Colorimetric and fluorometric determination of uric acid by a suspension-based assay using enzyme-immobilized micro-sized particles.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.

Development of a C-reactive protein quantification method based on flow rate measurement of an ink solution pushed out by oxygen gas generated by catalase reaction.

A novel determination method for protein biomarkers based on on-chip flow rate measurement was developed using a microchip with organic photodiodes (OPDs). This quantitative method is based on the flow rate measurement of an ink solution pushed out by oxygen gas generated through catalase reaction. The amount of oxygen gas generated in the sample reservoir is dependent on the concentration of the analyte; therefore, the flow rate of the ink solution is also dependent on the concentration of the analyte. The concentration of the analyte can thus be estimated by measurement of the ink solution flow rate. The ink solution flow rate was estimated by measuring the migration time of the ink solution between two points using two OPDs placed below the microchannel. The principle of this method was demonstrated by the measurement of catalase using the microchip. In addition, the developed method was applied to the determination of C-reactive protein (CRP), a biomarker of inflammation, based on a catalase-linked immunosorbent assay (C-LISA). The limit of detection for CRP was 0.20 µg/mL. The method was also applied to the determination of CRP in human serum, and the quantitative values obtained by this method were in excellent agreement with those obtained by the conventional enzyme-linked immunosorbent assay (ELISA) method. The developed method does not require a photodetector with high sensitivity and is thus capable of downsizing; therefore, this will be useful for on-site analyses such as point-of-care testing and field measurements.

Development of small-sized fluorescence detector for pipette tip-based biosensor for on-site diagnosis.

A small-sized fluorescence detector (referred to as a pipette tip [PT]-reader) was developed for a pipette tip-based biosensor. The PT-reader allows us to measure the fluorescence intensity of a solution in a truncated cone-shaped pipette tip with only the tip inserted into the PT-reader. A pipette holder made from a mixture of polydimethylsiloxane (PDMS) and carbon black was capable of the rigorous position arrangement of a truncated cone shaped-pipette tip and the prevention of stray light. The detection performance of the PT-reader was evaluated by measurement of resorufin. The limit of detection (LOD; 3σ) and the relative standard deviation (RSD, n = 4) were estimated to be 0.46 µM and 0.47-4.1%, respectively. This performance was comparable to that of a desktop-type fluorescence microplate reader. In addition, the PT-reader was applied to the quantification of immunoglobulin A (IgA), and the LOD (3σ) of IgA was estimated to be 1.0 ng/mL. The quantitation values of IgA in human saliva obtained by the PT-based enzyme-linked immunosorbent assay (PT-ELISA) were in agreement with those obtained by conventional ELISA. The PT-reader is expected to be useful for low-cost and user-friendly measurements, and the technique of device development proposed in this study will contribute to the progress of on-site medical diagnosis.

Development of a colorimetric assay for quantification of favipiravir in human serum using ferrihydrite.

We developed a colorimetric analytical method for favipiravir (FPV), a promising treatment for COVID-19. FPV forms yellow complexes with ferrihydrite (Fh) by a ligand substitution reaction with the iron (III) hydroxyl surface groups in Fh. Fh-coated microbeads were packed into a capillary tube with an inner diameter of 1 mm. FPV-spiked serum after pretreatment was drawn into the capillary packed with Fh-coated microbeads. The intensities of the yellow-color complexes on the microbeads were transformed into red-green-blue (RGB) pixels using Image-J software 1.8, and the difference between green and blue was used as the analytical signal. The signal increased with increasing FPV concentration. The proposed method showed good linearity (R2 = 0.9907) over a concentration range of 25-200 µg/mL. The RSD (n = 3) and the LOD (3σ) values were estimated to be 2.8-15.4% and 16.91 µg/mL, respectively. The proposed method enables us to quantify FPV rapidly and easily without the need for expensive equipment.

Development of a surface plasmon resonance sensor using an optical fiber prepared by electroless displacement gold plating and its application to immunoassay.

