Enzyme-linked amperometric electrochemical genosensor assay for the detection of PCR amplicons on a streptavidin-treated screen-printed carbon electrode.

A general purpose enzyme-based amperometric electrochemical genosensor assay was developed wherein polymerase chain reaction (PCR) amplicons labeled with both biotin and fluorescein were detected with peroxidase-conjugated antifluorescein antibody on a screen-printed carbon electrode (SPCE). As a proof of principle, the response selectivity of the genosensor was evaluated using PCR amplicons derived from lolB gene of Vibrio cholerae. Factors affecting immobilization, hybridization, and nonspecific binding were optimized to maximize sensitivity and reduce assay time. On the basis of the background amperometry signals obtained from nonspecific organisms and positive signals obtained from known V. cholerae, a threshold point of 4.20 microA signal was determined as positive. Under the optimum conditions, the limit of detection (LOD) of the assay was 10 CFU/mL of V. cholerae. The overall precision of this assay was good, with the coefficient of variation (CV) being 3.7% using SPCE and intermittent pulse amperometry (IPA) as an electrochemical technique. The assay is sensitive, safe, and cost-effective when compared to conventional agarose gel electrophoresis, real-time PCR, and other enzyme-linked assays for the detection of PCR amplicons. Furthermore, the use of a hand-held portable reader makes it suitable for use in the field.

Allele-specific genotyping by using guanine and gold electrochemical oxidation signals.

Electrochemical DNA biosensors have become strong candidates for DNA based analysis. Allele-specific genotyping is also one of the important research areas, where electrochemical approaches provide promising advances. Recently reported two methods based on electrochemical guanine and colloidal gold (Au) nanoparticle oxidation signals are reviewed and compared with the existing genotyping methods in this report.

Electrochemical genosensor for mitomycin C-DNA interaction based on guanine signal.

The interaction of mitomycin C (MC) with fish sperm or calf thymus DNA immobilized onto carbon screen-printed electrodes (CSPE) and carbon paste electrode (CPE) have been studied by using electrochemical techniques as square wave voltammetry (SWV) and differential pulse voltammetry (DPV). After the interaction was occurred between DNA and MC on electrode surface, it was observed that the guanine signal was higher with bare electrode than DNA-modified one. The changes in the experimental parameters such as the concentration of MC, and the accumulation time of MC were studied by using SWV and DPV. In addition, reproducibility, and detection limit parameters were determined using both electrodes. The partition coefficient of MC was also calculated before and after interaction of MC with dsDNA at CPE surface. These results showed that these two different DNA biosensors could be used for the sensitive, rapid and cost effective detection of MC-DNA interaction.

Detection of achondroplasia G380R mutation from PCR amplicons by using inosine modified carbon electrodes based on electrochemical DNA chip technology.

The detection of Achondroplasia G380R mutation from real samples was performed by monitoring the oxidation signal of guanine. Inosine-substituted 12-mer capture probes related to the homozygous or heterozygous alleles of Achondroplasia G380R mutation were adsorbed onto carbon paste electrode (CPE) surface. No guanine signal was obtained from the capture probes, since the inosine bases were electroinactive. Then, these probes were hybridized with the denatured PCR samples on the CPE surface. The hybridization between the probe and target sequences was determined by using the oxidation signal of guanine in connection with differential pulse voltammetry (DPV). The oxidation signal of guanine was observed as a result of the specific hybridization between the probe and PCR sample. The changes in the peak height of the guanine signal provided the information whether the PCR sample contained heterozygous allele or homozygous allele. Numerous factors affecting the hybridization and nonspecific binding events were optimized to detect down to 41.24 fmol/ml target DNA. The electrochemical detection of Achondroplasia G380R mutation from PCR samples greatly shortened and simplified the diagnosis for the homozygous mutant type, which is lethal for the newborn.

Electrochemical genosensor based on colloidal gold nanoparticles for the detection of Factor V Leiden mutation using disposable pencil graphite electrodes.

Electrochemical genosensors for the detection of the Factor V Leiden mutation from polymerase chain reaction (PCR) amplicons using the oxidation signal of colloidal gold (Au) is described. A pencil graphite electrode (PGE) modified with target DNA, when hybridized with complementary probes conjugated to Au nanoparticles, responded with the appearance of a Au oxide wave at approximately +1.20 V. Specific probes were immobilized onto the Au nanoparticles in two different modes: (a) Inosine-substituted probes were covalently attached from their amino groups at the 5' end using N-(3-dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent onto a carboxylate-terminated l-cysteine self-assembled monolayer (SAM) preformed on the Au nanoparticles, and (b) probes with a hexanethiol group at their 5' phosphate end formed a SAM on Au nanoparticles. The genosensor relies on the hybridization of the probes with their complementary targets, which are covalently immobilized at the PGE surface. Au-tagged 23-mer capture probes were challenged with the synthetic 23-mer target, 131-base single-stranded DNA or denatured 256-base polymerase chain reaction (PCR) amplicon. The appearance of the Au oxidation signal shortened the assay time and simplified the detection of the Factor V Leiden mutation from PCR amplified real samples. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the Au signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized. The detection limit for the PCR amplicons was found to be as low as 0.78 fmol; thus, it is suitable for point-of-care applications.

Allele-specific genotype detection of factor V Leiden mutation from polymerase chain reaction amplicons based on label-free electrochemical genosensor.

