Development of electroanalytical methods for studying microbes
Cheng, Ming Soon
Date of Issue2014
School of Physical and Mathematical Sciences
This thesis explores the utility of electrochemical biosensors for studying and detecting microbes. It consists of five chapters: a mini-review and four independent experimental works. Chapter 1 reviews the current state of novel biosensing techniques for ultrasensitive detection of microbes with highly promising application for disease diagnosis. Because of their remarkable specificity, sensitivity and response time, biosensors often present potential and powerful tools for quantitative and accurate detection of microbes. Recent advancements in nanotechnology, transduction system and genetic engineering provide various strategies to improve the detection performance of biosensors. In this chapter, I summarize concise and comprehensive lists of the recent bacteria and virus biosensors, and their analytical performance. In addition, I also include a brief summary of state-of-the-art molecular technologies for the detection of microbes. In Chapter 2, a sensitive and specific electrochemical membrane-based nanobiosensor is reported for quantitative and label-free detection of Escherichia coli cells and analysis of the viable but nonculturable state of E. coli cells that remain mostly undetected using conventional methods. The sensing mechanism depends on the blocking of nanochannels of an alumina-modified platinum wire electrode coated with a layer of anti-E. coli polyclonal antibody (isotype: IgG), upon the formation of immunocomplexes at the nanoporous alumina membrane. The resulting obstacle to diffusive mass transfer of a neutral redox probe, ferrocenemethanol, toward the underlying platinum electrode decreases the Faradaic current response of the nanobiosensor, measured using cyclic voltammetry. Experimental parameters including loading amount of antibody and pH are optimized. The membrane-based nanobiosensor gives a low limit of detection of 22 cfu mL-1 over a wide linear working range from 10 to 106 cfu mL-1 (R2 = 0.999). The nanobiosensor is specific toward E. coli with negligible cross reactivity to two other Gram-negative bacteria, Serratia marcescens and Salmonella typhimurium. Relative standard deviation for triplicate analysis of 2.5 % indicates good reproducibility. Differentiation of live, viable-but-non-culturable and dead E. coli cells are performed by monitoring of the bacterial enzyme catalytic activity using ferrocenemethanol as the alternative electron acceptor to oxygen, in the presence of glucose. In Chapter 3, the same design of membrane-based nanobiosensor is employed for ultrasensitive detection of dengue type 2 virus (DENV-2). Anti-DENV-2 monoclonal antibody (clone 3H5, isotype IgG) is used as the biorecognition probe in this work. The stepwise construction of nanobiosensor and detection of DENV-2 are characterized using differential pulse voltammetry. A low limit of detection of 1 pfu mL-1 with linear working range from 1 to 103 pfu mL-1 (R2 = 0.976) can be achieved by the nanobiosensor. The nanobiosensor is specific toward DENV-2 with minimal cross reaction with other non-specific viruses such as Chikungunya virus, West Nile virus, and dengue type 3 virus. Relative standard deviation for triplicate analysis of 5.9 % reveals reasonably useful level of reproducibility. Additionally, I demonstrate the direct quantification of DENV-2 load in whole mosquito vector, Aedes aegypti using the nanobiosensor. Chapter 4 reports the real-time monitoring of filamentous bacteriophage M13 infection of E. coli cells using an impedimetric microbial biosensor constructed from a gold electrode covalently coated with a self-assembled monolayer of anti-E. coli polyclonal antibody. After phage infection, damage to the lipopolysaccharide layer on the outer membrane surface of E. coli cells causes changes to its morphology and surface charge, resulting in the aggregation of anionic redox probe, ferri/ferrocyanide at the electrode surface and thereby increases the electron-transfer rate. The consequent decrease in electron-transfer resistance in the presence of phage is monitored using the electrochemical impedance spectroscopy. The filamentous phage-bacterium interaction, which is hardly observable using the conventional microscopic methods, is detected within 5 h using this impedimetric microbial sensor, thus demonstrates its superior performance in terms of analysis time, ease and reduced reliance on the labeling steps during in-situ monitoring of phage infection process. In Chapter 5, an impedimetric cell-based biosensor is fabricated from a poly-L-lysine-modified screen-printed carbon electrode to monitor the real-time DENV-2 infection of surface-immobilized baby hamster kidney fibroblast cells. Based on the described platform, DENV-2 induced cytopathic or cytopathogenic effects (degenerative morphological change, detachment, membrane degradation and death of host cells), which are indicated by a drastic decrease in impedance signal response, can be detected within ~ 35 hours post infection. A parameter that describes the kinetics of cytopathogenesis, CIT50 (time taken for 50 % decrease in impedance signal response) reveals an inverse linear relationship to the logarithm of virus titer. It is also reported that CIT50 values are delayed by 31.5 h for each order of magnitude decrease in multiplicity of infection. Therefore, the virus titer of given samples can be determined based on the measurement of impedance signal response and analysis of CIT50. Finally, the concluding remarks of these experimental works and the future outlook for biosensing techniques are included in the conclusion section.