A nanoporous membrane based electrochemical biosensor for bacteria detection with nanoparticle tag amplification

Abstract

Nowadays, biosensing has attracted tremendous interests for its versatility in many fields including food safety, medical diagnosis, and prognosis, as well as environmental detection. Food poisoning is a critical issue which threatens lives through pathogenic bacteria which are naturally infectious or releasing toxins. Conventional detection methods consist of culture & colony counting method, electron microscopy method, immunological reaction based method and polymerase chain reaction (PCR).These methods have been demonstrated outstanding for detection of pathogenic bacteria or characteristic virulence genes expressed in them. However, these traditional methods have drawbacks such as long testing time, complex working procedures and high cost. Hence, the development of new detection methods is of great importance. Recently, various biosensors including optical biosensors, piezoelectric biosensors, and electrochemical biosensors have been developed for pathogenic bacteria detection. Among these three types of biosensors for pathogenic bacteria detection, electrochemical biosensors are very promising with advantages of low-cost, ease to operate, rapid response and portability, which highly meets the requirement of on-site bacteria detection in food or water. Moreover, the recent rapid development of nanomaterials injects new energy into electrochemical biosensors with better performance such as high sensitivity and specificity. The research of this thesis is mainly focused on designing and fabricating nanoporous membrane based electrochemical biosensors with nanocatalyst-based signal amplification for rapid and sensitive detection of pathogenic bacteria or characteristic virulence genes expressed in pathogenic bacteria. Nanoporous alumina membrane is an excellent biosensing platform for various biological species detection due to its tunable nanopore size as well as the high surface area to volume ratio which allows numerous oligonucleotide probes and antibody immobilization. Nanoporous alumina membrane based microfluidic chambers are designed as the electrochemical detecting platform. E. coli O157:H7 genes and Salmonella enteritidis bacteria are chosen as the representative food-borne pathogens to testify the functionality of this biosensor. The first part of this thesis is to develop a nanoporous alumina membrane based electrochemical biosensor with platinum nanoparticle(PtNP) tags for E.coli O157:H7 gene detection.Firstly,(3-glycidoxypropyl) trimethoxysilane (GPMS) silane was immobilized onto nanoporous alumina membrane surface followed by further immobilization of oligonucleotide probes through covalent binding. After oligonucleotide probes immobilization, nanoporous alumina membrane was integrated with a PDMS microfluidic chamber. The solution with target E. coli O157:H7 gene was then dropped into the detection chamber and hybridized with oligonucleotide probes. Platinum nanoparticles (PtNPs) modified with secondary oligonucleotide probes were added and conjugated with target E.coli O157:H7 gene to form sandwich structures.PtNPs then catalyzed soluble 4-Chloro-1-naphthol (4-CN) in the solution into insoluble products which were deposited on the nanoporous membrane, leading to blockage of nanopores and impedance signal increase. By electrochemical impedance spectroscopy (EIS), the impedance signal increase was then measured. Various characterization methods including fluorescent labelling, electron microscopy and Zeta-potential measurement were used to confirm experimental steps. The hybridization time between target genes and probe genes as well as the precipitation catalyzing time were investigated by EIS. The specificity was also studied using non-target oligonucleotides and 6 bases mismatched oligonucleotides. As a result, this biosensor showed a good sensitivity and selectivity with a limit of detection (LOD) as low as 94 pM. The second part of this thesis was to develop a nanoporous alumina membrane based electrochemical biosensor with graphene oxide (GO)/Hemin-Antibody composite amplification for the whole-cell bacterial detection of Salmonella enteritidis. Nanoporous alumina membrane was firstly integrated into microfluidic chambers and then Salmonella enteritidis ompC antibodies were immobilized onto membrane surface with GPMS silane as the chemical linker. Target Salmonella enteritidis bacteria were then captured by these antibodies on nanoporous membrane. Here, GO/Hemin-antibody was chosen as amplification tag was composites instead of PtNPs because of the high loading capacity of graphene oxide and the easy modification of hemin molecules and antibodies onto GO surface. Precipitation of 4-CN was then performed, leading to a significant increase of impedance for signal amplification. Both the detection time and specificity of this biosensor was studied for Salmonella enteritidis. The amplification effect on detection sensitivity was also explored. As a result, this platform showed a good sensitivity and selectivity with a limit of detection (LOD) as low as 12 CFU/mL.