Study On Novel Electrochemical Biosensors And Their Applications In Biological Analysis

A biosensor is described as a detection device that incorporates a) a living organism or product derived from living systems (e.g., an enzyme or an antibody) and b) a transducer to provide an indication, signal, or other form of recognition of the presence of a specific substance in the environment. The concept of biosensor was originated from the paper by Clark and Lyons in 1962, in which they reported the first glucose biosensor. So far, biosensors have developed to be a frontier and newly-interdisciplinary covering chemistry, biology, medical science and electronics. Due to its good selectivity, high sensitivity, fast response time and miniature size, biosensors have attracted immense interest and thus played a significant analytical role in clinical diagnosis, environmental analysis, drug analysis, food safety inspection and other fields.Nanomaterials, with at least one of their dimensions ranging in scale from 1 to 100 nm, display unique properties such as large surface area, catalytic properties, biological compatibility and so on. In recent decades, the rapid growth of nanomaterials has brought a great momentum to biosensing technology and opened new horizons for highly sensitive detection.The main work of this dissertation is focused on the construction of electrochemical biosensors with ultra-high sensitivity, high selectivity and stability via the study on both the biorecognition molecule and the transducer. Some novel electrochemical biosensors were developed by using different kinds of materials as supporting matrix. Scanning electronic microscopy (SEM) and transmission electron microscopy (TEM) were used for characterization of the modified biosensors. The electrochemical behaviors of the biosensors were characterized by electrochemical impedance spectroscopy, cyclic voltammetry and differential pulse voltammetry. The designed biosensors were successfully applied to determine the Escherichia coli in water, acetylcholinesterase inhibition and glycan expression on living cancer cells. The biosensing technology and nanotechnology have been well combined in our work to provide new methods for the biological analysis. The primary research work is as follows:Chapter 1. OverviewIn this chapter, a detailed outlines and reviews with regard to the development of biosensor is given, which introduced the concepts, fundamental principles and applications of the biosensors. According to the difference of transducers, biosensors can be classified into several types and the characteristics of electrochemical biosensor were exploited. Then, the development and application of nanotechnology in biosensors was highlighted. We also reviewed briefly the current development of rapid detection methods of E.coli and introduced neurodegenerative disorders. Finally, we emphatically pointed out the purpose and significance of the dissertation, its innovation spot and content as well.Chapter 2. Optimized Ferrocene-Functionalized ZnO Nanorods for Signal Amplification in Electrochemical Immunoassay of Escherichia coliA novel strategy using ferrocene (Fc)-functionalized ZnO nanorods (NRs) for the amplified electrochemical immunosensor was developed in the present work. The detection antibody (dAb) and Fc were immobilized onto the surface of ZnO NRs, denoted as{dAb-ZnO-Fc} bioconjugates. Highlight of this work was to investigate the amount of dAb and Fc in the bioconjugates using the copper reduction/bicinchoninic acid reaction (BCA protein assay) and inductive coupled plasma-atomic emission spectroscopy (ICP-AES), respectively. Greatly amplified signal was achieved in the sandwich-type immunosensor when the dAb and Fc linked to ZnO NRs at a proper ratio. Using Escherichia coli (E. coli) as a model antigen, the designed immunoassay showed an excellent analytical performance, and exhibited a wide dynamic response range of E. coli concentration from 102 to 106 cfu/mL with a detection limit of 50 cfu/mL (S/N=3). By introducing a pre-enrichment step, the detection of 5 cfu/10mL E. coli in hospital sewage water was realized. This proposed signal amplification strategy was promising and could be easily extended to monitor other biorecognition events.Chapter 3. Study on Acetylcholinesterase Inhibition Induced by Endogenous Neurotoxin Based on a Novel Three-Dimensional Ordered Macroporous (3DOM) Composite Electrochemical BiosensorAcetylcholinesterase (AChE) is a well-known'serine hydrolase, and the dysfunction of AChE could disturb cholinergic neurotransmission and thus participate in the pathogenesis of neurodegenerative disorders, such as in Parkinson's disease (PD). In this chapter, a novel AChE biosensor was developed for the assay of AChE inhibition induced by endogenous neurotoxin. The biosensor, based on three-dimensional ordered macroporous (3DOM) composite, was firstly fabricated by electropolymerization of aniline in the presence of ionic liquid (IL) on a sacrificial silica nanospheres template to form IL-doped polyaniline (IL-PANI) film. After that, AuNPs were decorated on the IL-PANI film, which have been demonstrated to accelerate the electron transfer for a good sensitivity. The synthesized AuNPs/IL-PANI composite presented a 3D porous and homogeneous morphology to entrap enzyme molecules, and thus offered an effective matrix for the immobilization of AChE with high stability and bioactivity. The immobilized AChE showed favorable affinity to substrate acetylthiocholine chloride (ATCh) and excellent catalytic effect on the hydrolysis of ATCh to produce thiocholine (Th), which can be detected electrochemically. The designed AChE biosensor was successfully applied to evaluate the AChE inhibition induced by endogenous neurotoxin 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)-NMSal], which has been shown to contribute to the onset and progression of PD via disrupting the balance between dopamine and acetylcholine result from the inhibition of AChE. The results demonstrate that (R)-NMSal exerted a considerable effect on the AChE activity, and the inhibition is reversible. The developed assay method offered a new approach for determination of AChE activity, which is of great benefit to understand the mechanism behind neurotoxin-induced PD.Chapter 4. Lectin-Based Biosensor Strategy for Electrochemical Assay of Glycan Expression on Living Cancer CellsIn this chapter, we report a novel lectin-based biosensor for electrochemical assay of cancer-associated glycosylation by comparative study of mannose and sialic acid expression on normal and cancer cells derived from human lung, liver and prostate. Using a sandwich format, high sensitivity and selectivity were achieved by combining the lectin-based biosensor with the{lectin-Au-Th} bioconjugates featuring lectin and thionine (Th) labels linked to gold nanoparticles (AuNPs) for signal amplification. The proposed strategy demonstrated that mannose exhibited high expression levels in both normal and cancer cells, while sialic acid was more abundant in cancer cells compared to normal ones. The results were in good agreement with those from Fluorescent Microscopy studies. The differences in the two glycan expression indicated that sialic acid could serve as a potential biomarker for early cancer detection. The lectin-based biosensor was also successfully used to quantify cancer cells and evaluate the average amount of sialic acid on single cell surface, which could supply significant information on glycan functions in cancer progression. Overall, the lectin-based electrochemical biosensor provides an effective pathway to analyze glycan expression on living cells, and may greatly facilitate the medical diagnosis and treatment in early process of cancer.