A simple and low-cost method of fabricating an optical fiber for a surface plasmon resonance (SPR) sensor was proposed. The method is based on the electroless nickel plating and subsequent displacement gold plating of the core of the optical fiber. The thickness of the nickel and gold thin films deposited on the core of the optical fiber could be controlled by measuring the reflected light intensity from the tip of the optical fiber during the plating processes. The sensitivity and resolution of the SPR sensor with the fabricated optical fiber in the refractive index range from 1.333 to 1.348 were 1324.3 nm/RIU and 7.6 × 10-4 RIU, respectively. The developed SPR sensor was successfully used in the determination of immunoglobulin A (IgA) in human saliva. The IgA quantification results obtained by the SPR sensor were in excellent agreement with those obtained by conventional enzyme-linked immunosorbent assay using a 96-well microtiter plate.

Development of a fluorescence microplate reader using an organic photodiode array with a large light receiving area.

We developed a small fluorescence microplate reader with an organic photodiode (OPD) array. The OPD array has nine OPDs that have a large light receiving area (9.62 mm2 per one OPD). Since the OPD array is fabricated on a flat glass plate, it can be placed just below microwells and can detect fluorescence emitted through the entire surface of the microwell bottom. The analytical performance of the developed plate reader was evaluated by measuring an aqueous solution of resorufin. The limit of detection (LOD) for resorufin (0.01-0.05 µM) was lower than that obtained with a plate reader equipped with nine inorganic photodiodes developed in a previous study (0.30 µM) and a commercially available microplate reader (0.16 µM). These results indicate that the large light receiving area improves the detection performance of the system. In addition, the developed reader was successfully used to quantify immunoglobulin A (IgA) in human saliva. The LOD for IgA was estimated to be 1.2 ng/mL, which is low enough to objectively evaluate human stress.

Film-Thickness-Controllable System for Preparing Silver Nanofilms through Absorbance Monitoring of the Thickness during a Silver-Mirror Reaction.

An innovative technique is proposed for forming silver thin films of nanometer-order thickness via a silver-mirror reaction. This approach is made possible by the real-time monitoring of the thickness of a silver thin film formed on the edge surface of a fiber core during the silver-mirror reaction using a homemade absorbance measurement system. The monitored absorbance value increases as silver plating progresses, and the relationship between the absorbance values and the thickness of the silver thin film is linear in the thickness range from approximately 30 to 60 nm. This technique was applied to the preparation of a fiber-optic surface plasmon resonance (FO-SPR) sensor. The sensor was successfully used to measure sucrose solutions with concentrations of less than 16% (w/v). The sensitivity of the sensor probe was estimated to be 2205 nm/RIU in the refractive index range of 1.333 - 1.357. The relative standard deviation of the wavelength shift obtained from measurements using different sensor probes was estimated to be less than 3.3%.

Quantification of CRP in human serum using a handheld fluorescence detection system for capillary-based ELISA.

We developed a handheld fluorescence detection system for capillary-based enzyme-linked immunosorbent assay (ELISA). The detection system implements both a long-pass filter and perpendicular optical arrangement, i.e., a power LED and a palm-sized spectrometer, to minimize background signals from the excitation light and optical scattering. The lower detection limit for resorufin was 0.13 µM. The detection system was applied to the quantification of C-reactive protein (CRP) in human serum with a capillary-based ELISA. The lower detection limit for CRP was 31 ng/ml, and the observed CRP levels in human serum were comparable to those obtained with a conventional ELISA system.

Development of an on-chip sample injection system with a 6-port valve incorporated in a microchip.

Micro-flow-injection analysis (µFIA) is amenable to high-throughput systems with lower consumption of sample and reagent volumes. On-chip sample injection methods are important to prevent reduced analytical performance associated with dead volumes and diffusion of sample solutions. In this study, we have developed an on-chip sample injection system with a small-sized 6-port valve incorporated on a microchip. The valve is made with a 3D printer and is a simple structure that can be easily operated manually. A sample solution in a loading channel can be injected by switching the valve from the load to injection position. Sample injection tests using resorufin solutions revealed that samples can be injected below 100 µL min-1, and the performance of the sample injection system is comparable to that of a commercially available injector. In addition, the sample injection system was successfully applied to a flow-based assay for hydrogen peroxide. The detection limit (3σ) of hydrogen peroxide was estimated to be 0.5 µM, and the assay time after sample injection was approximately 100 s. The developed sample injection system will be useful for various microfluidic-based analyses including µFIA.

