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 μg ml^-1) 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 10⁴ to 10¹⁰ copies per μl 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.

Fig. 1. Characterization of GOQDs. (a) AFM image of the GOQDs deposited on mica substrates. (b) Height profile of the GOQDs. (c) Height distribution of the GOQDs. (d) High-resolution XPS C 1s spectra of the GOQDs. (e) FT-IR spectra of GOQDs. (f) UV-vis absorption spectrum of GOQDs.

Fig. 2. Fluorescence quenching ability of GOQDs. (a) Fluorescence quenching of 6-FAM by GOQDs. (b) Fluorescence quenching of JOE by GOQDs. (c) Fluorescence quenching of Cy3 by GOQDs.

Fig. 3. Screening the optimal concentration of GOQDs and the sensitivity and specificity of qRT-PCR. (a) qRT-PCR amplification curve of 106 bp plasmid DNA after addition of 26 μg ml⁻¹, 11 μg ml⁻¹, 5.3 μg ml⁻¹, 2.2 μg ml⁻¹ and 1.5 μg ml⁻¹ GOQDs, the inset figure was the result of agarose gel electrophoresis. (b) qRT-PCR amplification curve of 65 bp DNA after addition of 26 μg ml⁻¹, 11 μg ml⁻¹, 5.3 μg ml⁻¹, 3.3 μg ml⁻¹ and 2.2 μg ml⁻¹ GOQDs, the inset figure was the result of agarose gel electrophoresis. (c) The amplification plots of 106 bp plasmid DNA. The inset figure is a standard curve of 106 bp plasmid DNA amplification with a determining coefficient of 0.9983. (d) The amplification plots of 65 bp DNA. The inset figure is a standard curve of 65 bp DNA amplification with a determining coefficient of 0.9925.

Fig. 4. Scheme of enhancing qRT-PCR specificity by GOQDs. The whole picture shows the schematic diagram of the hypothesis of enhancing qRT-PCR specificity by GOQDs.

Fig. 5. Detection of two different lengths of DNA sequences by qRT-PCR based on GOQDs. (a) qRT-PCR amplification curve of 106 bp plasmid DNA after addition of 5.3 μg ml⁻¹ GOQDs, the inset figure was the result of agarose gel electrophoresis. (b) Fluorescence quenching results of Probe 1 with 5.3 μg ml⁻¹ GOQDs. (c) qRT-PCR amplification curve of 65 bp DNA after addition of 5.3 μg ml⁻¹ GOQDs, the inset figure was the result of agarose gel electrophoresis. (d) Fluorescence quenching results of Probe 2 with 5.3 μg ml⁻¹ GOQDs.