Ju-Young Byun

A regeneratable, label-free, localized surface plasmon resonance (LSPR) aptasensor for the detection of ochratoxin A.

Binding of an analyte on the surface of a nanoparticle typically promotes a change in the local refractive index, which gives rise to a shift in the wavelength of the localized surface plasmon resonance (LSPR) absorption band. The magnitude of the LSPR wavelength change is dependent on both the location of the analyte relative to the surface of the nanoparticle and the degree of alteration of the refractive index. We have employed this phenomenon as the basis for designing a new, label-free approach for the detection of the toxic mold mycotoxin, ochratoxin A (OTA) that employs a gold nanorod (GNR) and an aptamer target binding mechanism. In this system, binding of OTA causes an accumulation of OTA and G-quadruplex structure of the aptamer. This process results in a longitudinal wavelength shift of the LSPR peak associated with a change in the local refractive index near the GNR surface. By using this method, OTA can be quantitatively detected at concentrations lower than 1 nM. In addition, the results of this effort show that aptamer functionalized GNR substrate is robust in that it can be regenerated for reuse over seven times by heating in methanol at 70 °C to remove OTA. Moreover, the proposed biosensor system exhibits high selectivity for OTA over other mycotoxins. Finally, the sensor can be employed to detect OTA in ground corn samples with excellent recovery levels.

A structure-switchable aptasensor for aflatoxin B1 detection based on assembly of an aptamer/split DNAzyme.

An ultrasensitive, colorimetric and homogeneous strategy for aflatoxin B1 (AFB1) detection, which uses a DNA aptamer and two split DNAzyme halves, has been developed. Split halves of a hemin-binding DNAzymes is combined with an AFB1 aptamer to generate a homogeneous colorimetric sensor that undergoes an AFB1 induced DNA structural change. In the absence of AFB1, the split probes have peroxidase mimicking DNAzyme activity associated with catalysis of a color change reaction. Specific recognition of AFB1 by the aptamer component leads to structural deformation of the aptamer-DNAzyme complex, which causes splitting of the DNAzyme halves and a reduction in peroxidase mimicking activity. Therefore, a decrease of colorimetric signal arising from the catalytic process takes place upon in the presence of AFB1 in a concentration dependent manner in the 0.1-1.0 × 10(4) ng/mL range and with a colorimetric detection limit of 0.1 ng/mL. The new assay system exhibits high selectivity for AFB1 over other mycotoxins and can be employed detect the presence of AFB1 in ground corn samples. Overall, the strategy should serve as the basis for the development of rapid, simple and low-cost methods for detection of mycotoxins.

Label-free homogeneous FRET immunoassay for the detection of mycotoxins that utilizes quenching of the intrinsic fluorescence ofantibodies

The phenomenon of fluorescence quenching of an antibody by a specific ligand was applied in developing a technique for detection of mycotoxins, such as aflatoxin B₁ (AFB₁), ochratoxin A, and zearalenone. Studies showed that the intrinsic fluorescence of tryptophan (Trp) residues in antibodies, promoted by excitation at 280 nm, is quenched upon binding of specific mycotoxin ligands. Fluorescence quenching in FRET system takes place in these systems as a consequence of the overlap of the emission spectra of antibody donors with the absorption spectra of the mycotoxins. Further studies focusing on the detection of AFB₁ revealed that the Fab fragment, the variable region of the antibody where specific binding of AFB₁ occurs, can be utilized to increase the sensitivity of the detection system. The results demonstrate that fluorescence of the Fab fragment is almost completely quenched by AFB₁ whereas emission from intact anti-AFB₁ is only partially quenched by this mycotoxin. The limits of detection (LODs) were found to be 0.85 and 0.09 ng mL⁻¹ for assays using the intact antibody and the Fab fragment, respectively, corresponding to a 10-fold enhancement. A practical application of the Fab fragment based assay system was demonstrated by its use in the detection of AFB₁ in spiked barley grain samples. The observations made in this effort show that the new, label-free, non-competitive, and homogeneous FRET immunoassay strategy, which requires a simple sample preparation procedure, serves as a powerful tool for the rapid and sensitive quantitative determination of organic substances such as mycotoxin.

The use of an engineered single chain variable fragment in a localized surface plasmon resonance method for analysis of the C-reactive protein

A gold nanorod (GNR) based LSPR sensor has been developed for label-free detection of C-reactive protein (CRP). The sensor utilizes a single chain variable fragment (scFv) as a receptor to bind CRP. The results of this effort show that CRP in human serum can be quantitatively detected at lower than 1 ng mL(-1).

High-sensitivity detection of ATP using a localized surface plasmon resonance (LSPR) sensor and split aptamers

A highly sensitive localized surface plasmon resonance (LSPR) aptasensor for detection of adenosine triphosphate (ATP) has been developed. The sensor utilizes two split ATP aptamers, one (receptor fragment) being covalently attached to the surface of a gold nanorod (GNR) and the other labeled with a random DNA sequence and TAMRA dye (probe fragment). In the presence of both ATP and the probe fragment, a significant shift takes place in the wavelength of the LSPR band. This phenomenon is a consequence of the fact that the split fragments assemble into an intact folded structure in the presence of ATP, which brings about a decrease in the distance between the GNR surface and TAMRA dye and an associated LSPR wavelength. By using this sensor system, concentrations of ATP in the range of 10 pM-10 μM can be determined. In addition, by taking advantage of its denaturation properties, the LSPR aptasensor can be reused by simply subjecting it to quadruple salt-addition/2M NaCl washing steps. That the new method is applicable to biological systems was demonstrated by its use to measure ATP concentrations in E. coli and, thus to determine cell concentrations as low as 1.0×10(3) CFU.