Real-time assay for exosome membrane fusion with an artificial lipid membrane based on enhancement of gramicidin A channel conductance.

We report that the channel activities of gramicidin A in a supported lipid bilayer (SLB) were modulated by membrane fusion with exosomes. The mechanism of the modulation was an increase in the number of exosomes inserting into the SLB membrane, rather than enhancements of the single channel activity of gramicidin A. The modulation of apparent channel activities was applicable to the exosome fusion assay. This assay revealed that the membrane fusion of HEK-293 and MCF-7 exosomes was enhanced at pH 6.0, and the initial rates of membrane fusion for MCF-7 exosomes were higher than those for HEK-293 cells.

Rapid ELISA Using a Film-Stack Reaction Field with Micropillar Arrays.

A film-stack reaction field with a micropillar array using a motor stirrer was developed for the high sensitivity and rapid enzyme-linked immunosorbent assay (ELISA) reaction. The effects of the incubation time of a protein (30 s, 5 min, and 10 min) on the fluorescence intensity in ELISAs were investigated using a reaction field with different micropillar array dimensions (5-µm, 10-µm and 50-µm gaps between the micropillars). The difference in fluorescence intensity between the well with the reaction field of 50-µm gap for the incubation time of 30 s and the well without the reaction field with for incubation time of 10 min was 6%. The trend of the fluorescence intensity in the gap between the micro pillars in the film-stack reaction field was different between the short incubation time and the long incubation time. The theoretical analysis of the physical parameters related with the biomolecule transport indicated that the reaction efficiency defined in this study was the dominant factor determining the fluorescence intensity for the short incubation time, whereas the volumetric rate of the circulating flow through the space between films and the specific surface area were the dominant factors for the long incubation time.

A Handy Field-Portable ELISA System for Rapid Onsite Diagnosis of Infectious Diseases.

Enzyme-linked immunosorbent assays (ELISAs) are considered the gold standard for the detection of various immunological reactions and can be used for the detection of infectious diseases during outbreaks or in the care of individual patients. To be useful in the timely implementation of prevention and control measures against infectious diseases, a diagnostic modality should be rapid, accurate, and affordable. In the current study, we demonstrate the efficiency (90% less time and volume consumption compared with those of a standard 96-well ELISA), detection capability, and ease of operation of a field-portable, battery-operated ELISA system, approximately the size of a cellular phone (12 × 6 × 5.5 cm), in the serological diagnosis of measles and rubella viruses that has the potential for onsite testing such as during disease outbreaks.

An integrated enzyme-linked immunosorbent assay system with an organic light-emitting diode and a charge-coupled device for fluorescence detection.

A fluorescence detection system for a microfluidic device using an organic light-emitting diode (OLED) as the excitation light source and a charge-coupled device (CCD) as the photo detector was developed. The OLED was fabricated on a glass plate by photolithography and a vacuum deposition technique. The OLED produced a green luminescence with a peak emission at 512 nm and a half bandwidth of 55 nm. The maximum external quantum efficiency of the OLED was 7.2%. The emission intensity of the OLED at 10 mA/cm(2) was 13 µW (1.7 mW/cm(2)). The fluorescence detection system consisted of the OLED device, two band-pass filters, a five microchannel poly(dimethylsiloxane) (PDMS) microfluidic device and a linear CCD. The fluorescence detection system was successfully used in a flow-based enzyme-linked immunosorbent assay on a PDMS microfluidic device for the rapid determination of immunoglobulin A (IgA), a marker for human stress. The detection limit (S/N=3) for IgA was 16.5 ng/mL, and the sensitivity was sufficient for evaluating stress. Compared with the conventional 96-well microtiter plate assay, the analysis time and the amounts of reagent and sample solutions could all be reduced.