Sensitive SNP Detection of KIF6 Gene by Quantum Dot-DNA Conjugate Probe-Based Assay

Abstract

In recent years, quantum dots (QDs) have been widely used in numerous biological processes and bio-analysis as novel fluorescence indicators. In the study, we developed a quantum dot-based bio-barcode amplification in combination with a Taq DNA ligase approach for the rapid detection of a polymorphism in kinesin-like protein 6 gene (KIF6), which significantly enhances the sensitivity. A ligand-exchange reaction was performed to prepare MPA-functionalized water-soluble QDs. The amine-modified oligonucleotides were coupled to QDs surface to form functional QDs-DNA conjugates. Meanwhile, the surface of gold nanoparticles (AuNPs) was modified with both the detection probe and bio-barcode oligonucleotides. Then, the amine-modified capture DNAs were immobilized onto the chip, followed by the addition of the modified AuNPs to the modified chip surface to form sandwiched hybridization. When the capture DNA and detection probe were perfectly matched to target DNA (KIF6), Taq DNA ligase in the solution would link the gap between the two strands, resulting in a covalent linkage. The preprepared QDs-DNA conjugation was subsequently added into the system to eventually form the fluorescence assembly. There was a good linear relationship between the target DNA (KIF6) concentration (1pM to 1fM) and ΔG of spot on chip. The ΔG of single base mismatch group was far less than that of completely complementary group. The method of QD-based bio-barcode amplification can provide a detection limit with low fM sensitivity for the detection of KIF6 DNA samples.