Colorimetric detection of PCR products of DNA from pathogenic bacterial targets based on a simultaneously amplified DNAzyme

AbstractA novel strategy was devised for colorimetric analysis of the products of the polymerase chain reaction (PCR). The method takes advantage of simultaneous amplification of a horseradish peroxidase-mimicking DNAzyme (HRPzyme) during the PCR process. It is performed using a DNA specific forward primer and a universal reverse primer containing a complementary HRPzyme sequence. The double-strand PCR products, which include the HRPzyme sequence, are treated with a mixture of hemin and TMB (3,3′,5,5′–tetramethylbenzidine) in the presence of hydrogen peroxide. The resulting HRPzyme/hemin complex then promotes a peroxidase mimicking reaction, which produces the blue colored oxidized TMB. This colorimetric method can be more easily performed than previously developed gel based detection procedures and, as a result, can be conveniently applied to the specific and sensitive colorimetric analysis of DNA sequences arising from pathogenic bacteria. The potentially broad applicability of the new method has been demonstrated by its use in the identification of the 16s rDNA of Salmonella Typhimurium. FigureA novel strategy was devised for simple colorimetric analysis of PCR products with amplification of a horseradish peroxidase-mimicking DNAzyme(HRPzyme). This colorimetric method can be much more easily performed than previously developed gel based detection procedures and potentially broad applicability for other DNA analysis.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

A colorimetric homogeneous immunoassay system for the C-reactive protein

The C-reactive protein (CRP), which has a five repeat pentameric structure, is known to be a marker for acute inflammation and a potential risk predictor for cardiovascular disease. A simple and rapid homogeneous assay method for the detection of CRP, based on a gold nanoparticle (AuNP) aggregation induced colorimetric response, has been developed. In the technique, aggregation of CRP antibody-conjugated AuNPs is induced by addition of CRP as a consequence of its unique pentameric structure. CRP-promoted aggregation of the antibody-conjugated AuNPs results in a change of the wavelength maximum in the UV/Vis-spectrum. This homogeneous assay displays a typical hook effect, in which the signal level is directly proportional to CRP concentration until a critical concentration of CRP (the hook point) is reached. Above this concentration, the signal level decreases as the CRP concentration increases. The maximum shift in the absorption maximum was found to occur when the CRP antigen concentration is 100 ng mL(-1). In order to improve the linearity of the method, we employed a procedure, which takes advantage of a saturation phenomenon that leads to the hook effect, to increase the dynamic range of the CRP assay. Specifically, the use of CRP pre-spiked serum promotes maximum aggregation at the low CRP concentrations and, as a result, leads to an increase in the dynamic range for CRP detection. The applicability of the new homogenous assay system was demonstrated by its utilization for qualitative analysis of CRP in serum samples. The combined observations made in this effort show that the method using CRP antibody-conjugated AuNPs is both rapid and simple and, consequently, it can potentially be applied to onsite diagnosis.

A Localized Surface Plasmon Resonance (LSPR)-based, simple, receptor-free and regeneratable Hg2+ detection system

A simple, receptor-free and regeneratable Hg(2+) sensor, which utilizes localized surface plasmon resonance (LSPR) shifts of a gold nanorod (GNR), has been developed. Precipitation induced by coordination of Hg(2+) to citrate alters the local refractive index (RI) around the GNR surface on glass slide, promoting a red-shift in its LSPR absorption peak. This phenomenon is used to design a sensor that enables quantitative detection of Hg(2+) in the 1nM to 1mM concentration range with good linearity (0.9507 correlation coefficient) and limit of detection (LOD) is reached to 0.38nM. A high selectivity of this sensor for Hg(2+) is demonstrated by the specific LSPR red-shift of 27.67nm promoted by Hg(2+) in comparison to those caused by other metal ions. In addition, the reusability of the new sensor chip is shown by its successful reuse eight-times following successive washing/precipitation steps. Lastly, the sensor displays excellent recoveries in spiking test with real water samples, such as tap water, lake and river. The simple combination of precipitation of Hg(2+)-citrate complex and the LSPR red-shift has led to the design of a novel sensing strategy for Hg(2+) detection.

Homogeneous Fluorescence Resonance Energy Transfer Immunoassay for the Determination of Zearalenone

This study demonstrates the use of antigen-antibody binding for the detection of zearalenone. Based on the principle of the fluorescence resonance energy transfer (FRET) phenomenon between antibody and antigen, an immunoassay, in which zearalenone coupled with the anti-zearalenone antibody, was developed, optimized, and applied. Owing to intrinsic fluorescence properties in basic pH conditions with the optimal cationic surfactant, anti-zearalenone and zearalenone played roles as the respective donor and acceptor in the FRET immunoassay. As the concentration of analyte increased, the antigen/antibody emission intensity ratio (I 430 nm/I 350 nm) was enhanced due to larger amounts of zearalenone/anti-zearalenone complexes. This assay, based on the ratio of intensities (I 430 nm/I 350 nm), displayed high specificity and sensitivity with a detection limit of 0.8 ng mL−1 for zearalenone. The results obtained from analysis of spiked wheat grain samples were found to be in good agreement with those obtained by employing a direct competitive enzyme-linked immunosorbent assay. The label-free, noncompetitive, and homogeneous FRET immunoassay strategy served as a powerful tool for the simple, rapid, and sensitive quantitative determination of zearalenone in food and feed matrices.