Electrochemical genosensors based on PCR strategies for microorganisms detection and quantification (original) (raw)
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Bioelectrochemistry (Amsterdam, Netherlands), 2007
Direct electrochemical genosensor was developed for the detection of a probe sequence relative position in a PCR amplicon for the optimum detection of bacterial and microbiological diseases, in this study. The genosensor relies on a label-free electrochemical detection. The amino-linked inosine modified (guanine-free) coequal capture probes which were chosen from different parts of a PCR amplicon, immobilized on to disposable pencil graphite electrodes (PGE) by electrostatically and covalently. As a model case Hepatitis B virus (HBV) genome amplicon was used for the detection and specification. Hybridization was occurred after surface coverage with denatured amplicons. After hybridization, optimum probe sequence position was identified by using the differences between the responses of guanine oxidation signals. The results of this study might have a great convenience for the microbiological diseases detection applications such as DNA micro arrays.
Electrochemical DNA hybridization sensors applied to real and complex biological samples
Biosensors and Bioelectronics, 2010
a b s t r a c t DNA hybridization biosensors, also known as genosensors, are analytical devices for the detection of specific DNA "target" sequences in solution, upon hybridization of the targets with complementary "probes" immobilized on a solid substrate. Electrochemical genosensors hold great promise to serve as devices suitable for point-of-care diagnostics and multiplexed platforms for fast, simple and inexpensive nucleic acids analysis. Although a lot of progress has been made in the past few years, the performance of genosensors in complex biological samples has been assayed in only a small fraction of published research articles. This review covers such a group of reports, from the year 2000 onwards. Special attention is played in the nature and complexity of the samples and in the way matrix effects were treated and specificity controls were performed.
Miniaturized electrochemical detector as a tool for detection of DNA amplified by PCR
ELECTROPHORESIS, 2008
This paper reports on the analysis of specific sequence of Phage Lambda DNA amplified by PCR. Agarose gel electrophoresis, gel electrophoresis on chip and stationary electrochemical instrument were employed for detection of amplicons obtained after 2, 4, 6, 8, 10, 15, 20, 25, 30 and 35 cycles. In the case of agarose gel electrophoresis the lowest detectable amount of DNA was obtained after 15 PCR cycles. Gel electrophoresis on chip offers higher sensitivity because the lowest detectable amount of amplicons by this technique was obtained after eight PCR cycles. Further we employed square wave voltammetry and various working electrodes (hanging mercury drop electrode, screen-printed carbon electrode and carbonnanotube-based screen-printed electrodes) to detect amplicons. Amplicons obtained even after two cycles were detectable at all electrodes. To improve the selectivity of electrochemical detection carbon nanoelectrodes were off-line coupled with gel electrophoresis. Into the agarose gel the electrodes were placed. Further the amplicons were loaded into agarose gel wells. DNA migrating to the detection place was electrochemically analyzed. Amplicons obtained after two cycles were detectable by this hyphenated technique.
Biosensors and Bioelectronics, 2005
This paper describes the optimisation and the analytical performances of an enzyme-based electrochemical genosensor, developed using disposable oligonucleotide-modified screen-printed gold electrodes. The immobilisation of a thiol-tethered probe was qualitatively investigated by means of faradic impedance spectroscopy. Impedance spectra confirmed that the thiol moiety unambiguously drives the immobilisation of the oligonucleotide probe. Furthermore, both probe surface densities and hybridisation efficiencies were quantified through chronocoulometric measurements. Electrochemical transduction of the hybridisation process was also performed by means of faradic impedance spectroscopy, after coupling of a streptavidin-alkaline phosphatase conjugate and bio-catalysed precipitation of an insoluble and insulating product onto the sensing interface. Chronocoulometric results allowed discussion of the magnitude of hybridisation signals in terms of probe surface densities and their corresponding hybridisation efficiency. The genosensor response varied linearly (r 2 = 0.9998) with the oligonucleotide target concentration over three orders of magnitude, between 12 pmol/L and 12 nmol/L. The estimated detection limit was 1.2 pmol/L (i.e., 7.2 × 10 6 target molecules in 10 L of sample solution). The analytical usefulness of the impedimetric genosensor was finally demonstrated analysing amplified samples obtained from the pBI121 plasmid and soy and maize powders containing 1 and 5% of genetically modified product. Sensing of such unmodified amplicons was achieved via sandwich hybridisation with a biotinylated signaling probe. The electrochemical enzyme-amplified assay allowed unambiguous identification of all genetically modified samples, while no significant non-specific signal was detected in the case of all negative controls.
