Yoo Min Park

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.

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.

Detection of CTX-II in serum and urine to diagnose osteoarthritis by using a fluoro-microbeads guiding chip

This study reports a new strategy for simultaneous detection of the C-telopeptide fragments of type II collagen (CTX-II) as a biomarker of osteoarthritis (OA) using a fluoro-microbeads guiding chip. As osteoarthritis progresses, the joint components including matrix and cartilage are degraded by proteases. The degraded products such as CTX-II are released into the serum and urine, and the CTX-II concentration in body fluids reflects OA progression. Because the CTX-II has heterogeneous epitope structure in serum (sCTX-II; homodimers) and urine (uCTX-II; monomers or variant monomers), a multiple-sensing device enabling both sandwich and competitive-type immunoassays is required. For multiple assessments of serum and urinary CTX-II, we designed a fluoro-microbeads guiding chip (FMGC) containing multiple sensing areas and connecting channels. Using the approach, the sandwich (sCTX-II) and competition (uCTX-II) assays could be simultaneously performed on a single chip. We designed a fluidic control device enabling selective control of the open-close function of FMGC channels. The immune-specific signal was quantitatively analyzed by counting the number of fluorescent microbeads from the registered images. The results from the developed FMGC assay showed high correlation with those obtained in ELISA. The completion time of the FMGC assay was 24-fold and 3.5-fold shorter than the ELISA for urinary and serum CTX-II. Taken together, it enabled the simultaneous detection of both sCTX-II and uCTX-II. This FMGC-based assay would be a promising tool for monitoring of osteoarthritis.

Quantitative lateral-flow immunoassay for the assessment of the cartilage oligomeric matrix protein as a marker of osteoarthritis

This study reports a simple and quantitative analytical method for detecting cartilage oligomeric matrix protein (COMP) using a lateral-flow immunoassay (LFI) format. An immuno-chromatographic assay was developed using a gold nanoparticle (AuNP)-conjugated monoclonal antibody (Ab) probe for the quantitative detection of COMP in human synovial fluid (SF). The detection antibody was conjugated with the AuNP (∼30 nm) to enable detection, and the capture antibody was immobilized within a multi-spot pattern in the test zone of a membrane strip. COMP in the human SF binds to the AuNP-conjugated detection antibody, and the COMP-Ab complex then binds to the capture antibody. The immunoassay test can be analyzed by visualizing the test strip with a commercial scanner and an imaging software program. The color density of the multi-spot pattern at the test zone increased in proportion to the concentration of COMP. The developed immunoassay exhibited a dynamic detection range from 0.6 to 20 μg/mL of COMP. The detection limit of the proposed LFI system was approximately 0.2 μg/mL. The test results showed good correlation with those obtained using a conventional enzyme-linked immunosorbent assay (ELISA) to detect COMP. We expect that the proposed immunoassay method, based on the quantification of COMP, is suitable for osteoarthritis diagnosis using SF samples.

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.

Optical immunosensor for quantifying C-telopeptide fragments of type II collagen as an osteoarthritis biomarker in urine

A fluorescence-based biosensor for measuring the quantity of C-telopeptide fragments of type II collagen (CTX-II) was developed as an osteoarthritis biomarker in urine. During osteoarthritis progression, joint components such as cartilage collagen are degraded by collagenase-protease and secreted into the serum as CTX-II. To detect CTX-II in urine (uCTX-II) having structural heterogeneity, conventional sandwich format assay does not provide sufficient accuracy. As an alternative, a competitive immunoassay was developed for the quantification of uCTX-II, which uses antibody-conjugated fluoro-microbeads to generate an optical signal. After preparing the biosensing surface, the uCTX-II sample and antibody-optical probe conjugates were reacted on the PEG4-EKGPDP exposed biosensing surface. The optical probes competed with the uCTX-II in the sample for binding to the immobilized PEG4-EKGPDP. This assay was able to detect uCTX-II concentrations between 200 ng/mmol (corrected value vs. creatinine) and 1,400 ng/mmol, encompassing the clinical detection ranges required for osteoarthritis diagnosis. The competitive immunoassay developed for uCTX-II detection was rapid and exhibited good correlation with conventional ELISA methods. This novel competition assay is a promising tool for the diagnosis and monitoring of osteoarthritis using urine samples.

Development of a matrix metalloproteinase-2 (MMP-2) biosensing system by integrating an enzyme-mediated color development reaction into a common electronics components setup

Matrix metalloproteinase-2 (MMP-2) is closely related to the proliferation and invasion of various types of cancers. The protease is secreted by malignant tumor cells, thus allowing the enzyme to serve as a biomarker for cancer diagnosis. Methods have been developed to analyze MMP-2 activities; however, their applications to disease diagnosis have not been widely demonstrated yet because of the need for highend analytical equipment and labor-intensive processes. In this study, we developed an MMP-2 activity assay system by integrating an engineered autoinhibited β-lactamase which can be activated by MMP-2 in an optical sensing system consisting of reassembled common electronic components, such as a laser diode, a solar cell, and a multimeter. The autoinhibited β-lactamase was immobilized on a polymeric biosensing channel by a polydopamine coating and self-assembled monolayer methods. In the presence of MMP-2, the immobilized autoinhibited β-lactamase was converted to an active form that hydrolyzed the chromogenic cephalosporin CENTA, thereby changing the substrate color from pale yellow (λmax=340 nm) to highly discernible chrome yellow (λmax=405 nm). By reading the interfered laser-light intensity, we were able to analyze MMP-2 activities precisely both with the samples prepared in a buffer solution and also those in urine. These results suggested that the developed system can be used for the quantitative analysis of enzyme activity related to cancer diagnosis.

An Optical Biosensing Strategy Based on Selective Light Absorption and Wavelength Filtering from Chromogenic Reaction

To overcome the time and space constraints in disease diagnosis via the biosensing approach, we developed a new signal-transducing strategy that can be applied to colorimetric optical biosensors. Our study is focused on implementation of a signal transduction technology that can directly translate the color intensity signals—that require complicated optical equipment for the analysis—into signals that can be easily counted with the naked eye. Based on the selective light absorption and wavelength-filtering principles, our new optical signaling transducer was built from a common computer monitor and a smartphone. In this signal transducer, the liquid crystal display (LCD) panel of the computer monitor served as a light source and a signal guide generator. In addition, the smartphone was used as an optical receiver and signal display. As a biorecognition layer, a transparent and soft material-based biosensing channel was employed generating blue output via a target-specific bienzymatic chromogenic reaction. Using graphics editor software, we displayed the optical signal guide patterns containing multiple polygons (a triangle, circle, pentagon, heptagon, and 3/4 circle, each associated with a specified color ratio) on the LCD monitor panel. During observation of signal guide patterns displayed on the LCD monitor panel using a smartphone camera via the target analyte-loaded biosensing channel as a color-filtering layer, the number of observed polygons changed according to the concentration of the target analyte via the spectral correlation between absorbance changes in a solution of the biosensing channel and color emission properties of each type of polygon. By simple counting of the changes in the number of polygons registered by the smartphone camera, we could efficiently measure the concentration of a target analyte in a sample without complicated and expensive optical instruments. In a demonstration test on glucose as a model analyte, we could easily measure the concentration of glucose in the range from 0 to 10 mM.