Byoung Yeon Won

Enzyme-catalyzed signal amplification for electrochemical DNA detection with a PNA-modified electrode

The signal amplification technique of peptide nucleic acid (PNA)-based electrochemical DNA sensor was developed in a label-free and one-step method utilizing enzymatic catalysis. Electrochemical detection of DNA hybridization on a PNA-modified electrode is based on the change of surface charge caused by the hybridization of negatively charged DNA molecules. The negatively charged mediator, ferrocenedicarboxylic acid, cannot diffuse to the DNA hybridized electrode surface due to the charge repulsion with the hybridized DNA molecule while it can easily approach the neutral PNA-modified electrode surface without the hybridization. By employing glucose oxidase catalysis on this PNA-based electrochemical system, the oxidized mediator could be immediately reduced leading to greatly increased electrochemical signals. Using the enzymatic strategy, we successfully demonstrated its clinical utility by detecting one of the mutation sequences of the breast cancer susceptibility gene BRCA1 at a sample concentration lower than 10(-9) M. Furthermore, a single base-mismatched sample could be also discriminated from a perfectly matched sample.

Investigation of the signaling mechanism and verification of the performance of an electrochemical real-time PCR system based on the interaction of methylene blue with DNA

The operation of an electrochemical real-time PCR system, based on intercalative binding of methylene blue (MB) with dsDNA, has been demonstrated. PCR was performed on a fabricated electrode-patterned glass chip containing MB while recording the cathodic current peak by measuring the square wave voltammogram (SWV). The current peak signal was found to decrease with an increase in the PCR cycle number. This phenomenon was found to be mainly a consequence of the lower apparent diffusion rate of the MB-DNA complex (D(b) = 6.82 × 10(-6) cm(2) s(-1) with 612 bp dsDNA) as compared to that of free MB (D(f) = 5.06 × 10(-5) cm(2) s(-1)). Utilizing this signal changing mechanism, we successfully demonstrated the feasibility of an electrochemical real-time PCR system by accurately quantifying initial copy numbers of Chlamydia trachomatis DNA templates on a direct electrode chip. A standard calibration plot of the threshold cycle (C(t)) value versus the log of the input template quantity demonstrated reliable linearity and a good PCR efficiency (106%) that is comparable to that of a conventional TaqMan probe-based real time PCR. Finally, the system developed in this effort can be employed as a key technology for the achievement of point-of-care genetic diagnosis based on the electrochemical real-time PCR.

A DNA intercalation-based electrochemical method for detection of Chlamydia trachomatis utilizing peroxidase-catalyzed signal amplification

A sensitive electrochemical DNA detection method for the diagnosis of sexually transmitted disease (STD) caused by Chlamydia trachomatis was developed. The method utilizes a DNA-intercalating agent and a peroxidase promoted enzymatic precipitation reaction and involves the following steps. After hybridization of the target C. trachomatis gene with an immobilized DNA capture probe on a gold electrode surface, the biotin-tagged DNA intercalator (anthraquinone) was inserted into the resulting DNA duplex. Subsequently, the polymeric streptavidin/peroxidase complex was applied to the biotin-decorated electrode. Peroxidase catalyzed 4-chloronaphthol to produce insoluble product, which is precipitated on the electrode surface in the presence of hydrogen peroxide. Cyclic voltammograms with the gold electrode exhibited a peak current of ferrocenemethanol in electrolyte, which decreased in a proportional way to increasing concentration of target DNA owing to insulation of electrode surface by the growing insoluble precipitate. Using this strategy, we were able to detect picomolar concentrations of C. trachomatis gene in a sample taken from a real patient.

A Touchscreen as a Biomolecule Detection Platform

In recent years, considerable effort has been dedicated to the development of a simple, instrument-free detection method that enables point-of-care testing (POCT) and decentralized clinical diagnosis of diseases. One of the most promising approaches to this goal involves the use of an electrical detection strategy. Owing to operational simplicity and ability to be miniaturized, this approach has been subjected to very extensive studies. Nevertheless, except for a glucose sensor or i-STAT blood analyzer, no practical portable POCT device has been developed to date because of the lack of success in readily integrating detection elements into electrical devices. A touchscreen is an input device, generally used in kiosk systems, PDAs (personal digital assistants), or smart phones, that can detect the presence and location of a touch within the device area. Among other touchscreen technologies, including those that employ resistive, infrared, and surface acoustic wave (SAW) signaling, the capacitive touchscreen has been widely used recently owing to the recent increasing popularity of smart phones. Basically, a capacitive touchscreen detects small capacitance changes of electrodes in the device that are induced by a human finger touching event in accord with the relationship shown in Equation (1):

Mismatch DNA-specific enzymatic cleavage employed in a new method for the electrochemical detection of genetic mutations

Utilizing enzymatic mismatched DNA-specific cleavage and electrocatalytic signaling, a new electrochemical method for the detection of DNA mutations was developed and successfully applied to detect various mutations in the BRCA1 gene.

