DNA Biosensors Based On Strand Displacement And Target Recycling Signal Amplification For The Detection Of DNA And MicroRNA

Biosensing technology is a novel technology developed by integrating with many disciplines including biology, chemistry, physics, optics, electronics, etc. In specific, the optical and electrochemical biosensors are widely used in various fields, because they are of good selectivity, high sensitivity, short analysis time, low cost, ability of on-site monitoring in complex system and so on. Recently, these biosensors have already introduced into many areas for application such as disease diagnosis, mutation analysis and gene therapy. This article is to combine toehold-mediated strand displacement reaction(TMSDR), three-way DNA junction and target recycling signal amplified technology to build some novel electrochemiluminescence(ECL), electrochemical and fluorescence DNA biosensors for DNA or miRNA detection.This article includes four chapters. Chapter One: Toehold-mediated strand displacement reaction, and locked nucleic acid(LNA) modified three-way DNA junction were using to develop an ECL biosensor for the detection of BRCA1 gene related to breast cancer. The principle of our sensor is illustrated as follow: in the absence of BRCA1, capture probe(Cp), assisted probe 1(Ap1) and assisted probe2(Ap2) will hybridize to form a ternary three-way DNA junction structure with an external toehold on the surface of GE. In this scenario, ferrocene(Fc)-labeled in assisted probe 1(Ap1) is close to the Ru(bpy)32+, resulting in very weak ECL intensity due to the high ECL quenching efficiency of Fc to Ru(bpy)32+. While in the presence of target DNA, it hybridizes with the external LNA modified toehold of Ap2 to initiate a strand displacement reaction, eventually resulting in the release of Fc-labeled Ap1. Without the quenching effect of Fc to Ru(bpy)32+, ECL intensityincreases significantly. The proposed sensing platform was able to detect the target DNA in the concentration range from 10 fM to 0.8 pM with the detection limit as low as 0.8 fM. The ECL biosensor has a good selectivity, can discriminate a single-base mismatched sequence, offer a promising approach toward the single-nucleotide polymorphism(SNP) detection of DNA.Chapter Two: An immobilization-free electrochemical impedance biosensor for microRNA detection was developed in this work, which was based on both the duplex-specific nuclease(DSN) assisted target recycling and capture probe(Cp)enriched from the solution to electrode surface via magnetic beads(MBs). In the absence of miR-21, Cp cannot be hydrolyzed due to the low activity of DSN against ssDNA. Therefore, the intact Cp could be attached to the surface of magnetic glass carbon electrode(MGCE), resulting in a compact negatively charged layer as well as a large charge-transfer resistance. While in the presence of miR-21, it hybridized with Cp to form a DNA-RNA heteroduplex. Due to the considerable cleavage preference for DNA in DNA-RNA hybrids, DSN hydrolyzed the target-binding part of the Cp while liberating the intact miR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. In this way, a single miR-21 was able to trigger the permanent hydrolysis of multiple Cp. Finally, all Cp were digested. Thus, the negatively charged layer could not be formed, resulting in a small charge-transfer resistance. By employing the above strategy, the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with a detection limit of 60 aM. Meanwhile, the method showed little cross-hybridization among the closely related miRNA family members even at the single-base-mismatched level.Successful attempts were made in applying the approach to detect miR-21 in human serum samples of breast cancer patients.Chapter Three: TMSDR assisted target recycling amplification and the enrichment/separation of magnetic beads(MBs) were used to develop an electrochemical sensor for the detection of target DNA. The principle of our sensoris illustrated as follow, hairpin probes were modified on MBs surface by specific recognition of biotin and streptavidin, the hybridization with target DNA(T)triggered strand displacement reaction, leading to the generation of three-way DNA junctions and release of T, which can further trigger a new cycle. Theoretically, one T would fabricate abundant three-way DNA junctions. The terminal of three-way DNA junctions labeled with Horseradish peroxidase(HRP), through biotin-streptavidin interaction. Subsequently, the magnetic adsorption of MBs enriched numerous HRP on its surface. Finally, HRP catalyzed hydrogen peroxide(H2O2) to oxidation 3, 3', 5, 5'-tetramethylbenzidine(TMB) to produce electrochemical signals. By chronoamperometry the sensor can realize sensitively detection of target DNA. It was able to detect T in the concentration range from 0.1to 1 nM with the detection limit as low as 33.4 pM, and showed a good selectivity even against single-base mismatched DNA.Chapter Four: This chapter used ThioflavinT(ThT) to develop an enzyme-free and label-free fluorescence sensor for the amplified detection of target DNA. In this strategy, the target sequence is repeatedly used to trigger continuous hybridization of the three hairpin structures, forming numerous three-way DNA junctions, leading to amplified fluorescence after ThT promoted the formation of G-quadruplex at each end of the three arms of the three-way DNA junctions. The proposed sensing platform was able to detect the target DNA in the concentration range from 0.5 to 100 fM with the detection limit as low as 0.42 fM. Due to the good discrimination ability of the hairpin structures, three-way DNA junctions and ThT, the biosensor also showed excellent selectivity even against single-base mismatched DNA.