Study On The Immunosensor Based On The Molecular Orientation And The Current Signal Amplification Technology For Brucella Detection

Brucella is a gram-negative rod-shaped bacterium.The bacterium is highly infectious with a high morbidity.Both humans and animals can be infected.After infection,it can cause brucellosis.It mainly damage the reproductive system of humans and animals.The traditional detection methods of brucellosis need a high time cost and can not meet the rapid detection requirements.The emergence of electrochemical biosensors provides a new method or tool to solve this problem.The immobilization of biomolecular membranes on electrode is an important factor affecting sensor performance in the process of biosensor construction.Oriented-molecules immobilization technology can avoid the immobilization of disordered biomolecules on the sensor surface,reduce the steric hindrance between biomolecules,and improve the immobilization efficiency and activity of biomolecules,thereby improve the performance of the biosensor.Also,nanomaterials are widely used in biosensors due to their small size effects,quantum size effects,surface and interface effects,et al.This paper attempts to combine the oriented immobilization of brucella antibodies(in order to improve the binding rate of antibody and antigen)with the electrical signal amplification of nanomaterials(including nanomaterials such as nanogold and other nanocomposites)to detect brucella antigens.Three immunosensors were constructed systematically and studied deeply.The gist of this doctoral dissertation and conclusions are as follows:(1)Study on immunosensor based on GO/nano-gold/recombinant protein G/screen-printed carbon electrodeIn this part,a screen-printed carbon electrode was used as the immunosensor substrate,and graphene oxide film and nano-gold film were prepared by electrochemical CV method on electrode surface.Nano-gold had good biocompatibility,which could make the immobilization of biological easy,also has a excellent property of electric conduction which could provide basic conditions for the amplification of electrical signals.Recombinant protein G has a cysteine at its C-terminus,and the sulfhydryl group on the cysteine can form gold-sulfur covalent bonds with nano-gold.Therefore,we successfully immobilized recombinant protein G based on the principle of gold-sulfur covalent bonding,and achieved the oriented immobilization of the Brucella antibody based on the protein G specifically binding the Fc region of the antibody.Finaly we got a new type immunosensor.The constructed biosensor was applied to test the impedance value in a series of concentrations of Brucella antigen solution,and the linear relationship between the dimensionless I=(RAg-RBSA)/RBSA value and the logarithm of the brucella antigen concentration was established.The linear correlation coefficient R was 0.9855,and the lowest detection limit(S/N=3)was 3.2×102cfu/m L.The result showed that the sensor had excellent performance in terms of specificity,reproducibility,and stability,and the spike recovery rate was in the range of 92.97% to 100.71%.(2)Study on a sandwich immunosensor based on Ppy/GO/screen-printed gold electrodesIn this part,the screen-printed gold electrode was used as the platform of the immunosensor.The graphene oxide(GO)and polypyrrole(Ppy)membrane were successfully prepared by the one-step electrochemical CV method in the composite electrolyte containing the graphene oxide and the pyrrole monomer.The Fc terminal hydroxyl group of the primary Brucella antibody(Ab1)was binded to the cyano group(CN-)in the polypyrrole ring under the effect of an electricfield,and achieved the goal of the oriented immobilization antibody on the electrode surface by the electrochemical method.In order to further amplify the electrical signal,we synthesized GO/Fe₃O₄/MB/Ab2 nano-biological probe,thereby realized our aim to make a sandwich immunosensor with the oriented combination of the antibody and with the signal amplification.In the series test of the brucella antigen concentration,the response peak current of the biosensor had a good linear relationship with the concentration logarithm,and the linear range is between 1.6×102¹.6×108 cfu/m L,and the lowest limit(S/N=3)was 2.2×102cfu/m L.In addition,the biosensor had good specificity,high recovery rate,good reproducibility and good stability.(3)Study on the immunosensor based on the cysteamine-functionalized nanogold/4-MBA/screen-printed gold electrodeIn this part,the screen-printed gold electrode was applied as the immunosensor substrate.The4-mercaptobenzoic acid was immobilized on the surface of the gold electrode by the gold-sulfur covalent bond.The free carboxyl groups on the 4-MBA can provide chemical bonding groups for the construction of biosensors.The chitosan was used to successfully prepare nano-gold with a particle size of about50 nm,and the nano-gold was functionalized with the cysteamine.The functionalized nano-gold was connected to the surface of the 4-MBA/gold electrode by the amide bond(-CO-NH-)under the action of the cross-linking agent EDC/NHS.After activating the carboxyl of the brucella antibody Fc region by EDC/NHS,the oriented antibody immobilization was realized on the surface of the gold nanoparticles by binding with the amino of the functionalized gold nanoparticles.In a series of concentrations of the brucella antigen solutions,the square wave voltammetry(SWV)was applied to test the performance of the biosensor.The linear relationship between the current change value(?I=IBSA-IAg)and the logarithm of the antigen concentration Log C was established,and the piecewise linear equation was fitted as y₁=5.9828+5.5397x₁,y₂=-32.2109+11.7698x₂,and the correlation coefficient R was 0.9923 and 0.9965 respectively,and the minimum detection limit(S/N=3)was 5.12×10² cfu/m L.After the SWV data was transformed by a second derivative,the minimal detection limit reduced from 5.12×10² cfu/m L to7.0×10¹ cfu/m L.