Study On Novel Electrochemical Biosensors Based On Nanomaterials And Their Applications In Biological Analysis

Electrochemical biosensors, combining informatics and bio techno to gy, serve as an interdisciplinary frontier related to chemistry, biology, physics and electronics. Owing to their high sensitivity and selectivity, fast responses and advantages in miniaturization and online detection potentials, electrochemical biosensors have been extensively studied and applied in clinical medicine, food inspection and environment protection. Recently, nanomaterials, due to their unique properties, are widely used for developing electrochemical biosensors. The application of nanomaterials has brought a great momentum to electrochemical biosensors and opens new horizons for highly sensitive detection, which provides an avenue for high-throughput analysis of biological components on living cells.In this dissertation, we studied on the development of the novel electrochemical biosensors based on nanomaterials, and their applications in the analysis of E. coli in water and glycans on living cells. The details are listed below:Part 1. Development of an Electrochemical Immunoassay for Rapid Detection of E. coli Using Anodic Stripping Voltammetry Based on Cu@Au Nanoparticles as Antibody LabelsA sensitive electrochemical immunoassay for rapid detection of E. coli has been developed by Anodic Stripping Voltammetry (ASV) based on Cu@Au nanoparticles (NPs) as anti-E. coli antibody labels. The characteristics of Cu@Au NPs before and after binding with antibody were confirmed by transmission electron microscopy (TEM). After Cu@Au-labeled antibody reacted with the immobilized E. coli on PS-modified ITO chip, Cu@Au NPs were dissolved by oxidation to the metal ionic forms, and the released Cu2+ were determined at Nafion/Hg-modified glassy carbon electrode (GCE/Nafion/Hg) by ASV. The utilization of GCE/Nafion/Hg could enhance the sensitivity for Cu2+ detection with a concentration as low as 9.0×10-12 M. Since Cu@Au NPs labels were only present when antibody reacted with E. coli, the amount of Cu2+ directly reflected the number of E. coli. The technique could detect E. coli with a detection limit of 30 cfu/mL and the overall analysis could be completed in 2 h. By introducing a pre-enrichment step, a concentration of 3 cfu/10 mL E. coli in surface water was detected by the electrochemical immunoassay.Part 2. A Supersensitive DNA Electrochemical Biosensor Based on Magnetic Beads for E. coli Detection by DNA HybridizationA new type of DNA sequence-specific electrochemical biosensor based on magnetic beads for the detection of E. coli is reported in the present work. Alginic acid-coated cobalt magnetic beads, capped with 5'-(NH2) oligonucleotide and employed not only for magnetic separation but also as the solid adsorbent, were used as DNA probes to hybridize with the target E. coli DNA sequence. This assay was specific for E. coli detection depending on the uid A gene, which encodes for the enzymeβ-D-glucuronidase produced by E. coli strains. When daunomycin (DNR) was used as DNA hybridization indicator, the target sequences of E. coli hybridized with the probes resulted in the decrease of DNR reduction peak current, which was proportional to the E. coli concentration. The optimization of the hybridization detection was carried out and the specificity of the probes was also demonstrated. This DNA biosensor can be employed to detect a complementary target sequence for 0.30 nM and denatured PCR products for 0.50 ng/μL. The linear range of the developed biosensor for the detection of E. coli cells was from 1.0×102 to 2.0×103 cfu/mL with a detection limit of 50 cfu/mL.Part 3. Optimized Ferrocene-Functionalized ZnO Nanorods for Signal Amplification in Electrochemical Immunoassay of E. coliA novel strategy using ferrocene (Fc)-functionalized ZnO nanorods (NRs) for the amplified electrochemical immunoassay 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. The amount of dAb and Fc in the bioconjugates was investigated 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 immunoassay when the dAb and Fc linked to ZnO NRs at a proper ratio. Using 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 1.0×102 to 1.0×106 cfu/mL with a detection limit of 50 cfu/mL (S/N=3). By introducing a pre-enrichment step, the detection of 5 cfu/10 mL E. coli in hospital sewage water was realized.Part 4. Carbohydrate Derivative-Functionalized Electrochemical Biosensor for Competitive Assay of Glycan Expression on Living Cancer CellsA novel carbohydrate derivative-functionalized electrochemical biosensor was developed for competitive analysis of glycan expression on living cancer cells. Mannose present on cancer cells derived from human lung, liver, and prostate was used as a model glycan. The biosensor was designed by employing the multiwalled carbon nanotube/Au nanoparticle (MWNT/AuNP) composite film to modify an electrode surface for thiomannosyl dimer ([mannose-S]2) assembling through Au-S bond. The{Con A-MWNT-HRP} bioconjugates, as recognition elements, were prepared by exploiting amplification effect of MWNTs for loading enormous horseradish peroxidase (HRP) labels and mannose-specific concanavalin A (Con A). Using a rapid and one-step competitive assay, the biosensor surface-confined thiomannosyl dimer competed with cell surface mannose to specifically recognize the {Con A-MWNT-HRP} bioconjugates. The proposed biosensor exhibited attractive performances for the analysis of cancer cells with wide linear ranges and low detection limits. The average amount of mannose on single cell surface was also detected to be 5.8×1010 molecules for A549 (lung cancer),1.3×1010 molecules for QGY-7703 (liver cancer), and 1.9×1010 molecules for LNCaP (prostate cancer). The carbohydrate derivative-functionalized electrochemical biosensor shows high sensitivity, selectivity and rapid response, and possesses promising application in the study of glycan changes on living cancer cells.Part 5. Lectin-Based Biosensor Strategy for Electrochemical Assay of Glycan Expression on Living Cancer CellsWe 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 differences in the two glycan expression indicated that sialic acid could serve as a potential bio marker 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.