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.

Elution behavior of drugs in high-speed counter-current chromatography using on-column complexation with metal ions.

In this study, determination of (nitrogen containing) drugs by on-column complexation with metal ions in high-speed counter-current chromatography (HSCCC) was investigated. Bromazepam (BMP) was strongly retained in the organic upper stationary phase (UP) of the two-phase solvent system composed of tert-butyl methyl ether-acetonitrile-water (2:2:3, v/v/v) by eluting the aqueous lower mobile phase (LP) at a flow rate of 2 mL min-1. On the other hand, BMP (200 µg mL-1) was eluted faster without retention to the organic UP with the two-phase system containing 100 µg mL-1 of copper ions (CuCl2) because a very polar BMP-Cu2+ complex was immediately formed in the aqueous LP. The dramatic change in the retention behavior of BMP resulted from on-column complexation. The on-column complexation in HSCCC was further investigated for five (nitrogen containing) drugs and seven metal ions. In the result, tizanidine and phentolamine formed complexes with Al3+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+, ambroxol formed complexes with Al3+, Fe2+, and Cu2+, but voriconazole formed no complexes with all metal ions tested.

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.

Synthesis and structure-activity relationship of violaceoid D, a cytotoxic alkylated phenol isolated from Aspergillus violaceofuscus Gasperini.

The enantioselective synthesis of violaceoid D, a cytotoxic phenolic compound isolated from the culture broth of Aspergillus violaceofuscus Gasperini, was achieved. The total synthesis involves stereoselective construction of the stereogenic center of violaceoid D via Sharpless asymmetric dihydroxylation, followed by Smiles rearrangement. The absolute configuration of natural violaceoid D was determined to be R from the specific rotation value. Synthesized violaceoid D and its analogs were evaluated for cytotoxicity against two human cancer cell lines, Jurkat and HCT116. Because the enantiomer of violaceoid D showed no cytotoxicity, it is plausible that violaceoid D binds selectively to specific target molecules, such as proteins in the cancer cells.

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.

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.

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%.

A Fluorinated Carbanionic Substituent for Improving Water Solubility and Lipophilicity of Fluorescent Dyes.

Installation of a carbanionic substituent, that is strongly stabilized by two (trifluoromethyl)sulfonyl (Tf=SO2 CF3 ) groups, into several fluorescence dyes including boron-dipyrromethenes (BODIPYs), fluoresceins, and aminocoumarins has been achieved by the 2,2-bis(triflyl)ethylation reaction of the dye frameworks with highly electrophilic Tf2 C=CH2 , followed by neutralization with NaHCO3 . Despite the contradiction between water solubility and lipophilicity, the carbanion-decorated dyes thus obtained showed significant enhancement of not only water solubility but also lipophilicity. This work clearly demonstrates that the fluorinated, highly stabilized carbanionic substituent is a new option for controlling the macroscopic property of chemical materials.

Fully Automated Molecular Diagnostic System "Simprova" for Simultaneous Testing of Multiple Items.

Nucleic acid amplification-based diagnostics is known as one of the molecular diagnostic systems that allows higher sensitive detection of pathogens than test methods such as immunoassay. However, it has not been widely used because it is complicated to use and takes a long time to generate results. On the other hand, development of fully automated molecular diagnostic systems has been growing around the world as demand for such systems from physicians and laboratory technicians has increased. To meet this demand, we have developed the "Simprova" fully automated molecular diagnostic system, which takes advantage of LAMP (Loop-mediated Isothermal Amplification), a method Eiken Chemical Co., Ltd. invented. Simprova comprises a master unit that controls the entire system and a test unit that extracts and purifies nucleic acid from samples (pretreatment), and uses the LAMP method to detect and amplify nucleic acid. Users can obtain test results automatically by simply installing a pretreatment cartridge, a multi-well testing chip and the sample in the test unit. The multi-well testing chip has 25 reaction wells connected by channels and enables simultaneous testing of multiple targets with one sample. Turnaround time for one test is approximately 30 minutes. Since a conventional extraction and purification method using magnetic-bead separation is used for the pretreatment, nucleic acid can be extracted from serum, plasma, whole blood, urine, and sputum, for example. In addition, the system can perform random-access testing by connecting four test units to the master unit to realize near-the-patient testing. Simprova is therefore a robust and useful system for a wide variety of applications.

