Biosensing Technology And Instrument For Rapid Detection Of Escherichia Coli O157:H7

This work aimed to develop novel biosensing methods for the detection of foodborne pathogens in agricultrual products by using the latest research achievements in the field of molecular biology, nanotechnology, material science, sensing technique, and instrumental analysis technique. The Escherichia coli O157:H7(E. coli O157:H7) was selected as the target bacteria. After the careful study on the current detection methods for E. coli O157:H7, we chose two kinds of unlabeled biosensing methods for E. coli O157:H7detection in our study, including surface plasmon resonance (SPR) based biosensing method and electrochemical impedance spectroscopy (EIS) based biosensing method. However, some limitations still remain as the challenges when dealing with large-scale applications. Here, we used novel detection mode, bio-recognition molecule (e.g. lectin) and nanomaterials (e.g. gold nanoparticles and graphene) to develop novel biosensing methods for rapid and sensitive detection of E. coli O157:H7. Furthermore, we developed a portable SPR instrument and a handheld impedance instrument for E. coli O157:H7detection. This work provides several effective and useful biosensing methods for the detection of E. coli O157:H7, which may hold great promising in routine sensing applications.Main results and conclusions are as follows:(1) SPR biosensing methods for E. coli O157:H7detectionSubtractive inhibition mechanism based SPR biosensing method for E. coli O157:H7detectionA novel SPR biosensing method by means of a subtractive inhibition assay was developed for the sensitive detection of E. coli O157:H7in order to solve the problems in direct SPR detection method. In the subtractive inhibition assay, E. coli O157:H7cells and goat polyclonal antibodies for E. coli O157:H7were firstly incubated for a while. Then, the E. coli O157:H7cells that bound with antibodies were removed by stepwise centrifugation process. The remaining free unbound antibodies were detected through interaction with rabbit anti-goat IgG polyclonal antibodies immobilized on the sensor chip using BIAcore3000biosensor. We used the relationship between SPR signal and concentrations of E. coli O157:H7to establish the detection model. Results show that the signal was inversely correlated with the concentrations of E. coli O157:H7cells in a range from3.0x104to3.0x108cfu mL-1with the detection limit of3.0x104cfu mL-1. This detection limit was lower than those obtained by direct SPR method (in which the antibodies was firstly immobilized on the chip surface and then to capture E. coli O157:H7) and ELISA. The subtractive inhibition mechanism based SPR biosensing method could solve the drawbacks in current direct SPR method (e.g. low sensitivity), which may provide a useful way to implement SPR technique for other pathogens detection.Lectin based SPR biosensing method for E. coli O157:H7detectionA novel SPR biosensing method using lectin as bio-recognition molecule was developed for the rapid and sensitive detection of E. coli O157:H7. The selective interaction of lectins with carbohydrate components on the surface of bacterial cells was used as the recognition principle for the detection of E. coli O157:H7. This novel bio-recognition molecule shows some intrinsic advantages over antibodies, such as inexpensive and physicochemical stable. In the detection procedure, lectin molecules were firstly immobilized onto the chip surface via molecular self-assembly layer. When the target bacteria was captured by the lectins immobilized on the chip, the SPR signal increased, which was correlated with the concentrations of bacteria cells. Five types of lectins from Triticum vulgaris, Canavailia ensiformis, Ulex europaeus, Arachis hypogaea, and Maackia amurensis, were employed to evaluate the selectivity of the approach for binding E. coli O157:H7effectively. A linear range of3×105-3×108cfu mL-1and a detection limit of3x103cfu mL-1were obtained for determination of E. coli O157:H7when using the lectin from Triticum vulgaris as the bio-recognition element. Furthermore, the proposed biosensor was used to detect E. coli O157:H7in agricultural products. The detection limits in case of E. coli O157:H7contaminated cucumber samples and ground beef samples were3.0×104and3.0×105cfu mL-1, respectively. This work firstly reveals that the lectin could be an effective and promising bio-recognition material for constructing SPR biosensors for pathogens detection, which may have wide applications in routine sensing applications.