Electrochemical Immunosensor Based On Functional Nanoprobes For Sensitive Detection Of S.aureus In Food

Electrochemical immunosensor is a new type of biosensor developed by combining electrochemical technology with immune technology.It can convert biochemical signal into electrical signal and achieve quantitative detection of the analyte based on the specific recognition between antigen and antibody by using electrochemical workstation.Due to the merits of electrochemical immunosensor such as instrument miniaturization,specificity and selectivity,high sensitivity and rapidity,it has become one of the important means of detection of Staphylococcus aureus(S.aureus).In recent years,research on signal amplification techniques for immunosensors has received much attention.These signal amplification methods have played a significant role in improving the sensitivity of the immunesensor.There are mainly two ways to realize the signal amplification strategy:one is to amplify the signal by increasing the amount of the capture antibody on eletrode;the other is to use the catalytic property of the nanoprobe to amplify the signal.Therefore,this paper constructed three kinds of signal amplification electrochemical immunosensors for the detection of S.aureu in food.The main research contents of this paper are as follows:1.Construction of electrochemical immunosensor based on functionalized carbon nanospheres with CdTe quantum dots for sensitive detection of S.aureusAn electrochemical immunosensor based on CdTe QDS functionalized carbon nanospheres(CNS)was constructed for highly sensitive detection of S.aureus.CdTe QDS and CNS with uniform morphology and uniform size were first synthesized.Negatively charged CdTe were functionalized to negatively charged CNS by using positively charged chitosan(CS).The antibody was coupled to CNS@CdTe activated with EDC/NHS to produce the CNS@CdTe-Ab nanoprobe.At the same time,gold nanoparticles were electrodeposited on the surface of the glassy carbon electrode to immobilize capture antibody through the Au-S bond.Based on the specific immune reaction between bacteria and antibody,the S.aureus in the sample was recognized on the electrode immobilized by the capture antibody,and then the synthesized nanoprobe was further recognized on the surface of the modified electrode to construct electrochemical immunnosensor.Finally,the Cd2+ in the nanoprobe was eluted for differential pulse voltammetry detection.We had optimized the parameters that affect the performance of the immunosensor,such as antibody concentration,immune reaction time and pH of the test solution.Under the optimal conditions,experimental results demonstrated that the detection signal of the immunno sensor linearly depended on the logarithmic value of S.aureus concentration from 1.21 ×102 to 1.21 ×107 CFU mL-1 with a detection limit of 47 CFU mL-1.The electrochemical immunno sensor showed good specificity for the detection of S.aureus compared with the detection of nontarget bacteria such as Escherichia coli,Actobacillus and Vibrio parahaemolyticus.In the real sample detection process,the recovery rates were in the range of 88.9%～109.8%and the relative standard deviation(RSD)was between 2.8%～6.3%,indicating a certain potential of the immuno sensor in practical applications.2.Synthesis of DNAzyme functionalized Gold/platinum nanoprobe and its application in highly sensitive detection of S.aureusAn electrochemical immuno sensor was prepared by using streptavidin and DNAzyme functionalized Au@Pt nanocomposite(Au@Pt)as nanoprobe.Firstly,urchin-like Au@Pt was synthesized.Because of the catalysis of Au@Pt and DNAzyme for H2O2,the dual-signal amplification nanoprobe could be prepared by functionalizing the streptavidin and the thiolated DNAzyme respectively on the surface of the Au@Pt.At the same time,the GO@AuNPs nanocomposite was modified on the electrode surface to immobilize streptavidin.Based on the affinity reaction between biotin and streptavidin,the complex of biotinylated antibody-bacteria was recognized on the surface of the modified electrode,and the nanoprobe was further identified on the biotinylated antibody on the surface of the bacteria to construct electrochemical immuno sensor.Finally,electrochemical detection was performed through the current-time curve.Under the optimal conditions,experimental results demonstrated that the current change of the immuno sensor linearly depended on the logarithmic value of S.aureus concentration from 1.52×102 to 1.52×107 CFU mL-1 with a detection limit of 25 CFU mL-1.In addition,the prepared immunosensor had better repeatability,specificity and stability.In the real sample detection process,the recovery rates ranged from 90.4%to 110.8%and the RSD was in the range of 4.6%～7.7%,indicating the highly sensitive detection of S.aureus in real samples.3.Construction of ferrocenedicarboxylic acid(Fc)functionalized polystyrene acrylic spheroid(PSA)mimic enzyme signaling amplification strategy and its application in electrochemical immunosensorAn electrochemical immunosensor based on PSA@Fc mimic enzyme signal amplification strategy was established and was used for the highly sensitive detection of S.aureus in food.PSA with uniform particle size and uniform dispersion was synthesized,and then Fc was polymerized on the PSA surface under light irradiation conditions to construct PSA@Fc.The antibody was coupled to PSA@Fc activated with EDC/NHS to prepare the PSA@Fc-Ab nanoporbe.The nanoprobe can be used as a mimic enzyme to catalyze hydrogen peroxide and achieve signal amplification.At the same time,gold nanoparticles were self-assembled on the surface of glassy carbon electrode to immobilze the antibody.Then the modified electrode of immobilized antibody was used to capture S.aureus in the sample,and the nanoprobe was further recognized on the surface of S.aureus to construct electrochemical immunosensor.Finally,electrochemical detection was performed by using the differential pulse voltammetry.Under the optimal conditions,the results showed that the detection signal of the immunnosensor was linear with the logarithmic value of S.aureus concentration from 6.5×101 to 1.3×107 CFU mL-1.The detection limit of the immunnosensor is 18 CFU mL-1.The immunosensor had good repeatability,stability and specificity for the detection of S.aureus.In the real sample detection,the recovery rates were between 89.2%and 111.6%and the RSD ranged from 2.51%to 7.32%,indicating that the immunosensor had better performance and can be used for detection of the real sample.