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.

Analysis of Complexation Interactions between Metal Ions and Drugs under Pseudo-physiological pH Conditions by a High-throughput Screening Method Using a Solid-phase Extraction Cartridge.

A high-throughput screening method for the complexation between metal ions and drugs was established by combining solid-phase extraction (SPE) using a nitrilotriacetic acid (NTA) modified silica spin cartridge with subsequent HPLC analysis. First, a test metal ion solution was passed through the NTA cartridge, then a test drug solution diluted in phosphate buffered saline (pH 7.4) was passed through the metal-chelated NTA cartridge. The complexation behavior between the metal and the drug on the NTA cartridge was evaluated by HPLC quantification of the drug in the SPE eluate. Comprehensive analysis of the complexation behavior between 11 different metal ions and 55 drugs showed that Cu2+, Ni2+, Co2+, Zn2+, Cr3+ and Fe3+ formed complexes with 12, 5, 4, 2, 1 and 1 kinds of drugs, respectively. Bromazepam selectively formed complexes with Cu2+, Ni2+ and Co2+.

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.

An enzyme-modified capillary as a platform for simultaneous fluorometric detection of d-glucose and l- lactate.

The preparation of a glass capillary pattered with lipid layers　on which lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH) were regionally adsorbed and its application for simultaneous detection of d-glucose and l-lactate in human serum is described. A lipid layer was formed on the surface of BSA-unabsorbed octadecyltrichlorosilane (OTS) inner wall of a glass capillary. The electrostatic charge of the lipid layer was a key factor for adsorbing the enzymes on the lipid layer. The fluorescence intensities were observed at each enzyme site in the presence of diaphorase (DIA), ß-nicotinamide-adenine dinucleotide oxidized (NAD), resazurin, d-glucose and l-lactate. The fluorescence intensities at each enzyme site increased with an increase in the concentration of d-glucose and l-lactate=with the detection limits of 32 µM and 4.9 µM, respectively.

A Planar Bilayer Lipid Membrane Sensor Using a Miniaturized Auto-patch System.

A bilayer lipid membrane sensor was constructed with a miniaturized auto-patch system. The performance of the patch system was optimized to obtain an analytically relevant signal. A biosensor based on an anti-BSA-antibody as a receptor and gramicidin as a signal transduction element was demonstrated. The sensor for BSA exhibited a detection limit of pg/mL level for BSA.

Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance sensor.

We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki) obtained by the SPR method was CA074Me≈Z-Phe-Phe-FMK < leupeptin. This order was different from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly.

Monitoring of cholesterol oxidation in a lipid bilayer membrane using streptolysin O as a sensing and signal transduction element.

Streptolysin O (SLO), which recognizes sterols and forms nanopores in lipid membranes, is proposed as a sensing element for monitoring cholesterol oxidation in a lipid bilayer. The structural requirements of eight sterols for forming nanopores by SLO confirmed that a free 3-OH group in the ß-configuration of sterols is required for recognition by SLO in a lipid bilayer. The extent of nanopore formation by SLO in lipid bilayers increased in the order of cholestanol

Giant unilamellar vesicles containing Rhodamine 6G as a marker for immunoassay of bovine serum albumin and lipocalin-2.

Functionalized giant unilamellar vesicles (GUVs) containing a fluorescence dye Rhodamine 6G is proposed as a marker in sandwich-type immunoassay for bovine serum albumin (BSA) and lipocalin-2 (LCN2). The GUVs were prepared by the electroformation method and functionalized with anti-BSA antibody and anti-LCN2 antibody, respectively. The purification of antibody-modified GUVs was achieved by conventional centrifugation and a washing step in a flow system. To antigen on an antibody slip, antibody-modified GUVs were added as a marker and incubated. After wash-out of excess reagents and lysis of the bound GUVs with Triton X-100, the fluorescence image was captured. The fluorometric immunoassays for BSA and LCN2 exhibited lower detection limits of 4 and 80 fg ml(-)(1), respectively.