gaseous formaldehyde monitoring.

An ultrahigh-sensitive fiber-optic biochemical gas sensor (bio-sniffer) for continuous monitoring of indoor formaldehyde was constructed and tested. The bio-sniffer measures gaseous formaldehyde as fluorescence of nicotinamide adenine dinucleotide (NADH), which is the product of formaldehyde dehydrogenase (FALDH) reaction. The bio-sniffer device was constructed by attaching a flow cell with a FALDH immobilized membrane onto a fiber-optic NADH measurement system. The NADH measurement system utilizes an ultraviolet-light emitting diode (UV-LED) with peak emission of 335 nm as an excitation light source. The excitation light was introduced to an optical fiber probe, and fluorescence emission of neighboring NADH, which was produced by applying formaldehyde vapor to the FALDH membrane, was concentrically measured with a photomultiplier tube. Assessment of the bio-sniffer was carried out using a standard gas generator. Response, calibration range and selectivity to other chemical substances were investigated. Circulating phosphate buffer, which contained NAD+, available for continuous monitoring of formaldehyde vapor. The calibration range of the bio-sniffer was 2.5 ppb to 10 ppm, which covers the guideline value of the World Health Organization (80 ppb). High selectivity to other gaseous substances due to specific activity of FALDH was also confirmed. Considering its high sensitivity, a possible application of the bio-sniffer is continuous indoor formaldehyde monitoring to provide healthy residential atmosphere.

Tonometric biosensor with a differential pressure sensor for chemo-mechanical measurement of glucose

A tonometric biosensor for glucose was constructed using a chemo-mechanical reaction unit and a differential pressure sensor. The reaction unit was fabricated by using both liquid and gas cells separated by an enzyme diaphragm membrane, in which glucose oxidase was immobilized onto the single (gas cell) side of the dialysis membrane. By applying glucose solution (0, 25.0, 50.0, 100, 150 and 200 mmol/l) into the liquid cell of the chemo-mechanical reaction unit, the pressure in the gas cell decreased continuously with a steady de-pressure slope because the oxygen consumption in the gas cell was induced by the glucose oxidase (GOD) enzyme reaction at the enzyme side of the porous diaphragm membrane. The steady de-pressure slope in the gas cell showed the linear relationship with the glucose concentration in the liquid cell between 25.0 and 200.0 mmol/l (correlation coefficient of 0.998). A substrate regeneration cycle coupling GOD with l-ascorbic acid (AsA: 0, 1.0, 3.0, 10.0 and 50.0 mmol/l; as reducing reagent system) was applied to the chemo-mechanical reaction unit in order to amplify the output signal of the tonometric biosensor. 3.0 mmol/l concentration of AsA could optimally amplify the sensor signal more than 2.5 times in comparison with that of non-AsA reagent.

2D spatiotemporal visualization system of expired gaseous ethanol after oral administration for real-time illustrated analysis of alcohol metabolism

A novel 2-dimensional spatiotemporal visualization system of expired gaseous ethanol after oral administration for real-time illustrated analysis of alcohol metabolism has been developed, which employed a low level light CCD camera to detect chemiluminescence (CL) generated by catalytic reactions of standard gaseous ethanol and expired gaseous ethanol after oral administration. First, the optimization of the substrates for visualization and the concentration of luminol solution for CL were investigated. The cotton mesh and 5.0 mmol L(-1) luminol solution were selected for further investigations and this system is useful for 0.1-20.0 mmol L(-1) of H(2)O(2) solution. Then, the effect of pH condition of Tris-HCl buffer solution was also evaluated with CL intensity and under the Tris-HCl buffer solution pH 10.1, a wide calibration range of standard gaseous ethanol (30-400 ppm) was obtained. Finally, expired air of 5 healthy volunteers after oral administration was measured at 15, 30, 45, 60, 75, 90, 105 and 120 min after oral administration, and this system showed a good sensitivity on expired gaseous ethanol for alcohol metabolism. The peaks of expired gaseous ethanol concentration appeared within 30 min after oral administration. During the 30 min after oral administration, the time variation profile based on mean values showed the absorption and distribution function, and the values onward showed the elimination function. The absorption and distribution of expired gaseous ethanol in 5 healthy volunteers following first-order absorption process were faster than the elimination process, which proves efficacious of this system for described alcohol metabolism in healthy volunteers. This system is expected to be used as a non-invasive method to detect VOCs as well as several other drugs in expired air for clinical purpose.