(2) EIS biosensing methods for E. coli O157:H7detectionDisposable nanocomposite sensor based EIS biosensing method for E. coli O157:H7detectionA novel EIS biosensing method using disposable nanocomposite sensor for the low-cost and high-sensitive detection of E. coli O157:H7was developed. The commercial screen-printed electrode was used as the substrate in the proposed sensor. Then, a graphene oxide film was formed on the surface of screen-printed electrode using simple drop-coating method. The reduction of graphene oxide into graphene was performed by amperometric i-t curve method. Then, one-step electrodeposition method was employed to prepare gold nanoparticles on the graphene film surface. Antibody was immobilized on the surface of gold nanoparticles through electrostatic adsorption, and then BSA was used to block the redundant active sites on gold nanoparticles. When E. coli O157:H7was captured by the disposable sensor, the electron transfer kinetics on electrode surface was hindered, inducing the increase on electron transfer resistance. Results show that the change of electron transfer resistance is directly proportional to concentrations of E. coli O157:H7. A wide linear range of1.5×103-1.5×107cfu mL-1and a low detection limit of1.5×103cfu mL-1were obtained using the developed biosensing method. Additionally, nontarget bacteria, such as E. coli DH5a, Listeria monocytogenes, Staphylococcus aureus, were used to demonstrate the high selectivity and specificity of the biosensor. High-performance, low cost, and easy operation make the developed EIS biosensing method very attractive in routine sensing applications.Graphene paper sensor based EIS biosensing method for E. coli O157:H7detection In this study, a low-cost and robust impedimetric immunosensor based on gold nanoparticles modified free-standing graphene paper electrode for rapid and sensitive detection of E. coli O157:H7was developed. Graphene paper was prepared by chemical reduction of graphene oxide paper obtained from vacuum filtration method. Scanning electron microscope, Raman spectroscopy and X-ray diffraction techniques were employed to investigate the surface morphology and crystal structure of the prepared graphene paper. The gold nanoparticles were grown on the surface of graphene paper electrode by one-step electrodeposition technique. The immobilization of anti-E. coli O157:H7antibodies on paper electrode were performed via biotin-streptavidin system. Electrochemical impedance spectroscopy was used to detect E. coli O157:H7captured on the paper electrode. Results show that the developed paper immunosensor possesses greatly enhanced sensing performance, such as wide liner range (1.5×102-1.5×107cfu mL-1), low detection limit (1.5×102cfu mL-1), and excellent specificity. Furthermore, flexible test demonstrate the graphene paper based sensing device has high tolerability to mechanical stress. The strategy of structurally integrating metal nanomaterials, graphene paper, and biorecognition molecules would provide new insights into design of flexible immunosensors for routine sensing applications.(3) Portable instruments for the detection of E. coli O157:H7Construction of portable SPR instrument for rapid detection of E. coli O157:H7The feasibility of the construction of portable SPR instrument for the rapid detection of E. coli O157:H7was investigated. This instrument is composed of commercial integrated SpreetaTM SPR sensor system and sensing control part developed by our laboratory. The sensing control part includes hardware and supported software. The stability and precision of the developed instrument was verified by testing the ethanol and glucose solutions. Furthermore, the developed instrument was employed to detection E. coli O157:H7by combining with the established SPR biosensing methods in previous work. Results show that the miniature SPR instrument could be used to detect E. coli O157:H7in the range of3.0×105-3.0×108cfu mL-1. Additionally, this instrument could achieve multi-channel simultaneous detection of various samples. Fast, less sample, and portable make the developed instrument as an effective tool for on-site and rapid detection of E. coli O157:H7.Construction of handheld impedance instrument for rapid detection of E. coli O157:H7The feasibility of the construction of handheld impedance instrument for the rapid detection of E. coli O157:H7was investigated. This instrument consists of nanomaterials modified screen-printed electrode and handheld impedance meter. The screen-printed electrode was prepared with the same method mentioned in the previous work. When E. coli O157:H7was captured by antibody modified electrode, the impedance at the frequency of1kHz measured by handheld impedance meter increased. The portable instrument was used for the detection of E. coli O157:H7at the range of1.5×104-1.5x107cfu mL-1and detection limit of1.5x104cfu mL-1. Excellent accuracy, precision, and portability make the developed instrument as an effective tool for on-site and rapid detection of E. coli O157:H7.