A novel electrochemical method to detect theophylline utilizing silver ions captured within abasic site-incorporated duplex DNA

We herein describe a novel and label-free electrochemical system to detect theophylline. The system was constructed by immobilizing duplex DNA containing an abasic site opposite cytosine on the gold electrode surface. In the absence of theophylline in a sample, silver ions freely bind to the empty abasic site in the duplex DNA leading to the highly elevated electrochemical signal by the redox reaction of silver ions. On the other hand, when theophylline is present, it binds to the abasic site by pseudo base pairing with the opposite cytosine nucleobase, which consequently prevents silver ions from binding to the abasic site. As a result, redox reaction of silver ions would be greatly reduced resulting in the accordingly decreased electrochemical signal. By employing this electrochemical strategy, theophylline was reliably detected at a concentration as low as 3.2 μM with the high selectivity over structurally similar substances such as caffeine and theobromine. Finally, the diagnostic capability of this method was also successfully verified by reliably detecting theophylline present in a real human serum sample with an excellent recovery ratio within 100±6%.

Fabrication of conductive oxidase-entrapping nanocomposite of mesoporous ceria–carbon for efficient electrochemical biosensor

A conductive nanocomposite containing an immobilized oxidative enzyme in the pores of mesostructured ceria (CeO2)–carbon was developed as an efficient electrochemical biosensing platform. The construction of the nanocomposite began with the incorporation of CeO2 in a carbon matrix by the co-assembly of cerium nitrate, resol, and triblock copolymer via a facile evaporation-induced self-assembly method, which resulted in the formation of mesoporous ceria–carbon (denoted as Meso-CeO2/C). Glucose oxidase (GOx) was subsequently immobilized in the vacant pores of the Meso-CeO2/C by using glutaraldehyde crosslinking to prevent enzyme leaching from the matrix. H2O2 generated by the catalytic action of GOx in proportion to the amount of target glucose was rapidly converted into hydroxyl radicals by the catalytic activity of CeO2, which induced subsequent anodic oxidation of Ce3+ into Ce(OH)22+ or Ce(OH)4 with the anodic current. The constructed Meso-CeO2/C exhibited higher resolution in electrochemical detection of H2O2 than pure mesoporous carbon without ceria owing to the catalytic activity of ceria. The anodic current responses by the nanocomposite containing GOx in Meso-CeO2/C resulted in a linear increase in the concentration of target glucose (0.25–5 mM), which is suitable to measure the serum glucose, with excellent storage stability of over two months at room temperature. The biosensor also exhibited a high degree of precision and reproducibility when employing real human blood samples. Based on these results, we anticipate that this novel biosensing format can be readily extended to other oxidative enzymes for the detection of various clinically important target molecules.

Label-Free Multiplex DNA Detection Utilizing Projected Capacitive Touchscreen

A novel strategy to achieve label-free multiplex DNA detection system based on the projected capacitive touchscreen is developed. Touchscreen panel is first fabricated by patterning the ITO (indium-tin-oxide) electrode array on the glass wafer, and the electrodes are modified with the respective capture probe DNA sequences complementary to hemagglutinin1 (H1), neuraminidase1 (N1), and matrix1 (M1) DNA to demonstrate the molecular diagnosis of H1N1 influenza virus as a model pathogen. DNA sample is applied to the electrodes to allow hybridization of target DNA with the corresponding complementary capture probe. As a result, the hybridization event significantly enhanced the capacitance on the electrode, which can be very conveniently detected by the projected capacitive touchscreen. Based on this design principle, the authors have successfully detected target regions of H1N1 influenza virus in a label-free multiplexed manner. This touchscreen-based detecting system would greatly benefit the point-of-care testing (POCT) in various diagnostic applications.