Facilitation of Dye-Based Quantitative Real-Time Polymerase Chain Reaction with Poly(ethylene glycol)-Engrafted Graphene Oxide

Abstract

Quantitative real-time polymerase chain reaction (qPCR) is an important and extensively utilized technique in medical and biotechnological applications. qPCR enables the real-time detection of nucleic acid during amplification, thus surpassing the necessity of post-amplification gel electrophoresis for amplicon detection. Despite being widely employed in molecular diagnostics, qPCR exhibits limitations attributed to nonspecific DNA amplification that compromises the efficiency and fidelity of qPCR. Herein, we demonstrate that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly improve the efficiency and specificity of qPCR by adsorbing single-stranded DNA (ssDNA) without affecting the fluorescence of double-stranded DNA binding dye during DNA amplification. PEG-nGO adsorbs surplus ssDNA primers in the initial phase of PCR, having lower concentrations of DNA amplicons and thus minimizing the nonspecific annealing of ssDNA and false amplification due to primer dimerization and erroneous priming. As compared to conventional qPCR, the addition of PEG-nGO and the DNA binding dye, EvaGreen, in the qPCR setup (dubbed as PENGO-qPCR) significantly enhances the specificity and sensitivity of DNA amplification by preferential adsorption of ssDNA without inhibiting DNA polymerase activity. The PENGO-qPCR system for detection of influenza viral RNA exhibited a 67-fold higher sensitivity than the conventional qPCR setup. Thus, the performance of a qPCR can be greatly enhanced by adding PEG-nGO as a PCR enhancer as well as EvaGreen as a DNA binding dye to the qPCR mixture, which exhibits a significantly improved sensitivity of the qPCR.

Poly(ethylene glycol)-Engrafted Graphene Oxide for Gene Delivery and Nucleic Acid Amplification

“…Biocompatible polymer PEG can be conjugated or attached to GO via covalent or noncovalent functionalization, respectively, for various applications (Table 1). Our group synthesized poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) for the enhancement of nucleic acid amplification and nucleic acid delivery [54,63,64]. Briefly, PEG-nGO was synthesized by adding NaOH and chloroacetic acid to the sonicated GO (conversion of −OH groups to −COOH groups).…”

“…Encouraged by the beneficial effect of PEG-nGO on end-point PCR assay, our group expanded the applicability of PEG-nGO by investigating the effect of PEG-nGO on quantitative fluorescence-based qPCR assay [64]. qPCR is the most availed tool in clinical diagnostics that offers the advantage of real-time detection of nucleic acid during the amplification process, eliminating the need for post-amplification gel electrophoresis for amplicon detection [89,90].…”