Research Of Sandwich-type Electrochemical Immunosensor For The Detection Of Escherichia Coli

Microbiological contamination caused by food-borne diseases has become a major public health problem in the world. The Centers for Disease Control and Prevention estimates that there are 76 million cases of food-borne illness each year in the United States. Among all pathogens, Escherichia coli(E. coli) is one of the most dangerous pathogens, which can cause hemorrhagic colitis with symptoms such as bloody diarrhea, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Therefore, there is an urgent need to develop simple,rapid and cost-efficient strategies for the sensitive and specific determination of E. coli.In this work, three types of electrochemical biosensors for highly sensitive and selective detection of E. coli were developed on the basis of composites, rolling circle amplification(RCA) and silver nanocluster(Ag NCs) nanowires. These sensors all exhibited low detectin limits and wide liner range for the detection of E. coli, and they also have many good performance such as high selectivity, good repeatability and excellent stability. In addition,these biosensor have been successfully applied to the quantitative assay of E. coli in synthetic samples. Specific research contents are as follows:1. A novel electrochemical immunosensor assay(EIA) for high sensitive and specific detection of Escherichia coli O157: H7 has been developed. This immunosensor is constructed by the assembly of capture antibody on SG-PEDOT-Au NPs composites modified glass carbon electrode. In the presence of target E. coli O157: H7, horse radish peroxidase(HRP)-labeled antibody is captured on the electrode surface to form a sandwich-type system via the specific identification. As a result, E. coli O157: H7 detection is realized by outputting a redox current from electro-reduction of hydrogen peroxide reaction catalyzed by HRP. In our assay, the combination of the unique properties of sulfonated graphene(SG) and gold nanoparticles(Au NPs) can not only accelerate electron transfer on electrode interface, but provide an excellent scaffold for the conjugation of capture antibody that significantly improves the target capture efficiency and enhances the sensitivity of the biosensor. The results reveal the calibration plot obtained for E. coli O157: H7 is approximately linear from7.8×10 to 7.8×106 colony-forming unit(cfu) m L-1 with the limit of detection of 3.4×10 cfu m L-1. In addition, the biosensor has been successfully applied to quantitative assay of E. coliO157: H7 in synthetic samples(spring water and milk). Hence, the developed electrochemical-based immunosensor might provide a useful and practical tool for E. coli O157: H7 determination and related food safety analysis and clinical diagnosis.2. In this work, a simple, label-free, low cost electrochemical biosensor for highly sensitive and selective detection of E. coli has been developed on the basis of rolling circle amplification(RCA) coupled peroxidase-mimicking DNAzyme amplification. A aptamer-primer probe(APP) containing anti-E. coli aptamer and a primer sequence complementary to a circular probe, which includes two G-quadruplex units, is used for recognizing target and triggering RCA-based polymerase elongation. Due to RCA coupled DNAzyme amplification strategy, the presence of target E. coli leads to the formation of numerous G-quadruplex oligomers on electrode, which folds into G-quadruplex/hemin complexs with the help of K+ and hemin, thus generating extremely strong catalytic activity toward H2O2 and giving a remarkably strong electrochemical response. As far as we know,this work is the first time that RCA coupled peroxidase-mimicking DNAzyme amplification technique have been integrated into electrochemical assay for detecting pathogenic bacteria.Under optimal conditions, the proposed biosensor exhibits ultrahigh sensitivity toward E. coli with detection limits of 8 cfu m L-1 and a detection range of 5 orders of magnitude. Besides,our biosensor also shows high selectivity toward target E. coli and has the advantages in its rapidness, low cost, simplified operations without the need of electrochemical labeling steps and additional labile reagents. Hence, the RCA coupled peroxidase-mimicking DNAzyme amplification-based electrochemical method might create a useful and practical platform for detecting E. coli and related food safety analysis and clinical diagnosis.3. In this work, a simple, label-free, low cost electrochemical biosensor for highly sensitive and selective detection of E. coli has been developed on the basis of DNA-stabilized silver nanocluster(Ag NCs) nanowires. To fabricate the Ag NCs nanowires, We assembled Ag NCs into nanowires by long repetitive DNA strands synthesized from rolling circle amplification(RCA). RCA reaction, which amplifies the single target binding events by a factor of hundreds to thousands produces a long DNA molecule. The functional oligonucleotide probe(FOP) integrates both recognition sequence for hybridization with RCA products and template sequence for synthesis of Ag NCs, which appears to possess exceptionalmetal mimic enzyme properties for catalyzing H2O2 reduction. With this RCA amplification strategy, the amount of Ag NCs is dramatically enhanced, thus generating extremely strong catalytic activity toward H2O2 and giving a remarkably strong electrochemical response.Furthermore, due to the high efficiency of Ag NCs nanowires in signal amplification, the proposed biosensor exhibit ultrahigh sensitivity for the specific E. coli detection with a wide linear range from 4.7×10 to 4.7×106 cfu m L-1 and a low detection limit of 41 cfu m L-1, which shows good performance in the complex samples. As far as we know, this work is the first time that Ag NCs nanowires amplification technique have been integrated into electrochemical assay for detecting pathogenic bacteria. In addition, the proposed strategy holds the potential of being extended for the detection of aptamer binding molecules and combined with other detection tools such as fluorescent assay. Hence, our biosensor may create a versatile and practical platform for detecting bacterial and related food safety analysis and clinical diagnosis.