An electrochemical genosensor for the genotype detection of allele-specific factor V Leiden mutation from PCR amplicons using the intrinsic guanine signal is described. The biosensor relies on the immobilization of the 21-mer inosine-substituted oligonucleotide capture probes related to the wild-type or mutant-type amplicons, and these probes are hybridized with their complementary DNA sequences at a carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using the oxidation signal of guanine in connection with differential pulse voltammetry (DPV). The guanine signal was monitored as a result of the specific hybridization between the probe and amplicon at the CPE surface. No label-binding step was necessary, and the appearance of the guanine signal shortened the assay time and simplified the detection of the factor V Leiden mutation from polymerase chain reaction (PCR)-amplified amplicons. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the guanine signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized to detect down to 51.14 fmol/mL target DNA. With the help of the appearance of the guanine signal, the yes/no system is established for the electrochemical detection of allele-specific mutation on factor V for the first time. Features of this protocol are discussed and optimized.

Electrochemical biosensor for the interaction of DNA with the alkylating agent 4,4'-dihydroxy chalcone based on guanine and adenine signals.

The interaction of an alkylating agent, 4,4'-dihydroxy chalcone (DHC) with calf thymus double stranded DNA (dsDNA) and calf thymus single stranded DNA (ssDNA) was studied electrochemically based on the oxidation signals of guanine and adenine by using differential pulse voltammetry (DPV) at carbon paste electrode (CPE). As a result of the alkylation of DHC between the base pairs in dsDNA, the voltammetric signal of guanine and adenine greatly decreased. After the interaction of DHC with ssDNA, a higher decrease in the oxidation signals of guanine and adenine was observed under the same conditions. The partition coefficients of DHC at dsDNA and ssDNA modified CPEs were calculated. The interactions of DHC with synthetic polynucleotides, such as polyguanylic acid and polyadenylic acid were also observed. In addition, the detection limit and the reproducibility were determined by using DPV. The interaction of DHC with dsDNA in solution-phase was also investigated and the results were compared with the ones obtained by surface immobilized dsDNA. The application of electrochemical DNA biosensor for monitoring the DNA-alkylating agent interactions was explored.

DNA and PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical label.

Described here are the electrochemical parameters for MB on binding to DNA at hanging mercury drop electrode (HMDE), glassy carbon electrode (GCE), and carbon paste electrode (CPE) in the solution and at the electrode surface. MB, which interacts with the immobilized calf thymus DNA, was detected by using single-stranded DNA-modified HMDE or CPE (ssDNA-modified HMDE or CPE), bare HMDE or CPE, and double-stranded DNA-modified HMDE or CPE (dsDNA-modified HMDE or CPE) in combination with adsorptive transfer stripping voltammetry (AdTSV), differential pulse voltammetry (DPV), and alternating current voltammetry (ACV) techniques. The structural conformation of DNA and hybridization between synthetic peptide nucleic acid (PNA) and DNA oligonucleotides were determined by the changes in the voltammetric peak of MB. The PNA and DNA probes were also challenged with excessive and equal amount of noncomplementary DNA and a mixture that contained one-base mismatched and target DNA. The partition coefficient was also obtained from the signal of MB with probe, hybrid, and ssDNA-modified GCEs. The effect of probe, target, and ssDNA concentration upon the MB signal was investigated. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.

DNA sensing on glassy carbon electrodes by using hemin as the electrochemical hybridization label.

The electrochemical behavior of hemin, an iron complex of porphyrin, on binding to DNA at a glassy carbon electrode (GCE) and in solution, is described. Hemin, which interacts with covalently immobilized calf thymus DNA, was detected by use of a bare GCE, a double-stranded DNA-modified GCE (dsDNA-modified GCE), and a single-stranded DNA-modified GCE (ssDNA-modified GCE), in combination with differential pulse voltammetry (DPV). The structural conformation of DNA was determined from changes in the voltammetric signals acquired on reduction of hemin. As a result of its large steric structure and anionic substitution on its porphyrin plane, hemin intercalates between the base pairs of dsDNA. A scan-rate study for hemin and the dsDNA-hemin complex were also performed to determine the electrochemical behavior of the complex. The partition coefficient was obtained from the peak currents measured when different concentrations of hemin were in the presence of dsDNA. By observing the oxidation signals of guanine, damage to DNA after reaction with hemin at the GCE surface was also detected. The electrochemical detection of hybridization between the covalently immobilized probe and its target sequence was detected by use of hemin. These results demonstrate the use of DNA biosensors in conjunction with hemin for electrochemical detection of hybridization and damage to DNA.

Electrochemical DNA biosensor for the detection of TT and Hepatitis B virus from PCR amplified real samples by using methylene blue.

DNA biosensors based on nucleic acid hybridization processes are rapidly being developed towards the goal of rapid and inexpensive diagnosis of genetic and infectious diseases. Electrochemical transducers are often being used for detecting the DNA hybridization event, due to their high sensitivity, small dimensions, low cost, and compatibility with microfabrication technology. In this study, an electrochemical biosensor for the voltammetric detection of DNA sequences related to the Hepatitis B virus (HBV) and TT virus (TTV) from polymerase chain reaction (PCR) amplified real samples is described for the first time. The biosensor relies on the immobilization of the 21- or 24-mer single stranded oligonucleotides (probe) related to the HBV and TTV sequences and hybridization of these oligonucleotides with their complementary sequences (target) at carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using square wave voltammetry (SWV) with moving average baseline correction and methylene blue (MB) as the hybridization indicator. As a result of the interaction between MB and the bound guanine bases of hybrid at CPE surface, the MB signal decreased, when it was compared with the MB signal, which was observed with probe modified CPE. The difference between the MB signals, obtained from the hybrid modified and the probe modified CPE is used to detect the DNA sequences of the infectious diseases from PCR amplified real samples. Numerous factors affecting the target hybridization and indicator binding reactions are optimized to maximize the sensitivity.