Electrochemical Detection Of Small Molecules And Analysis Of Enzyme Activity Based On Molecular Probe And Gold Nanoparticles

Biosensors are important topic in the analytical chemistry and biochemistry area, and have a huge application prospect in the medical treatment, medicine, biological engineering, environmental protection, food industry, and so on. Electrochemical biosensors, based on the specific recognition of biomaterials and electrochemical signal, is one of the most important biosensors and present some outstanding advantages including high sensitivity, nice selectivity, low cost, and easy to be miniaturized. In addition, nanomaterials have won wide attention from scientific researchers because of its small size, large specific surface area, and nice biocompatible properties. Research shows that the application of nanomaterials could greatly improve the performance of the biosensors. In our work, we aim to develop new types of electrochemical biosensors by means of the combination of biochemistry, nanotechnology and electrochemical methods for highly sensitive detection of the small biomolecules and enzyme. The details are summarized as follows:1. Electrochemical detection of nicotinamide adenine dinucleotide based on molecular beacon-like DNA and E.coli DNA ligaseAn electrochemical method for NAD+ detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and E. Coli DNA ligase. In this method, MB-like DNA labeled with 5’-SH and 3’-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD+, E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD+-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchanged freely with the electrode, then the generated electrochemical signals can be measured (eT ON). Under optimized conditions, a linear response to NAD+ range from 3 nM to 5μM and a detection limit of 1.8 nM were obtained. And the proposed strategy had sufficient selectivity to discriminate NAD+ from its analogues.2. Electrochemical detection of telomerase activity based on gold nanoparticles amplificationIn this study, we have developed an electrochemical biosensor for highly detection of telomerase. First, based on the telomere can be used as the template for reverse transcription, the template DNA containing the telomerase substrate primer was immobilized on the gold nanoparticles (AuNPs) modified Au electrode. The signal DNA probes labeled with methylene blue can be hybrized with the template DNA and the electrons transimission can be produced between the methylene blue and the surface of the electrode. In the presence of telomerase and dNTPs, telomerase substrate primer performed extension reaction, the expension product was hybridized with the template DNA, then methylene blue-labeled probe was dissociated from the electrode surface, and result in a weak electrochemical signal. This is a signal“On”to“Off”prosess. This method can detect the telomerase activity from 2×102 cultured/ mL Hela cells.3. Electrochemical detection of DNA polymerase KF- activity based on gold nanoparticles amplificationAn electrochemical method for detection of KF- was developed based on the unique advantages of gold nanoparticle (AuNPs). In this method, the primer DNA was immobilized on the Au electrode, the report DNA was tethered with AuNPs and the template DNA could hybridize with both of primer DNA and report DNA. When put the modified electrode into the electrolyte containing the [Ru(NH3)6]3+ (RuHex), the RuHex quantitatively binds to the DNA via electrostatic interaction, and produce electrochemical signals. In the presence of dNTPs and KF-, the extension reaction was triggered, and the report DNA was released. Therefore, the electrochemical signals decreased duing to the release of aboundant RuHex. This fabrication biosensor can detect the KF- as low as 5 U/mL.