Detection of Single Nucleotide Polymorphisms by Fluorescence Embedded Dye SYBR Green I Based on Graphene Oxide

Abstract

The detection of single nucleotide polymorphisms (SNPs) is of great significance in the early diagnosis of diseases and the rational use of drugs. Thus, a novel biosensor based on the quenching effect of fluorescence-embedded SYBR Green I (SG) dye and graphene oxide (GO) was introduced in this study. The probe DNA forms a double helix structure with perfectly complementary DNA (pcDNA) and 15 single-base mismatch DNA (smDNA) respectively. SG is highly intercalated with perfectly complementary dsDNA (pc-dsDNA) and exhibits strong fluorescence emission. Single-base mismatch dsDNA (SNPs) has a loose double-stranded structure and exhibits poor SG intercalation and low fluorescence sensing. At this time, the sensor still showed poor SNP discrimination. GO has a strong effect on single-stranded DNA (ssDNA), which can reduce the fluorescence response of probe DNA and eliminate background interference. And competitively combined with ssDNA in SNPs, quenching the fluorescence of SG/SNP, while the fluorescence value of pc-dsDNA was retained, increasing the signal-to-noise ratio. At this time, the sensor has obtained excellent SNP resolution. Different SNPs detect different intensities of fluorescence in the near-infrared region to evaluate the sensor's identification of SNPs. The experimental parameters such as incubation time, incubation temperature and salt concentration were optimized. Under optimal conditions, 1 nM DNA with 0–10 nM linear range and differentiate 5% SNP were achieved. The detection method does not require labeling, is low cost, simple in operation, exhibits high SNP discrimination and can be distinguished by SNP at room temperature.

Mutations in microRNA-128-2-3p identified with amplification-free hybridization assay

“…However, the methods is not applied for detection of specific miRNAs SNPs in the biological samples and are only analyzing the wild-type miRNA [ 40 , 41 ]. A recent study by Xia et al [ 43 ] applied the SYBR green fluorophore for SNP detection. In the assay, graphene oxide was added to prevent SYBR green dsDNA insertion, quenching the fluorescent signal and thus allowing SNP detection.…”

Recent biomedical advancements in graphene oxide- and reduced graphene oxide-based nanocomposite nanocarriers

“…Biosensors containing either GO or rGO generally exhibit fluorescence and fluorescence-quenching properties [ 35 , 36 ]. However, high conductivity of rGO makes it useful for EC or ECL assays [ 37 , 38 ].…”

“…However, high conductivity of rGO makes it useful for EC or ECL assays [ 37 , 38 ]. Moreover, GO is often used in nucleotide detection owing to its strong interactions with ssDNA, allowing for detection of specific sequences [ 36 , 182 ]. Although several applications involving GO/rGO in biosensing exist, those that uses of them as nanocarriers are limited [ 183 , 184 ].…”

“…Xia et al [ 36 ] detected single nucleotide polymorphisms (SNPs) by embedding them in SYBR Green I (SG) before adding GO particles. Subsequently, fluorescence from SG in unstable SNPs was highly quenched by GO within 3 min, whereas fluorescence from perfectly complementary dsDNA was comparatively high.…”

“…Although other methods will be discussed, GO/rGO-based materials have been particularly popular in bioimaging because of their fluorescent emission under the right excitation wavelength [ 33 , 34 ]. Meanwhile, GO-/rGO-based biosensors use their fluorescence quenching abilities [ 35 , 36 ] although high electrical conductivity of rGO makes it a suitable candidate for electrochemical (EC) or electrochemiluminescence (ECL) assays [ 37 , 38 ]. Furthermore, GO/rGO is known to promote stem cell proliferation and differentiation, which has encouraged research in their use in tissue engineering, particularly that of cardiac and nerve tissues, which improves in the presence of a conductive material [ 39 – 41 ].…”