A strategy for preparing non-fluorescent graphene oxide quantum dots as fluorescence quenchers in quantitative real-time PCR

Abstract

In recent years, graphene oxide quantum dots (GOQDs) have emerged as novel nanomaterials for optical sensing, bioimaging, clinical testing, and environmental testing. However, GOQDs demonstrate unique photoluminescence properties, with GOQDs having quantum limitations and edge effects that often affect the accuracy of the test results in the sensory field. Herein, GOQDs with a large content of hydroxyl groups and low fluorescence intensity were first prepared via an improved Fenton reaction in this study, which introduces a large amount of epoxy groups to break the C-C bonds. The synthesized GOQDs show no significant variation in the fluorescence intensity upon ultraviolet and visible light excitations. We further utilized the GOQDs as fluorescence quenchers for different fluorescent dyes in real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and verified that the addition of GOQDs (5.3 mg ml À1 ) into a qRT-PCR system could reduce the background fluorescence intensity of the reaction by fluorescence resonance energy transfer (FRET) during its initial stage and its non-specific amplification, and improve its specificity. In addition, the qRT-PCR method could detect two different lengths of DNA sequences with a high specificity in the 10 4 to 10 10 copies per ml range. It is of paramount importance to carry out further investigations to establish an efficient, sensitive, and specific RT-PCR method based on the use of GOQD nanomaterials as fluorescence quenchers.

Multifunctional carbon nanomaterials for diagnostic applications in infectious diseases and tumors

“…primers) through π-π stacking and hydrogen bonding, thereby allowing the matched primer-template complexes to be detached from its surface for subsequent PCR reactions, while the primers remain adsorbed on its surface when mismatched. The above findings encouraged our group to introduce in 2019 GOQDs into the qRT-PCR system to improve its performance [ 161 ]. GOQDs can adsorb primers and TaqMan probes by π-π stacking and hydrogen bonding, thereby reducing the background fluorescence intensity of TaqMan probes, reducing the non-specific amplification in PCR reactions and improving the specificity of the detection.…”

Role of Förster Resonance Energy Transfer in Graphene-Based Nanomaterials for Sensing

“…Reprinted from [76] with permission from the American Chemical Society. Xiao and He et al [77] fabricated GOQDs (G20) for the detection of DNA sequences (Figure 22). Briefly, G20 was treated with three different fluorescent dyes to check the photoluminescence properties and quenching effect.…”

Graphene Oxide-Based Highly Sensitive Assay of Circulating MicroRNAs for Early Prediction of the Response to Neoadjuvant Chemotherapy in Breast Cancer

“…Graphene oxide (GO) is an oxidized form of graphene that contains numerous oxygen-containing functional groups on its surface because of the powerful effects of certain oxidizing agents, which render GO hydrophilic and biocompatible for a wide range of biomedical applications. − Previous studies have demonstrated that GO could adsorb single-stranded DNA through π–π stacking and hydrogen bonding, while dsDNA bases were shielded by negatively charged phosphate groups and had a low affinity for GO. − The use of GO in the PCR system can promote the formation of primer–template complexes and accelerate PCR rates. A previous study has reported on the introduction of graphene oxide quantum dots into the qRT-PCR system to improve its performance . In another study, a newly established GO-based qRT-PCR method was used to detect ovarian cancer-associated miRNAs .…”

“…A previous study has reported on the introduction of graphene oxide quantum dots into the qRT-PCR system to improve its performance. 35 In another study, a newly established GO-based qRT-PCR method was used to detect ovarian cancer-associated miRNAs. 36 GO was further used in qRT-PCR, and it was found that the pre-adsorption of primers onto the GO surface could improve the efficiency of primer−template hybridization and reduce nonspecific amplification, thus improving the sensitivity of qRT-PCR.…”