Soft contact-lens biosensor for real-time tear sugar monitoring at the eye

A soft contact-lens (SCL) biosensor for in-situ tear sugar monitoring was fabricated and tested. In contrast to the previously reported continuous glucose monitoring (CGM) devices, we focused on the relationship between blood glucose and tear glucose. The SCL biosensor was constructed by applying microelectromechanical systems (MEMS) techniques to functional polymers. The sensor has film electrodes on the surface of a rounded surface of polydimethyl siloxane (PDMS) contact-lens and glucose oxidase (GOD) was immobilized on the sensing region of the electrodes. Sufficiently adhesive and flexible electrodes were formed on the polymer device (PDMS and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer). In the in-vitro characterization, the SCL-biosensor showed excellent relationship between the output current and glucose concentration from 0.03 to 10.0 mmol/L, which included the reported tear glucose concentrations (0.14 mM) of humans. Based on the result, in-situ tear glucose monitoring with the SCL-biosensor was carried out. The output current of the SCL-biosensor was sufficiently stable and reflected the tear glucose levels. The static glucose level in the basal tear of a rabbit was measured and estimated to 0.13 mmol/L. Since the biosensor was also possible to be sotored for more than 50 days, daily disposable application was expected with the SCL biosensor.

An autonomous drug release system based on chemo-mechanical energy conversion “Organic Engine” for feedback control of blood glucose

A novel autonomous drug release system was fabricated and tested. The system consists of two integrated units: decompression unit and drug release unit. The decompression unit was fabricated by separating a cylindrical cell into a top cell (gas phase) and a bottom cell (liquid phase) by glucose oxidase (GOD) enzyme immobilized membrane. The enzyme membrane recognizes glucose and converts chemical energy found in glucose to mechanical energy. The linear correlation between glucose concentration and de-pressure slope of the top cell was revealed as applying glucose solution to the bottom cell. Afterward, the drug release unit which utilizes the energy of the decompression unit as a power source was fabricated and evaluated by recording its release actions. The drug release unit was made to release at a constant quantity of drug in the liquid phase. The system was then fabricated by combining the decompression unit and the drug release unit. And it was evaluated in an open loop and in a closed loop by applying a mixture of glucose solution (100 mmol/l) and NADH(+) using glucose dehydrogenase enzyme (GDH) as a glucose reducer. Glucose concentration decreased gradually in the closed loop and, as a consequence, interval time of the GDH release became longer. In other words, an inverse correlation between actuation interval of the system and glucose concentration was shown. As a result, the possibility of feedback control of glucose concentration by the drug release system without external energy was confirmed.

A highly sensitive and temporal visualization system for gaseous ethanol with chemiluminescence enhancer

A two-dimensional gaseous ethanol visualization system has been developed and demonstrated using a horseradish peroxidase-luminol-hydrogen peroxide system with high-purity luminol solution and a chemiluminescence (CL) enhancer. This system measures ethanol concentrations as intensities of CL via the luminol reaction. CL was emitted when the gaseous ethanol was injected onto an enzyme-immobilized membrane, which was employed as a screen for two-dimensional gas visualization. The average intensity of CL on the substrate was linearly related to the concentration of standard ethanol gas. These results were compared with the CL intensity of the CCD camera recording image in the visualization system. This system is available for gas components not only for spatial but also for temporal analysis in real time. A high-purity sodium salt HG solution (L-HG) instead of standard luminol solution and an enhancer, eosin Y (EY) solution, were adapted for improvement of CL intensity of the system. The visualization of gaseous ethanol was achieved at a detection limit of 3 ppm at optimized concentrations of L-HG solution and EY.

