Electrochemical Polymerase Chain Reaction Using Electroactive Graphene Oxide Nanoparticles as Detection Labels

Abstract

Electroactive nanocarbon tags are used in this work to label the DNA primers for the polymerase chain reaction (PCR) amplification of Cauliflower Mosaic Virus 35S promoter sequence, one of the most common markers for the detection of genetically modified organisms (GMOs). The PCR product carrying the electrochemical label can be directly detected on miniaturized electrodes, with the working signal being correlated to the reduction of oxygen-containing groups on the nanocarbon surface. A linear relationship was first established between the electrochemical signal and the nanomaterial concentration, both for the unconjugated electroactive nanocarbon and the conjugates with single-stranded and double-stranded DNA. After which, PCR amplification using a modified sense-primer was performed, and discrimination between amplified products from positive samples (GMO maize) and negative controls (non-GMO maize) was achieved successfully. After the optimization of PCR experimental conditions using the electroactive nanocarbon label, the electrochemical signal recorded as a function of PCR cycle number showed an exponential increase, very similar to that obtained in optical-based real-time PCR. From that, the extrapolated cycle threshold value showed a linear relationship with the initial number of copies of target DNA. Through the findings, electroactive nanocarbon material demonstrated high potential as electrochemical label for PCR, with the electrochemical signals produced directly correlated to the amount of PCR product. This work will serve as a stepping stone for the development of a robust, efficient, and portable electrochemical PCR system, with a reduced cost considering the wide availability and suitability of carbon nanomaterials for mass production, and the cost-effective electrochemical detection.

The potential of electrochemistry for the detection of coronavirus-induced infections

“…Adapted with permission from Ref. [ 54 ]; (C) Electrochemical PCR scheme using graphene oxide nanoparticles as labels for DNA primers: (i) Electrochemical detection on miniaturized electrode; (ii) nanographene oxide reduction current vs PCR cycle number for positive (red) and negative control (blue) [ 26 ]. Reproduced with permission from Ref.…”

“…Our group has recently made use of electrochemical PCR to successfully detect genetically modified organisms by amplifying the Cauliflower Mosaic Virus 35S promoter sequence with the help of electroactive nanographene oxide nanoparticles as labels for the DNA primers used in PCR amplification step [ 26 ]. Electrochemical PCR has been already employed for the detection of Influenza A (AH1pdm) virus [ 58 ] using methylene blue as electroactive intercalator, which electrochemical signal is inversely correlated to the amount of PCR product [ 58 ].…”

“…Besides electrochemical sensors and biosensors for point-of-care diagnosis, electrochemistry was also recently used as an alternative to conventional optical PCR, whereby electroactive indicators or detection labels were employed to monitor the progress of the PCR reaction, with the measured oxidation or reduction signal being correlated to the amount of the PCR-amplified product [ 26 ]. Without the need for fluorescent labels and optical detection, electrochemical PCR can represent the cost-effective and portable option sought for COVID-19 intensive testing.…”

A CRISPR/Cas12a-Mediated Dual-Mode Electrochemical Biosensor for Polymerase Chain Reaction-Free Detection of Genetically Modified Soybean

“…Electrochemical biosensors are one of the earliest and most mature DNA detection technologies with unique advantages. , Because signals can be measured directly without requiring an expensive, complicated, and fault-prone transduction device, electrochemical biosensors are of simple operation, high sensitivity, low cost, and easy portability, providing a promising approach for the field detection of GMOs. − However, sensitivity and specificity need to be further improved because the analyte concentration is usually very low without PCR amplification. Obviously, to construct an electrochemical biosensor for GMOs with high sensitivity and specificity, a novel signal amplification strategy and a new recognition system are greatly demanded.…”

Advances on the Use of Graphene as a Label for Electrochemical Biosensors

“…Plenty of works are shown in the literature whereby DNA oligonucleotides, proteins, antibodies and enzymes used as bio‐recognition elements are immobilized onto the graphene surface not only by physical adsorption, but also through covalent conjugation with the OCGs . Furthermore, it has been shown that OCGs can play a role in the generation of the working signal by exploiting their intrinsic optical and electrochemical properties . However, on the other hand, the electrical conductivity of graphene is largely impaired when significant amounts of OCGs are introduced, and this will have detrimental effects on the use of the graphene material especially for electrochemical applications including biosensing.…”