Study On Immunosensors For Rapid Detection Of Pathogenic Microorganism

Foodborne disease not only undermined the health of human being heavily, but also made the huge economic loss. It has been one of the most critical international public health problems. Contamination caused by pathogenic microorganism poses the most severe threat to foodsafety. The conventional culturing and plating method is typically time-consuming and labor-intensive though able to test microorganisms in complicated food sample with low detection limit. Other detection methods, such as PCR and ELISA, are less time-consuming, however, they involve costly equipment and complicated sample pretreatment. Biosensor technologies play an increasingly important role in the detection of pathogenic microorganism because of their great potential to satisfy the practical need for rapid, portable, low-cost, and on-line or in-field detection of foodborne microorganism.Among biosensors, immunosensors are broadly investigated for microorganism detection due to their specific advantages, such as specific affinity reaction, simple fabrication, and sensitive recognition. This work focused on methods for detection of the typical foodborne pathogenic bacteria Escherichia coli O157:H7 and avian influenza virus (AIV) H5N1 using piezoelectric immunosensor and electrochemical immunosensor in combination with nano technology and electrochemical impedance spectroscopy (EIS).The main contents and results are summarized as follows.(1) Piezoelectric immunosensor based on 3-mercaptopropionic acid (MPA) self-assembled monolayers (SAMs) was developed for E. coli O157:H7 detection combining flow injection analysis. A linear relationship between the frequency shift and logarithm value of E. coli O157:H7 concentrations was found in the range of 2.2×105～2.2×108 CFU/mL. The regression equation was y=-6.7 LogN+22.8 with correlation coefficient of 0.9171. The low detection limit was 2.2×105 CFU/mL. Different MPA modification times were compared using cyclic voltammetry (CV). The experimental results indicated that 12 h was sufficient to coat the electrode surface with MPA completely. Quartz crystal microbalance (QCM) and CV were also employed to characterize the stepwise assembly of the immunosensor and the detection procedure. (2) A piezoelectric immunosensor based on gold nanowire was studied for E. coli O157:H7 detection. Gold nanowires were immobilized onto a monolayer of 1.6-hexanedithiol self-assembled on the electrode surface. Antibody immobilization was completed through protein A adsorption onto the gold nanowires. It was found that gold nanowires could be immobilized onto the electrode surface well through immersion method in gas phase other than injection method in flow cell. Hexadecyl trimethyl ammonium bromide in gold nanowire solution inhibited the adsorption of protein A onto the gold nanowire layer, which resulted in the low amount of immobilized antibodies. The tests showed that the hexadecyl trimethyl ammonium bromide could not be washed away from the immunosensor. SEM image demonstrated that the signal of hexadecyl trimethyl ammonium bromide was large enough to cover the target signal.(3) A piezoelectric immunosensor based on gold nanoparticles was developed for E. coli O157:H7 detection. It was based on antibodies immobilization onto 16-mercaptohexadecanoic acid (MHDA) SAMs on the gold nanoparticles layer which was modified onto the electrode surface through a self-assembled monolayer of 1,6-hexanedithiol. The results indicated that cleaning the QCM electrode with super Piranha improved the gold nanoparticles immobilization. The immunosensor responded the target bacteria better in lower concentrations than high concentrations. E. coli O157:H7 solution without centrifugation could cause obvious non-specific signal due to the substance in the culture. Compared with the control tests, the immunosensor improved the detection limit for one log, and it has achieved the lowest detection limit in theoretical calculation. The immobilization of gold nanoparticles and the capture of E. coli O157:H7 onto the QCM electrode surface were demonstrated through SEM.(4) Magnetic nanobeads amplification based QCM immunosensor was developed as a new application for detection of avian influenza (AI) H5N1 virus. Polyclonal antibodies against Al H5N1 virus surface antigen HA (Hemagglutinin) were oriented on the surface of the QCM gold electrode through self-assembled monolayer of MHDA. Magnetic nanobeads (30 nm) coated with anti-H5 antibodies through biotin-streptavidin binding were used for further amplification of the binding reaction between the antibody-antigen(virus). Experimental results indicated that the nonspecific signal was negligible using D-biotin and BSA together to block the nanobeads surface. The magnetic nanobeads amplified the target signal much better in lower titers than high titers of the virus. And the amplification signal improved two logs of the detection limit. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent. A linear relationship between the logarithmic value of H5N1 virus titers and frequency shift was found ranging from 0.128 to 12.8 HA unit. The corresponding equation wasΔf=-37.67 log N-44.295 (R2=0.99). The detection limit of 0.0128 HA was obtained in 2 hours of detection time. The immunosensor was evaluated with tracheal swab samples. A linear relationship between the frequency shift and logarithmic value of AI H5N1 virus titers was found in a range of 0.128-12.8 HA unit. The regression equation wasΔf=-15.835 logN-23.969 with correlation coefficient of 0.87. The detection limit for the swab samples was 0.128 HA unit. No significant interference was observed from non-target subtypes such as AI subtypes H3N2, H2N2, and H4N8. Both bindings of target H5N1 viruses and magnetic nanobeads onto the QCM electrode surface were further confirmed by environmental scanning electron microscope (ESEM).(5) The feasibility of a label-free electrochemical immunosensor employing a quartz crystal Au electrode as working electrode was demonstrated for E. coli O157:H7 detection using Faradaic impedance spectroscopy. A proper equivalent circuit, including ohmic resistance of the electrolyte (Rs), double-layer capacitance (Cdl), electron-transfer resistance (Ret), and Warburg impedance, was introduced for modeling the performance of immunosensor. Among these impedance components, the greatest change was found in electron-transfer resistance due to the binding of E. coli cells. A linear relationship betweenΔimpedance (Zcells—Zatubody) and the logarithmic value of E. coli O157:H7 concentrations was found, y=244.31 LogN—739.96, with a correlation coefficient of 0.98. The detection limit of the immunosensor was 103 CFU/mL. Both EIS and CV were employed to characterize modifications of the electrode and the immunoreaction.(6) A label-free capacitive immunosensor based on quartz crystal Au electrode was developed for detection of E. coli O157:H7. Antibodies were immobilized onto SAMs of MPA on the sensor surface. The experimental results indicated that the capacitance change was correlated with the concentration of E. coli O157:H7. A linear relationship between the capacitance shift and logarithm value of E. coli O157:H7 concentrations was found in the range of102～105 CFU/mL:y=-41.65 LogN+56.75 (R2=0.98). The detection limit was 102 CFU/mL. An equivalent circuit was introduced to simulate the immunosensor, and the double layer capacitance (Cdl) consisted of Cins,Crec,CGC in series. Milk, ground beef, and spinach were selected as food samples to evaluate the capacitive immunosensor with detection limit of 103, 103. and 104 CFU/mL respectively. CV and EIS were employed to characterize the stepwise assembly of the immunosensor.