The Research Of Electrochemical Biosensors For POCT Application

In this dissertation,we aim to develop novel highly sensitive and specific detection methods by combining the advantages of electrochemical sensors with DNA nanostructures and inorganic nanomaterials based interface modification and enzyme signal amplification technology. Then, coupling with the multivariate analysis capability of screen print electrodes and integrated device to develop fast, simple, portable and low-cost detection device for Point of care test of biomolecules. Based on the demand of bacterial detection in medical diagnostics and gender identification in forensic analysis, herein, we developed a series of ectrochemical biosensing methods, the details are given as following:Firstly, in order to achieve high sensitive detection and specific phenotyping of bacteria, we designed specific and universal primers for the asymmetric RT-PCR of 16 s rRNA sequence and the probes for capture and detection of PCR product. In this study, the electrode interface was modified with DNA tetrahedron structure probes, which provided a rigid structure and solution-phase-like environment for the improved hybridization efficiency of the capture probe with the target sequence. In addition, The gap probe can not only open the secondary structure of target sequences, but also can improve the base stacking force to improve the hybridization efficiency and stability of the hybrid product, resulting in high sensitivity for DNA detection. By combining the gap-based sandwich strategy with the polyHRP-based signal amplification, the limit of detection(LOD) of synthetic DNA can achieve 100 aM,we also can realize rapid and sensitive detection and identification of bacteria using PCR product derived from 9 types of bacterias. This method enables single bacteria detection, and was not limited by complex samples. Moreover, compared to the real-time quantitative PCR, our method was more specific for the identification of bacteria. The only drawback is that this method required PCR, which increased the operating procedure and result in the specifictity for bacteria identification was not excellent.Secondly, in order to achieve fast, simple and more specific detection and identification of bacteria. We designed the specific and unversal probes according to the conserved and variable region sequence of high abundance 16 srRNA for the detection and phenotype of bacteria based on the PCR-free bactarial lysate. Combining the efficient lysis of bacterial and the DNA tetrahedron structure probes modified interface with the gap-based sandwich strategies and enzyme-signal amplification method, we can achieve sensitive and specific detection of bacterial lysates. By the introducing of gap probe, the probe can hybridized with the 16 srRNA to form a complete double-stranded structure, which can effectively prevent the degradation of 16 srRNA and improve the stability of the detection signal. Compared to the PCR-based detection, this proposed method can not only achieved single bacteria detection from 100 ul whole blood samples, but also it had a good specificity for bacteria idenification. In addition, by using gold nanopatical modified screen printing electrode and combining the advantages of DNA tetrahedron probe with the screen printing electrode array, the limit of detection of bacteira can reach up to 1-100 CFU with excellent accuracy. Based on the electrode array, simple operation, we can realize simultaneous detection and identification of bacteria in short time(2h). More over, due to the low cost, ease of integration and miniaturization of the screen printed electrodes, it is expected to combine microfluidics with electrochemical biosensor to integrate sample preparation and detection procedure in one device for POCT application.Finally, with response to the lack of portable equipment for on-site forensic analysis of biological sample, we have developed a smart phone integrated electrochemical sensor device and a method for gender verification based on enzyme cascade detection. The interface of SPCE chip was modificated with carbon nanotubes and functionalized with polyMB to achieve superior performance for the highly sensitive and rapid detection of enzyme levels in serum. By combination of the CK and ALT enzyme cascade reaction,we can improve the distinction between male group and female group. Compared to the optical method which is susceptible to interference color of the biofluid, this proposed method had a good stability in serum and serum stain. The testing can be completed within 20 minutes, and the portability of the smart phone interfaced electrochemical device was very suitable for POCT application. And there was large potential to expand the device for on-site or remote application in clinical diagnosis, public health, food safety and environmental monitoring field.