Environmental and Toxicology Management
The quest for alternative methods is driven by the need to provide expertise in real time in biological fields such as medicine, pathogenic bacteria and viruses identification, food protection, and quality control. Polymerase Chain Reaction (PCR) and Enzyme Linked Immunosorbent Assay (ELISA) are examples of traditional methods that have some limitations and lengthy procedures. Biosensors are the most appealing option because they provide easy, dependable, fast, and selective detection systems compared to conventional methods. This review provides an overview of electrochemical genosensor based biosensor diagnostic system for infectious diseases detection as well as their applications, demonstrating their utility as a fast and responsive tool for detecting pathogenic bacteria, viruses, GMOs, and human diseases.
DNA Electrochemical Behaviors, Recognition and Sensing by Combining with PCR Technique
Sensors, 2003
The electrochemical studies on the behaviors and recognition of DNA have attracted considerable attention. DNA biosensors based on nucleic acid hybridization process are rapidly being developed towards the goal of rapid and inexpensive diagnosis of genetic and infectious diseases. This brief review focuses on the current state of the DNA electrochemical sensors with emphasis on recent advances, challenges and trends. The works on DNA electrochemical behaviors, recognition and detection in our group in the last three years are also introduced.
Biosensors and Bioelectronics, 2007
This paper deals with the use of an electrochemical genosensor array for the rapid and simultaneous detection of different food-contaminating pathogenic bacteria. The method includes PCR amplification followed by analysis of the amplicons by hybridisation with toxin-specific oligonucleotide probes. A screen-printed array of four gold electrodes, modified using thiol-tethered oligonucleotide probes, was used. Unmodified PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes and biotinylated signaling probes. The resulting biotinylated hybrids were coupled with a streptavidin-alkaline phosphatase conjugate and then exposed to an ␣-naphthyl phosphate solution. Differential pulse voltammetry was finally used to detect the ␣-naphthol oxidation signal. Mixtures of DNA samples from different bacteria were detected at the nanomolar level without any cross-interference. The selectivity of the assay was also confirmed by the analysis of PCR products unrelated to the immobilised probes.
Biosensors and Bioelectronics, 2008
A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng l −1 and 0.45 ng l −1 of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes.
Indicator-Free Electrochemical Dna Biosensor for the Detection of Hybridisation Reaction
Sensors and Microsystems, 2002
A novel electrochemical hybridization biosensor protocol without using an external indicator is described. The oxidation signals of adenine and guanine from calf thymus double-stranded DNA (dsDNA) and calf thymus singlestranded DNA (ssDNA) was studied by using differential pulse voltammetry (DPV) at carbon paste electrode (CPE). The oxidation signals of adenine and guanine obtained from the ssDNA modified CPE was higher than that from the dsDNA modified CPE due to the accessible unbound adenine and guanine bases. The electrochemical determination of hybridization between native capture DNA probe and target oligonucleotides and polynucleotides such as poly[G], poly[C], poly[I] and poly[A], poly[T] were also accomplished. The dependence of the peak heights of guanine and adenine signals on the number of the respective bases in oligonucleotides was observed by means of DPV. The dependence of the guanine signal upon the concentration of the target and the noncomplementary DNA sequences was also observed. The use of the intrinsic DNA electrochemical signals for monitoring hybridization events offers several advantages over the common use of carcinogenic external indicators and expensive inosine substituted capture DNA probes, such as a shorter assay time and cost-effective procedure. Performance characteristics of the biosensor are described, along with future prospects.
Biosensors and Bioelectronics, 1999
A disposable electrochemical sensor for the detection of short DNA sequences is described. Synthetic single-stranded oligonucleotides have been immobilized onto graphite screen printed electrodes with two procedures, the first involving the binding of avidinbiotinylated oligonucleotide and the second adsorption at a controlled potential. The probes were hybridized with different concentrations of complementary sequences. The formed hybrids on the electrode surface were evaluated by differential pulse voltammetry and chronopotentiometric stripping analysis using daunomycin hydrochloride as indicator of hybridization reaction. The probe immobilization step, the hybridization event and the indicator detection, have been optimized. The DNA sensor obtained by adsorption at a controlled potential was able to detect 1 g/ml of target sequence in the buffer solution using chronopotentiometric stripping analysis.