Research On The Application Of Electro-analytical Methods For The Rapid Detection Of Escherichia Coli

E. coli is the most common intestinal gram-negative bacterium, which naturally occurs in the intestinal tract of humans and warm-blooded animals. In particular, E. coli is often preferred as an indicator organism because it is specific for and reliably reflects fecal contamination. Some strains E. coli can cause diarrhea, urinary tract infections, inflammations and peritonitis in immunosupressed patients as children and elderly people. In many countries, health departments routinely detect E. coli in water or food because E. coli conveys the potential presence of pathogens. Therefore, a novel, rapid and more practical technique for the detection of E. coli is required for safety and hygiene reasons. The development of new methods for E. coli detection is essential in the fields of food industry, environmental monitoring, clinical diagnostics and so on.Conventional methods for the detection of E. coli are multiple-tube fermentation (MTF), membrane filter (MF), plate count and so on. For example, plate count is one of the most widely used methods. Although it has the advantage of high accuracy, its incubation period is 24 to 48h, and it typically requires long time from initiation to readout when it is used in food and sample matrices. Hence, there have been many attempts to improve the traditional bacteria detection techniques by employing electrochemistry, immunoassay and optical technology.In recent years, nano-materials have played an important role in the field of electro-analytical chemistry because have advantages of big surface area and electrochemical catalytic activity. Immunosensors and DNA electrochemical biosensor also have attracted a considerable attention. We prepared IrO₂-Pd chemically modified electrode as the electro-detector in flow injection analysis. The detection limit of this method to E. coli was 150cfu/mL and the complete assay could be performed in 3h. An electrochemical impedance immunosensor for E. coli detection was developed by immobilizing anti-E. coli antibodies at an Au electrode. With preconcentration and pre-enrichment steps, it was possible to detect E. coli concentration as low as 50 cfu/mL in river water samples. A novel, sensitive electrochemical biosensor based on magnetic beads for E. coli detection by DNA hybridization was also described. The details are listed below:Part 1. Rapid Detection of Escherichia coli by Flow Injection Analysis coupled with Amperometric Method Using an IrO₂-Pd Chemically Modified ElectrodeAn amperometric method for the rapid detection of Escherichia coli (E. coli) by flow injection analysis (FIA) using an IrO₂-Pd chemically modified electrode (CME) was developed in this paper. The method is based on a good markerβ-D-galactosidase which was found in E. coli strains.β-D-galactosidase was produced by the induction of isopropylβ-D-thiogalactopyranoside (IPTG) and released from E. coli cells through the permeabilization of both polymyxin B nonapeptide and lysozyme to E. coli cells wall. The releasedβ-D-galactosidase could catalyze the hydrolysis of the substrate p-aminophenylβ-D-galactopyranoside (PAPG) in the culture medium to produce 4-aminophenol which was proportional to the concentration of E. coli. Hence, E. coli could be detected by the determination of 4-aminophenol. An IrO₂-Pd CME, which showed high sensitivity in determination of 4-aminophenol, was prepared as the electro-detector in FIA. The amplified response current of 4-aminophenol obtained at the IrO₂-Pd CME was linear with the concentration of E. coli ranging from 2.0×10² to 1.0×10⁶cfu/mL, the detection limit of this method to E. coli was 150cfu/mL and the complete assay could be performed in 3h.Part 2. Self-Assembled Monolayers-Based Immunosensor for Detection of Escherichia coli Using Electrochemical Impedance SpectroscopyAn electrochemical impedance immunosensor for the detection of Escherichia coli (E. coli) was developed by immobilizing anti-E. coli antibodies at an Au electrode. The immobilization of antibodies at the Au electrode was carried out through a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies reproducibly and densely on the self-assembled monolayer (SAM). The surface characteristics of the immunosensor before and after the binding reaction of antibodies with E. coli were characterized by atomic force microscopy (AFM). The immobilization of antibodies and the binding of E. coli cells to the electrode could increase the electro-transfer resistance, which was directly detected by electrochemical impedance spectroscopy (EIS) in the presence of Fe(CN)₆^3-/ Fe(CN)₆^4- as a redox probe. A linear relationship between the electron-transfer resistance and the logarithmic value of E. coli concentration was found in the range of E. coli cells from 3.0×10³ to 3.0×10⁷cfu/mL with the detection limit of 1.0×10³ cfu/mL. With preconcentration and pre-enrichment steps, it was possible to detect E. coli concentration as low as 50 cfu/mL in river water samples.Part 3. A supersensitive DNA electrochemical biosensor based on magnetic beads for E. coli detection by DNA hybridizationA novel, sensitive electrochemical biosensor based on magnetic bead for E. coli detection by DNA hybridization is described. This Escherichia coli-specific assay was based on the uid A gene, which encodes for the enzymeβ-D-glucuronidase mainly produced by E. coli strains. Alginic acid-coated magnetite cobalt nanoparticles, capped with 5'-(NH₂) oligonucleotide, are used as DNA probe to hybridized with target E. coli DNA sequence. Differential pulse voltammetric technique was utilized for detection in the presence of daunomycin as a DNA hybridization indicator. Optimization was carried out and specificity was also tested. This DNA biosensor can be employed to detect a complementary target probe (3×10¹⁰mol/L), denatured PCR products (0.5ng/uL) and E. coli cells (50cells/mL).