Yong Duk Han

Paper-based glucose biosensing system utilizing a smartphone as a signal reader

A simple paper-based optical biosensor for glucose monitoring was developed. As a glucose biosensing principle, a colorimetric glucose assay, using glucose oxidase (GOx) and horseradish peroxidase (HRP), was chosen. The enzymatic glucose assay was implanted on the analytical paper-based device, which is fabricated by the wax printing method. The fabricated device consists of two paper layers. The top layer has a sample loading zone and a detection zone, which are modified with enzymes and chromogens. The bottom layer contains a fluidic channel to convey the solution from the loading zone to the detection zone. Double-sided adhesive tape is used to attach these two layers. In this system, when a glucose solution is dropped onto the loading zone, the solution is transferred to the detection zone, which is modified with GOx, HRP, and chromogenic compounds through the connected fluidic channel. In the presence of GOx-generated H2O2, HRP converts chromogenic compounds into the final product exhibiting a blue color, inducing color change in the detection zone. To confirm the changes in signal intensity in the detection zone, the resulting image was registered by a digital camera from a smartphone. To minimize signal interference from external light, the experiment was performed in a specifically designed light-tight box, which was suited to the smartphone. By using the developed biosensing system, various concentrations of glucose samples (0–20 mM) and human serum (5–17 mM) were precisely analyzed within a few minutes. With the developed system, we could expand the applicability of a smartphone to bioanalytical health care.

Multienzyme-modified biosensing surface for the electrochemical analysis of aspartate transaminase and alanine transaminase in human plasma

AbstractWe investigated the electrochemical detection of aspartate transaminase (AST) and alanine transaminase (ALT) by using a multienzyme-modified electrode surface. Determination of the activities of transaminases in human serum is clinically significant because their concentrations and ratios indicate the presence of hepatic diseases or myocardial dysfunction. For electrochemical detection of AST and ALT, enzymes that participate in the reaction mechanism of AST and ALT, such as pyruvate oxidase (POX) and oxaloacetate decarboxylase, were immobilized on an electrode surface by using an amine-reactive self-assembled monolayer and a homobifunctional cross-linker. In the presence of suitable substrates such as l-aspartate (l-alanine) and α-ketoglutarate, AST and ALT generate pyruvate as an enzymatic end product. To determine the activities of AST and ALT, electroanalyses of pyruvate were conducted using a POX and ferrocenemethanol electron shuttle. Anodically generated oxidative currents from multienzyme-mediated reactions were correlated to AST and ALT levels in human plasma. On the basis of the electrochemical analysis, we obtained calibration results for AST and ALT concentrations from 7.5 to 720 units/L in human plasma-based samples, covering the required clinical detection range. FigurePOX-OAC calatytic cycles for AST and ALT analysis

Bioelectrocatalytic detection of glycated hemoglobin (HbA1c) based on the competitive binding of target and signaling glycoproteins to a boronate-modified surface

We developed an electrochemical glycated hemoglobin (HbA(1c)) biosensor for diagnosing diabetes in whole human blood based on the competitive binding reaction of glycated proteins. Until now, no studies have reported a simple and accurate electrochemical biosensor for the quantification of HbA(1c) in whole blood. This is because it is very difficult to correctly distinguish HbA(1c) from large amounts of hemoglobin and other components in whole blood. To detect glycated hemoglobin, we used electrodes modified with boronic acid, which forms a covalent bond between its diol group and the cis-diol group of the carbohydrate moiety of glycated proteins. For accurate HbA(1c) biosensing, we first removed blood components (except for hemoglobin) such as glycated proteins and blood glucose as they interfere with the boronate-based HbA(1c) competition analysis by reacting with the boronate-modified surface via a cis-diol interaction. After hemoglobin separation, target HbA(1c) and GOx at a predetermined concentration were reacted through a competition onto the boronate-modified electrode, allowing HbA(1c) to be detected linearly within a range of 4.5-15% of the separated hemoglobin sample (HbA(1c)/total hemoglobin). This range covers the required clinical reference range of diabetes mellitus. Hence, the proposed method can be used for measuring %HbA(1c) in whole human blood, and can also be applied to measuring the concentration of various glycated proteins that contain peripheral sugar groups.