NADH-Fluorometric Biochemical Gas Sensor (Bio-Sniffer) for Evaluation of Indoor Air Quality

A fiber-optic biochemical gas sensor (bio-sniffer) for assessment of indoor formaldehyde (FA) is fabricated and tested in monitoring of FA degradation by TiO2. The biosniffer measures FA vapor as fluorescence of reduced nicotinamide adenine dinucleotide (NADH), which is the product of enzymatic reaction of FA dehydrogenase (FALDH). Usually, an enzyme loses its specific activity in the gas phase. This makes biochemical gas monitoring difficult. We employ a microflow-cell with a FALDHimmobilized membrane to prevent the FALDH from deactivation at the optode. An ultraviolet light-emitting diode (UV-LED) with peak emission of 335 nm is employed as an excitation light source. Emission of the UV-LED is introduced to the optode through an optical fiber and fluorescence of NADH is picked up coaxially at the optode. To improve the sensitivity, a multi-LED light source is introduced instead of the previously reported system. A photomultiplier tube is utilized as a photodetector. Continuous FA monitoring with biochemical method is successfully conducted with high selectivity at subppb level. Owing to the improved excitation source, the detection limit is improved to 750 ppt. A real-sample test is also carried out using a photocatalytic titania. A standard gas is flowed into a cell, in which photocatalyst glasses are located, and the FA level is measured using the biosniffer. Therefore, degradation of FA by the catalytic reaction of titania is monitored as reduction of FA in real time. According to the results, it is expected to be useful in fast and convenient monitoring of indoor FA.

Amperometric Biosensor Based on Enzyme Immobilization with Post Process for Medical and Multiple Applications

An amperometric biosensor with a laminar structure for various analytes was fabricated and tested. The biosensor was composed of two platinum thin-film electrodes and a hydrophilic polytetrafluoroethylene membrane. This structure has the advantage of enzyme immobilization for varying purposes in a post-process. The biosensor was fabricated using a simplified four-step coating process: polydimethylsiloxane was applied to the hydrophilic polytetrafluoroethylene membrane as an insulation coating, followed by platinum sputtering to form two thin-film electrodes. An adhesive coating was applied to form an inactive platinum surface and an enzyme immobilization region on the sensing region of the biosensor. The platinum electrodes were used for amperometric measurements. They were useful for the determination of 2.00 to 200 µmol/l of NADH with good precision. The applicability of the biosensor for immobilizing each enzyme: aldehyde dehydrogenase, formaldehyde dehydrogenase, and glucose oxidase in post process was investigated. The calibration ranges of the biosensors for acetaldehyde solution, formaldehyde vapor, and glucose solution were 1.00 to 200 µmol/l, 2.0 to 12 ppm, and 0.100 to 10.0 mmol/l, respectively. The results indicate that the structure and fabrication process of the amperometric biosensor is useful for multipurpose applications.

Fiber-Optic Fluoroimmunoassay System with a Flow-Through Cell for Rapid On-Site Determination of Escherichia coli O157:H7 by Monitoring Fluorescence Dynamics

Dynamic fluoroimmunoassay with a flow-through system using optical fiber probes consisting of polystyrene was developed and applied to a quantitative detection of E. coli O157:H7. The system measures E. coli as fluorescence of sandwich-type immune complexes formed by capture antibodies immobilized on the surface of the probe, E. coli cells, and fluorescently labeled detection antibodies. Excitation was carried out using an evanescent wave from the probe. Resulting fluorescence recoupled into the probe was detected by a photodiode. The assay system was constructed with a flow cell which was available for sequential injection of experimental reagents. In vitro characterization was performed using the flow cell, and the calibration range of E. coli O157:H7 was from 103 to 107 cells/mL. The measurement for each sample was completed within 12 min. Furthermore, it was also possible to estimate the concentrations of E. coli O157:H7 by the increasing rate of fluorescence during binding reaction of detection antibodies to antigens. This minimized the time for measurement down to 6 min. The system is suitable for rapid and direct determination for microorganisms or bacteria in food, clinical, and environmental sources.