Electroactive nanocarbon materials as signaling tags for electrochemical PCR

Abstract

Electrochemical polymerase chain reaction (PCR) represents a valid alternative to the optical-based PCR due to reduced costs of signaling labels, use of simpler instrumentation, and possibility of miniaturization and portability of the systems, which can facilitate decentralized detection. The high intrinsic electroactivity and strong linear relationship between the material concentration and its redox signal suggest a possible use of oxidized nanocarbon materials as electroactive tags for PCR. Herein, we compared three different nanographene oxide materials namely nGO-1, nGO-2 and nGO-3 as signaling tags for the detection of genetically modified organisms (GMO) by electrochemical PCR. The three materials differ in size, chemical composition as well as type and amount of oxygen functionalities verified by extensive characterization with X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), transmission electron microscopy (TEM) and electrochemical methods. A sense primer sequence belonging to the Cauliflower Mosaic Virus 35S promoter (a common genetic marker for GMO screening) was used to conjugate to the nanocarbon materials by carbodiimide chemistry before PCR amplification with a biotinylated antisense strand. Finally, the amplified electroactive PCR product was detected, where the reduction signal derived from the electrochemically reducible oxygenated functionalities on the nanocarbon material surface was directly correlated to the presence of GMO. Overall, we were able to correlate the different material characteristics with their performance as electroactive labels and identify the nanocarbon material that exhibits the highest potential to be used as innovative electroactive label for PCR in the amplification and detection of the selected target sequence.