Use of a Columnar Metal Thin Film as a Nanosieve with Sub-10 nm Pores

A columnar-structured nanosieve is unique in the sense that it is a general thin film formed by physical vapor deposition (PVD). Instead of additional processes to make nanopores, the numerous voids naturally formed among columnar grains during PVD are used as nanopores. Since the thin film formed by PVD has vertically grown columnar grains, the fabricated nanosieve has numerous straight-opened nanopores, which is an ideal structure for a nanosieve.

Chip-based cartilage oligomeric matrix protein detection in serum and synovial fluid for osteoarthritis diagnosis

We have developed a method to detect cartilage oligomeric matrix protein (COMP) as a specific biomarker of osteoarthritis (OA). In pathological conditions of the cartilage, COMP is released first into the synovial fluid (SF) and from there into the blood. Thus, measurement of COMP in the blood and SF facilitates OA diagnosis. To determine COMP, we developed a fluoro-microbead guiding chip (FMGC)-based immunoassay. The FMGC has four immunoreactive regions, each with five patterns, to allow multiple assays. A COMP-specific capture antibody was immobilized to the FMGC surface to create a self-assembled interfacial layer. SF or serum samples from patients with OA possessing the target COMP were applied to the COMP-sensing monolayer. To generate binding signal, COMP detection antibody-conjugated fluoro-microbeads were applied and the numbers of fluoro-microbeads bound specifically were counted to determine COMP concentrations. This FMGC-based immunoassay clearly distinguished immunospecific from nonspecific binding by comparing optical signals from inside and outside of the patterns. The optical signals showed linear correlations with serum and SF COMP concentrations. Optical detection and quantification of COMP using fluorescence microscopy correlated well with results from commercial enzyme-linked immunosorbent assay (ELISA). This FMGC-based immunoassay offers a new approach for detecting a clinically relevant biomarker for OA in human blood and SF.

The transformation of common office supplies into a low-cost optical biosensing platform

By reassembling common office supplies, an optical biosensing system was developed. A laser pointer and the solar cell from a calculator were utilized in the developed optical biosensing system as the light source and signal transducer, respectively. For intuitive signal evaluation, a multimeter was used. The following two types of conventional enzymatic colorimetric assays were employed with the optical biosensing system: (i) the Trinder׳s reaction-based enzymatic assay; and (ii) the competitive enzyme-linked immunosorbent assay. These colorimetric assays were performed in reaction channels made from transparent polymer and glass. By matching the maximum absorption spectra of the colored end products from the assays with the emission spectra of the laser diodes, the biochemical reaction rate was manifested as a change in the intensity of the laser beam. This change was then converted by the solar cell into voltage and displayed on the connected multimeter. To verify the detection performance of the system, glucose and an osteoarthritis biomarker (urinary collagen type II C-telopeptide fragments [uCTX-II]) were quantified. With glucose, the voltages registered were linearly correlated with the glucose concentration, from 0 to 10 mM. Using a competitive immunoassay for uCTX-II, the system exhibited a calibration curve with a dynamic detection range between 1.3 and 10 ng/mL uCTX-II. Given the advantages of the proposed biosensing system, including its high sensitivity, facile fabrication, and the high obtainability and cost-effectiveness of the components used to make it, we expect that this study will provide a basis for the production of a low-cost optical biosensor.

Ambient light-based optical biosensing platform with smartphone-embedded illumination sensor.

We present a hand-held optical biosensing system utilizing a smartphone-embedded illumination sensor that is integrated with immunoblotting assay method. The smartphone-embedded illumination sensor is regarded as an alternative optical receiver that can replaces the conventional optical analysis apparatus because the illumination sensor can respond to the ambient light in a wide range of wavelengths, including visible and infrared. To demonstrate the biosensing applicability of our system employing the enzyme-mediated immunoblotting and accompanying light interference, various types of ambient light conditions including outdoor sunlight and indoor fluorescent were tested. For the immunoblotting assay, the biosensing channel generating insoluble precipitates as an end product of the enzymatic reaction is fabricated and mounted on the illumination sensor of the smartphone. The intensity of penetrating light arrives on the illumination sensor is inversely proportional to the amount of precipitates produced in the channel, and these changes are immediately analyzed and quantified via smartphone software. In this study, urinary C-terminal telopeptide fragment of type II collagen (uCTX-II), a biomarker of osteoarthritis diagnosis, was tested as a model analyte. The developed smartphone-based sensing system efficiently measured uCTX-II in the 0-5ng/mL concentration range with a high sensitivity and accuracy under various light conditions. These assay results show that the illumination sensor-based optical biosensor is suitable for point-of-care testing (POCT).

