Whole-cell Electrochemical Biosensor Based On Bacterial Inward Electron Flow:System Development,Principle And Application

In recent years, biosensors, which are capable simple and fast biochemical analyses, draw extensive applications in industrial production. Based on the adopted recognition element, biosensors are mainly classified into enzyme sensor, tissue sensor, cell-based biosensor or whole-cell sensor. Whole-cell biosensors overmatch the others due to the low cost, simple operation and bioavailability information. However, current whole-cell biosensors usually use fluorescence or enzyme activity as output signal, which limits their application. Therefore, design the whole-cell electrochemical biosensor system is of great significance as it would impressively simplify the procedures for biosensor signal output and processing. Among them, integrating the electroactive bacteria which is capable of multiple extracellular electron transfer pathways into the whole-cell biosensor is the probably successful attempt as these electroactive bacteria meet all the requirements for electrochemical biosensors.In this presented thesis, whole-cell electrochemical biosensing systems were developed based on inward electron flow of Shewanella oneidensis MR-1 in three-electrode system. The influence of electron acceptor, electron shuttle and outer-membrane cytochrome of Shewanella on the inward electron flow efficiency were studied in details. The feasibility for electron acceptor and electron shuttles quantitative determination were investigated by adjusting the rate-limited step in the electron transfer with proper biosensor design. Furthermore, novel bioelectrochemical methods were developed for fumarate, nitrate, nitrite and riboflavin detections. Main research achievements are as follows:1)Low-cost whole-cell electrochemical biosensing systems were successfully developed based on inward electron flow of Shewanella oneidensis MR-1 in three-electrode system. The electroactive bacteria Shewanella cells were served as recognition and transduction element to produce current as output signals. The successful attempts broaden the choice for electrochemical biosensor design.2)Novel bioelectrochemical sensor for fumarate(biomarker for cancer and indicator for spoiled food) detection was developed. The output current exhibits excellent linear relationship(R2=0.9997)with the fumarate concentration increase from 2 mM to 10 m M. The limit of detection(LOD)(S/N=3)was 0.83 mM, while the limit quantification(LOQ)(S/N=10)was 1.2 mM. This biosensor system displayed remarkable selectivity, stability and reproducibility, with successfully application for fumarate detection in real samples. Meanwhile, bioelectrochemical sensors for environment pollutants(nitrate and nitrite) detection were developed as well, and the LOD were 41.5 n M or 40 n M, respectively.3) Bioelectrochemical sensor for riboflavin detection was developed. Considerable low LOD(S/N=3)as 1.3 n M was achieved. Additionaly, it was successfully applied to detect riboflavin in real samples.