Electrochemical Biosensoring Technique Based On Gold Nanoparticles Modified Electrode

Electrochemical biosensor analysis has the following characteristics, simplicity, specific recognition and requiring small amount of sample, thus offering advantages over commonly used assays. There's great significant to establish a rapid screening method based on biosensor for medicine study and environmental pollution monitoring. Nanomateriales have unique physical and chemical superiority, like enhancing electrons transferring, keeping biological activity of biomoleculars, activating the electrode surface and so on. With the development of nanotechnology, it's a promising study field to apply nanomaterials for electrochemical biosensor device.In this article, three novel electrochemical biosensors were fabricated based on Au nanoparticles modified electrode. The main contents are listed as the following:(1) An electrochemical biosensor was developed to directly detect and screen estrogenic substances through estrogen receptor binding based on lipid bilayers modified by Au nanoparticles. The biosensor is a rapid and simple method to detect estrogen, and is able to detect the EEQ of estrogenic compounds in environmental sample.(2) An immunosensor for detection Mycobacterium tuberculosis was fabricated based on the nanosturcture film formed by chitosan, Au nanoparticles and ALP-IgG on the electrode surface.(3) A DNA-Au nanoparticles composite electrochemical biosensor was developed for rapid bio-analysis of compounds with genetoxic effect without microbe or cell culture. The procedures for preparation of biosensor are straightforward, fast and cost effective, and the biosensor is able to detect compounds with genetoxic, which could be useful as early warning tool for environment risk assessment.Part I:Electrochemical biosensor based on lipid bilayers modified by Au nanoparticles for detection of estrogenic substancesObjection:There is a growing demand for new technologies that are capable of screening the wide variety of xenoestrogens in environment. The aim of this part is to develop a nanostructure electrochemical biosensor for directly detection estrogenic substances based on estrogen receptor (ER) binding without the use of radio- or enzyme-labeled compounds. Method:In the present work,17β-estradiol was used as the standard solution. Firstly, the biosensor was fabricated by immobilization bilayer lipid membrane (s-BLM) on the pt disk electrode, followed by electrochemically deposit Au nanoparticle through s-BLM to form Au/s-BLM/Pt. Then, ER was immobilized into the membrane of s-BLM modified with Au nanoparticles to fabricate the electrode ER/Au/s-BLM/Pt for detection estrogen. After 17β-estradiol binding to ER/Au/s-BLM/Pt, the change of electron-transfer resistance of the electrode would be proportional to the quantity of 17β-estradiol. Therefore, the dose-response relationship curve about the 17β-estradiol concentration against the change of electron-transfer resistance was obtained. The electrochemical characteristics of the modified electrode during the self-assembled process were characterized by using cyclic voltammetry and electrochemical impedance spectroscopy. This method was also applied for detection of other known xenoestrogen. Moreover, the EEQs estrogen compounds in the water samples (from Yangtze River and Han River in Wuhan) were analyzed by the ER/Au/s-BLM/Pt biosensor and MCF-7 cell proliferation assay. Results:(Ⅰ) Cyclic voltammetric behavior and electrochemical impedance spectroscopy of the modified electrode implied that the modification process was successful. (Ⅱ) 0.3 nM was chosen as the appropriate concentration of ER for the preparation of the biosensor and the optimal incubation time for 17β-estradiol was 20 mins. (Ⅲ) The biosensor was able to detect the natural estrogen 17(3-estradiol with a linear correlation ranging from 5 to 150 ng/L with the correlation parameter r=0.996, and the detection limit was 1 ng/L. (Ⅳ) This method was also applied to detect other known xenoestrogen, such as bisphenol A and 4-nonylphenol, and satisfied sensitivity and quantitative results were obtained. (Ⅴ) Compared with the electrode of ER/s-BLM/Pt without Au nanoparticles modification, the sensitivity of ER/Au/s-BLM/Pt for 17β-estradiol was higher. And the repeatability, selectivity and stability of the biosensor were also studied. (Ⅵ) The results of samples obtained from ER/Au/s-BLM/Pt biosensor were consistent with those from MCF-7 cell proliferation assay. Conclusion:The biosensor is a rapid and simple method to detect estrogen, and is able to