Electrochemical DNA Sensor Based On In-situ Labeling Of Electroactive Molecular On The Molecular Beacon

Compared with the traditional linear probe-based DNA biosensor, the molecular beacons (MBs)-based hybridization biosensing technology shows the advantages of stronger specificity, higher selectivity, more simple operability and more comprehensive application. However, there are still some disadvantages for this technology, for instance, the labeling and separating processes for the electroactive MBs probe are often complicated, the type of the signal molecule labeled on MBs probe is few and signal intensity in the classical MBs-based biosensor is limited. For improveing the above lack, the main idea of the research was to construct the electrochemical biosensor for detecting of DNA based on a novel approach to in-situ labele electroactive signal molecule on the terminal of MBs, which immobilized on the electrode. The main contents are as follows:(1) Melamine (Mel) was grafted on the free terminal of carboxyl group modified MBs as a multi-amino platform, which is used to coupled with2,6-pyridinedicarboxylic acid (PCA), then make use of Cu2+coordinate with PCA yielding copper complex as an electroactive tag. Therefore, a novel electrochemical DNA sensor based on in-situ labeling of PCA-Cu2+was constructed. The layer-by-layer self-assembly process was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Meanwhile, the condition of assembly and analysis detection was optimized. The hybridization experiments shown that the proposed sensor exhibited good specificity for target DNA in the kinetic range from1×10-14～1×10-7mol L-1with a detection limit of1.4×10-15mol L-1. In addition, the selectivity experiment also showed that the biosensor can well distinguish the target DNA from the non-complementary sequences.(2) AuNPs self-assembled by Au-NH on the NH2-terminal of MBs probe which had immobilized on electrode surface. Then Mel and Cu2+were, in turn, grafted on AuNPs through Au-NH assembly and coordination reaction, respectively, yielding an electroactive MB probe with AuNPs amplification platform and Mel-Cu2+complex as the signal source. The selectivity experiment also showed that the biosensor can well distinguish the target DNA from the non-complementary sequences and exhibited good specificity for target DNA in the kinetic range from1.0×10-18～1.0×10-12mol L-1with a detection limit of1.2×10-19mol L-1. Meanwhile, the developed biosensor showed good reproducibility and robustness due to the stable inorganic composition of Mel-Cu2+complex.(3) Construction of a high signal-to-noise ratio electrochemical impedance-based DNA sensor employing AuNPs tag. The developed sensor was fabricated via using carbodiimide crosslinking agent (EDC/sulfo-NHS) to activated-COOH group of thioglycolic acid (TGA), which was conjugated to3’-end of an amino-modified MBs on the electrode surface through carboxylic-ammonia covalent coupling. Then the modified electrode incubated into freshly prepared AuNPs and AuNPs tag was labeled on the free terminal of MBs through self-assembling with the-SH of TGA. EIS was used to characterize the property of the biosensor, the hybridization specificity experiments showed that the sensing system can accurately discriminate complementary sequence from mismatch and noncomplementary sequences. A wide linear range of1.0×10-17～1.0×10-11mol L-1for detecting target DNA, with a low detection limit of1.7×10-18mol·L-1. The proposed sensor was exhibited good reproducibility and robustness.