Construction And Application Of Electrochemical Biosensor Based On Silver Nanoparticles

A biosensor is generally defined as an analytical device which converts a biological response into a quantifiable and processable signal by using biological element (e.g. nucleic acids, tissue, microorganisms, enzymes, antibodies, antigen etc.) as bioreceptors. As an important branch of biological sensors, electrochemical biosensor combines the electrochemical method with the biological sensing technology, which has the advantages of both electrochemical and sensor technology. Up to now, electrochemical biosensors have attracted considerable attentions as a new kind of biosensors for clinical diagnosis, food safety, drug analysis, microbial and environmental testing owing to their distinctive merits such as rapidity, high sensitivity, wide detection range, convenient operation and simplified optical setup. With the development of nanoscience and nanotechnology, nanomaterials have been used widely in electrochemical biosensors. Taking advantage of excellent structure, catalytic and electrochemical characteristics of nanomaterials and the introduction of nanomaterials with different composition (metal, metal oxides, carbon materials, etc.) and morphology (nanoparticles, nanowires, nanotubes and nano microsphere, etc.) for the construction of sensing interface, can greatly improve the analytical performance of electrochemical biosensor. Therefore, finding suitable nanomaterials to fabricate desired electrochemical biosensor with excellent analytical performance has become a hotspot in biosensing field and one of the main goals of analysts.By using Ag nanoparticles (AgNPs) with good electrochemical activity as signal molecules, nanomaterials and biological reactions for signal amplification, several electrochemical biosensors have been developed and successfully applied to the detection of DNA, thrombin and Escherichia coli with satisfactory results. This dissertation is divided into five parts, a total of six chapters. The specific content is as follow:1. A new amplification strategy based on the signal of DNA probe modified Ag nanoparticle aggregates through biotin-streptavidin (B-SA) interaction was developed to determine the specific DNA squence of Bacillus thuringiensis (Bt). The novel tag was detected through the characteristic of solid-state Ag/AgCl redox process, which was subsequently used to quantify the amount of Bt transgenic sequence. A detection limit of 10 fM was achieved with this new DNA biosensor. The new electrochemical DNA biosensor not only exhibited excellent sensitivity, but also showed good selectivity. We believe that the preliminary work will promote further study on electrochemical DNA biosensor of transgenic sequence and impact actively on the applications of electrochemical DNA biosensor.2. Monodisperse AgNPs with uniform particle sizes were successfully prepared via reduction by using sodium citrate and NaBH4 as co-reductants. Based on the hybridization chain reaction (HCR) and B-SA for double-assisted signal amplification, a high sensitive and universal DNA electrochemical sensor was developed, which can be used to detection the concentration of specific DNA at fM level. The developed sensor possessed not only high sensitivity, but also good selectivity, which can readily distinguish completely complementary DNA sequences and one-base mismatched DNA sequences. Furthermore, this biosensor was successfully used to monitor the level of Bt transgenic sequence. It is worth mentioning that if the part of initiator hairpin DNA was replaced by complementary DNA sequences of target DNA, the biosensor could be used to detect all of the DNA sequence. In particular, our sensing strategy would open new avenues on the design of HCR-based DNA biosensors.3. Based on the aptamer and silver enhancement technology, a rapid and highly sensitive electrochemical biosensor for thrombin detection was developed. When the solid-state Ag/AgCl process was used for determination, the current signal could be greatly enhanced 252-fold by the increased mounts of AgNPs through silver enhancement compared to the unamplified method. By coupling with the good selectivity of aptamer and the high sensitivity of solid-state Ag/AgCl process, the present biosensor showed an excellent analytical performance such as high sensitivity, good selectivity, stability and reproducibility. The deficiency is to use the silver enhancement technique can lead to a stronger background current, so the background current should be deducted in the process of analysis.4. Taking into consideration the advantages of the promoting electron transfer rate of P-GE-Au and the DNA induced Ag aggregation, an electrochemical biosensor with amplification techniques for rapid, sensitive and selective determination of biomarker. Moreover, this biosensor displayed an excellent analytical performance by combining with the advantages of the specificity of aptamer and sensitivity of solid-state Ag/AgCl voltammetry.5. With the signal amplification of the prompting electron transfer rate of AuNPs and the increasing AgNPs loading of P-GO-Ag-Ab probes, a new electrochemical immunoassay for E. coli has been proposed. In this protocol, the solid-state voltammetric technique was first introduced to the detection of bacteria. Integrating the advantages of sensitive solid-state voltammetric detection, specific immunoreaction with nanoparticle amplification technique, the constructed immunosensor showed a good analytical performance for E. coli. Real samples have been performed and the results obtained are in good agreement with the plate counting methods. Based on these results, it is demonstrated that the developed immunoassay could be used for the detection of E. coli in water samples. We anticipated that our sensing strategy would be extended for determination of other bacterium and open a new avenue on the design of electrochemical sensors.