Retroreflective Janus Microparticle as a Nonspectroscopic Optical Immunosensing Probe

We developed retroreflective Janus microparticles (RJPs) as a novel optical immunosensing probe for use in a nonspectroscopic retroreflection-based immunoassay. By coating the metals on the hemispherical surface of silica particles, highly reflective RJPs were fabricated. On the basis of the retroreflection principle, the RJPs responded to polychromatic white light sources, in contrast to conventional optical probes, which require specific monochromatic light. The retroreflection signals from RJPs were distinctively recognized as shining dots, which can be intuitively counted using a digital camera setup. Using the developed retroreflective immunosensing system, cardiac troponin I, a specific biomarker of acute myocardial infarction, was detected with high sensitivity. On the basis of the demonstrated features of the retroreflective immunosensing platform, we expect that our approach may be applied for various point-of-care-testing applications.

An immunoblot-based optical biosensor for screening of osteoarthritis using a smartphone-embedded illuminometer

We report a new smartphone-based immunosensing system that integrates an immunoblotting assay and a built-in illumination sensor to assay an osteoarthritis marker. The simple optical biosensing system developed in this study effectively uses smartphone-embedded components such as a white light-emitting diode and an illumination sensor as the light source and optical receiver, respectively. In contrast to conventional optical sensors, which utilize a specific spectrum and focused wavelength, the illumination sensor sensitively responds to the variations in external light intensity over a wide range of wavelengths. This functionality of the illumination sensor in the smartphone was employed as a signal transducer in the optical system. The immunoblotting technique, which uniformly changes the intensity of light because of the precipitation reaction, was introduced into the developed optical system. The horseradish peroxidase-induced insoluble precipitate interferes with the penetration of incident light, thereby facilitating the variation of applied light intensity. Subsequently, the quantity of light passing through the biosensing channel was immediately analysed using a lux meter in the mobile application. Herein, the urinary C-terminal telopeptide fragment of type II collagen (uCTX-II) was selected as an osteoarthritis biomarker and analysed to demonstrate the feasibility of the developed illumination sensor. The results indicate an obvious change in the lux value in accordance with the uCTX-II concentration ranging from 0 to 10 ng mL−1. The results were highly reproducible and sensitive to the variations in the concentration of the analyte. This suggests the potential use of the developed illumination sensor as a promising tool for the quantitative diagnosis of target analytes and point-of-care testing.

Microchip-Based Organophosphorus Detection Using Bienzyme Bioelectrocatalysis

We have developed a microsystem for the detection of organophosphorus (OP) compounds using acetylcholine esterase (AchE) and choline oxidase (ChOx) bienzyme bioelectrocatalysis. Because AchE is irreversibly inhibited by OP pesticides, the change in AchE activity with OP treatment can be traced to determine OP concentration. Polymer-associated ChOx immobilization on the working electrode surface and magnetic microparticle (MP)-assisted AchE deposition methods were employed to create an AchE–ChOx bienzyme-modified biosensing system. ChOx was immobilized on the micropatterned electrodes using poly(L-lysine), glutaraldehyde, and amine-rich interfacial surface. AchE was immobilized on the MP surface via Schiffs base formation, and the enzyme-modified MPs were deposited on the working electrode using a magnet under the microfluidic channel. The bioelectrocatalytic reaction between AchE–ChOx bienzyme cascade and the ferrocenyl electron shuttle was successfully used to detect OP with the developed microchip. This provides a self-contained and relatively easy method for OP detection. It requires minimal time and a small sample size, and has potential analytic applications in pesticides and chemical warfare agents.