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.

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%.

An electrochemical one-step system for assaying methyltransferase activity based on transport of a quantum dot signaling tracer.

A one-step, electrochemical method for assaying methyltransferase (MTase) activity, based on the convective transport of a quantum dot (QD) signaling tracer, has been developed. The assay chip used in this system was prepared by modifying a gold matrix with CdSe/ZnS QD-tagged dsDNA, which contains a specific methylation site (5'-GATC-3') recognized by MTase. Treatment of the chip with DNA adenine methylation (Dam) MTase, generates a methylated sequence (5'-GAmTC-3') within the dsDNA. The methylated dsDNA is then subjected to a cleavage reaction, induced by DpnI, which leads to release from the gold matrix of a DNA fragment tethered to a QD. Detection of the released QD, using square wave anodic stripping voltammetry (SWASV) on a glassy carbon (GC) electrode, enables the reliable quantitation of the methylated DNA. Because it is accomplished in a simple and convenient one step and does not require any complicated secondary or tedious washing steps, the new assay method holds great promise for epigenetic analysis in facility-limited environments or point-of-care testing (POCT) applications.

A one-step, electrochemical biosensing strategy that is based on transport of signaling CdS nanoparticles controlled by biomolecules.

A novel, one-step electrochemical biosensing technique has been developed by utilizing a strategy in which a biomolecule controls transport of CdS-signaling nanoparticles to the surface of an electrode. The viability of this approach was explored using DNA as a model target biomolecule. The capture and signaling probes both contain nucleic acid sequences that are complementary to the target DNA. The detection chamber consists of a gold matrix modified with the capture probe on the bottom, a glassy carbon (GC) working electrode on the top, and a buffered electrolyte containing the signaling probe conjugated with the CdS nanoparticle. When target DNA is not present in the chamber, the CdS-signaling probe is freely transported to the GC electrode where CdS accumulates during the preconcentration step and undergoes electrochemical anodic stripping voltammetry (ASV) that subsequently generates a current signal during the following oxidative stripping step. On the other hand, target DNA present in the sample undergoes simultaneous hybridization to both the capture and signaling probes in a sandwich-like manner. This phenomenon leads to fixation of the CdS nanoparticles on the bottom of the chamber, thus preventing their electrochemical reduction on the GC electrode. As a result, the electrochemical signal is reduced in the presence of target DNA. Based on the reduction of the current signal, target DNA from C. trachomatis was successfully detected without the need for any complicated secondary procedures. This electrochemical one-step detection method could serve as a conceptually new technology enabling highly convenient biosensing that is applicable to point-of-care testing (POCT).

A one-step electrochemical method for DNA detection that utilizes a peroxidase-mimicking DNAzyme amplified through PCR of target DNA.

A novel one-step electrochemical method for DNA detection is described. The procedure utilizes a reaction catalyzed by a peroxidase-mimicking DNAzyme to produce a product, which forms an insoluble precipitation layer on the surface of an electrode. A rationally designed forward primer, conjugated with a peroxidase DNAzyme complementary sequence at its 5'-end, is used for PCR amplification of target DNA. As a result, the DNAzyme sequence is produced by amplification only when the target DNA is present in the sample. The PCR product is then subjected to the precipitation reaction on the electrode surface using an electrolyte assay buffer containing 4-chloronaphthol, hydrogen peroxide, ferrocenemethanol, hemin, and 5'-lambdaexonuclease. Finally, analysis is carried out using Faradaic impedance spectroscopy. The impedance value was found to greatly increase when target DNA is present owing to the formation of a precipitation layer on the electrode surface caused by the catalytic action of the DNAzyme. In contrast, no impedance increase is observed when a control sample not containing target DNA is utilized. By employing this strategy, target DNA from Chlamydia trachomatis was reliably detected within a 10 min period following precipitation without the need for complicated secondary procedures. This effort has led to the development of a highly convenient electrochemical one-step method for DNA detection that utilizes a peroxidase-mimicking DNAzyme, which is specifically designed to undergo amplification during PCR of target DNA.

Electrochemical detection of DNA mutations on a PNA-modified electrode utilizing a single-stranded DNA specific endonuclease.

Utilizing a peptide nucleic acid (PNA)-modified electrode and a single-stranded DNA specific endonuclease, a novel electrochemical method to identify DNA mutations has been developed and represents a totally new strategy for the electrochemical diagnosis of human genetic mutations.

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.

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 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.

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.

Bioelectrocatalytic signaling from immunosensors with back-filling immobilization of glucose oxidase on biorecognition surfaces.

We report a novel method of electrochemical signaling from antigen-antibody interactions at immunoelectrodes with bioelectrocatalyzed enzymatic signal amplification. For the immunosensing surface construction, a poly(amidoamine) G4-dendrimer was employed not only as a building block for the electrode surface modification but also as a matrix for ligand functionalization. As a model biorecognition reaction, the dinitrophenyl (DNP) antigen-functionalized electrode was fabricated and an anti-DNP antibody was used. Glucose oxidase (GOX) was chosen to amplify electrochemical signal by enzymatic catalysis. The signal amplification strategy introduced in this study is based on the back-filling immobilization of biocatalytic enzyme to the immunosensor surface, circumventing the use of an enzyme-labeled antibody. The non-labeled native antibody was biospecifically bound to the immobilized ligand, and the activated enzyme (periodate-treated GOX) reacted and "back-filled" the remaining surface amine groups on the dendrimer layer by an imine formation reaction. From the bioelectrocatalyzed signal registration with the immobilized GOX, the surface density of biospecifically bound antibody could be estimated. The DNP functionalization reaction was optimized to facilitate the antibody recognition and signaling reactions, and approximately 6% displacement of surface amine to DNP was found to be an optimum. From quartz crystal microbalance measurement, immunosensing reaction timing and the surface inertness to the nonspecific biomolecular binding were tested. By changing the surface functionalization level of DNP in the calibration experiments, immunosensors exhibited different dynamic detection ranges and limits of detection, supporting the capability of parameters modulation for the immunosensors. For the anti-DNP antibody assay, the fabricated immunosensor having 65% functionalization ratio exhibited the linear detection range of 10(-4) to 0.1 g/L protein and a limit of detection around 2 x 10(-5) g/L.