detect the EEQ of estrogenic compounds in environmental sample, which can be performed in common laboratories. And Au nanoparticles enhanced the sensitivity and stability of the biosensor.PartⅡ:Rapid detection of Mycobacterium bovis by nanobiosensorObjection:Mycobacterium bovis (M. bovis) infection of cattle is causing considerable economic losses worldwide, and posing treats to human health. Accurate and rapid identification methods can positively enhance outbreak investigation and aid infection control. In this study, the objection is to develop an amperometric immunosensor for detection of M. bovis in milk. Method:The immunosensor was fabricated firstly by dropping the mix solution of chitosan (Ch), Au nanoparticles (AuNPs) and alkaline phosphatase labeled goat anti-mouse IgG (ALP-IgG) on glassy carbon electrode (GCE) to form a robust film with good biocompatibility, then monoclonal mouse antibody against Mycobacterium tuberculosis (anti-M.TB), which could identify M. bovis, was immobilized on the composite film through the reaction with ALP-IgG to construct an anti-M.TB/ALP-IgG/AuNPs/Ch/GCE immunosensor. The ALP-IgG on the modified GCE led to an amperometric response of the substrate 1-naphthyl phosphate. After the immunosensor was incubated with M. bovis, the access of electro-catalytic behavior center of the immunosensor to 1-naphthyl phosphate was partly inhibited, which was reflected by diminishing amperometric response as M. bovis concentration increased gradually, under differential pulse voltammetry (DPV). The electrochemical characteristics of the modified electrode during the self-assembled process were characterized by using cyclic voltammetry and electrochemical impedance spectroscopy. Moreover, this method was applied for detection milk samples. Results:(Ⅰ) Cyclic voltammetric behavior and electrochemical impedance spectroscopy of the modified electrode implied that the modification process was successful. (Ⅱ) The optimal incubation time of 30 mins for M. bovis was determined, and pH=7.8 was chosen as the appropriate pH for testing buffer. (Ⅲ) The detection limit of the immunosensor for M. bovis dissolved in PBS solution was 0.5×103 cfu/mL (S/N=3) and with a linear range 104～106 cfu/mL. (Ⅳ) The repeatability, selectivity and stability of the biosensor were also studied. (Ⅴ) This method was also applied to milk samples spiked with M. bovis, the results were consistent with PBS samples. Conclusion:The immunosensor is a rapid and simple method to detect M. bovis. When it's applied for milk, good results are also obtained.PartⅢ:DNA-Au nanoparticle composite biosensor as a tool for detection of toxicants in waterObjection:It's very important to establish a rapid screening method for environmental pollution. The aim of this part is to develop a DNA-Au nanoparticle composite electrochemical biosensor for rapid bio-analysis of compounds with genetoxic effect without microbe or cell culture. Method:In this study, 2-aminoanthracene was used as the standard solution. Firstly, Au nanoparticles were prepared by chloroauric acid through being reduced by oleylamine. Au nanoparticles were characterized by UV-Vis and transmission electron microscope (TEM). Then, the biosensor was fabricated by using the self-assembly of Au nanoparticles on the electrode surface, followed by directly adsorption of DNA onto the Au nanoparticles. The samples interaction with DNA has been measured through the variation of the electrochemical signal of guanine by square wave voltammetric scans. After 2-aminoanthracene interaction with DNA on the electorde, the decreas percentage of guanine peak height would be proportional to the quantity of 2-aminoanthracene. This method was also applied for detection pollutants in the water samples, and the resultes were compared with single cell gel eletrophoresis (SCGE) assay. Results:(Ⅰ) UV-Vis results and TEM image of Au nanoparticles implied that Au nanoparticles were stable and distributing uniformly. (Ⅱ) The biosensor was able to detect 2-aminoanthracene with a linear correlation ranging from 50 to 1.25×103 nM with the correlation parameter r=0.966, and the detection limit was 10 nM. (Ⅲ) This method was also applied for environmental water samples. The results obtained from DNA-Au nanoparticle were consistent with those from SCGE assay. Conclusion:The procedures for preparation of DNA-Au nanoparticle composite biosensor are straightforward, fast and cost effective. And the biosensor is able detect compounds with genetoxic in environment water samples, which could be useful as an early warning tool for environment risk assessment.