Study On Electrochemical Impedance Bacteria Biosensor With Gold Nanoparticle Signal Amplification

Bacteria exist in our lives everywhere, but pathogens affect human life and health seriously. In recent years, the diseases and losses caused by foodborne pathogens are continuous increasing all over the world. EHEC O157:H7 is one of the most serious foodborne pathogens, which can cause hemorrhagic colitis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, other diseases and even death. Therefore, biosecurity has become an increasingly important element in the world.Generally, the increasing demand for detection of ultralow amount of anlytes is pushing the enhancement of detection sensitivity by selecting different signal amplification strategies. Nanoparticles, which have good physical and chemical properties, have been widely applied for amplification in biosensor. Aiming for the detection of Escherichia coli O157:H7 (E. coli O157:H7), many strategies have been proposed. Electrochemical impedance spectroscopy (EIS) is considered as one of the most appealing techniques in terms of rapid, high sensitivity and low consumption. Therefore, developing a sensitive and rapid assay for bacteria analysis is urgently needed. This paper focuses on construction of high sensitive immunosensor for detection of E. coli O157:H7 using electrochemical impedance spectroscopy and differential pulse voltammetry.This paper consists of the following four chapters:Chapter I:IntroductionFirstly, we introduce a brief overview of pathogens and describe the biological characteristics and pathogenicity of EHEC O157:H7. Then the detection methods of pathogens including traditional detection methods and some new detection methods were introduced. And an overview of electrochemical biosensors is summarized, which focuses on electrochemical impedance biosensors for the detection of bacteria. Also nanomaterials are widely applied in electrochemical impedance bacterial biosensors. Finally, the background, purpose and contents of this paper are described.Chapter Ⅱ:The research of bonding degree between E. coli O157:H7 and the different functions of gold nanoparticlesThe situation about E. coli O157:H7 combined with different functions of the gold nanoparticles (AuNPs) are studied in this chapter. Firstly,4-mercaptobenzoxazole acid (MPBA) functionalized AuNPs under different conditions and concanavalin A (Con A) functionalized AuNPs are prepared. Then, MPBA functionalized AuNPs, Con A functionalized AuNPs and unmodified bare AuNPs are respectively mixed with E. coli O157:H7 in accordance with certain proportions. Finally, the results prove that the capacity of unmodified bare AuNPs binding with E. coli O157:H7 is strongest by TEM chart comparison assay.Chapter III:Highly sensitive and signal-off detection of E. coli O157:H7 using impedimetric immunosensor based on strong interactions between bacteria and naked gold nanoparticlesIn this study, a signal-off impedimetric immune-biosensor based on naked gold nanoparticle (AuNP)-mediated electron transfer (ET) across a self-assembled monolayer (SAM) was developed for highly sensitive detection of E. coli O157:H7 bacteria. The gold electrode was functionalized with-COOH by a mixed self-assembly of 11-mercaptoundecanoic acid (MUA) and 6-Mercapto-l-Hexan (MH). And then, the biosensor was fabricated through covalently grafting the SAM modified gold electrode with specific antibody. A great background impedance of the biosensor was obtained because of the modification of the electrode with SAM and immobilized antibody. After bacteria capture using the immobilized antibody, the biosensor was immersed into AuNP solution. As a result, AuNPs were attached to the surface of the bacteria based on the strong interaction between naked AuNP and bacteria. AuNPs attached on the bacteria surface mediated ET across an SAM, decreasing the electron transfer resistance (Ret) of the biosensor using the [Fe(CN)6]3-/4- redox probe. This consequent decrease in Ret can be easily monitored using electrochemical impedance spectroscopy. Based on AuNP-mediated ET, the biosensor shows a decrease in Ret, and a linear relationship was found in the range 300 to 1×105 cfu/mL. Furthermore, the biosensor showed differentiation abilities for other bacteria.Chapter FV:Immunosensor for the detection of Escherichia coli O157:H7 using electrochemical method based on AuNPs signal amplificationIn this study, a cheap and high sensitive amperometric immunosensor has been developed for the detection of E. coli O157:H7. This biosensor was prepared as follows. Firstly, polydopamine film was formed on self-made and disposable screen-printed carbon electrode (SPCE) surface by a method of electrochemical polymerization. Next, E. coli O157:H7 antibody (anti-E. coli O157:H7) was covalently bound to the PDA on SPCE surface by Michael addition reaction and the Schiff base reaction between quinonyl of polydopamine film surface and amino group of antibody surface. Then, E. coli O157:H7 antibody can recognize and capture E. coli O157:H7. Finally, the above electrode was immersed in the solution of AuNPs which could be adsorbed by E. coli O157:H7. AuNPs stripping signal was measured by the differential pulse voltammetry (DPV), which could be used for quantitative detection of E. coli O157:H7 according to the linear relationship between the peak current and concentration of E. coli O157:H7 suspension. This sensor could successfully detect E. coli O157:H7 with high specificity.