Development Of CRP Electrochemical Immunosensor Based On Synergistic Signal Amplification

C-reactive protein（CRP）is the most sensitive response marker of acute phase proteins in human body,which is very important for the detection of various diseases.At present,the detection methods of CRP include turbidimetry,immunochromatography,chemiluminescence,fluorescence,electrochemical method and so on.The electrochemical sensor has the advantages of fast,sensitive,simple and easy to carry.Therefore,this paper studies the construction of electrochemical immunosensor to detect CRP.In this paper,an electrochemical immunosensor was constructed based on synergistic signal amplification to detect CRP,that is,indole and Fe（CN）₆^3-/4-were used as electrochemical probes,spherical nano gold（Au NPs）and nanocomposite pyrrole-gold（Py-Au）were used as signal amplification elements,and screen printed carbon electrode（SPCE）and glass carbon electrode（GCE）were used as substrates,two electrochemical immunosensors BSA/Anti-CRP/indole/Au NPs/SPCE and BSA/Anti-CRP/Py-Au/GCE were formed by immobilizing Anti-CRP and incubating bovine serum albumin（BSA）in turn to realize the electrochemical detection of CRP.The main research contents are as follows:（1）BSA/Anti-CRP/indole/Au NPs/SPCE electrochemical immunosensor was constructed to detect CRP.With Au NPs as amplification signal element and indole as modification probe,the oxidation potential in DPV diagram was near 0.6 V and the current value increased about 7 folds.SEM,EDS and electrochemical methods were used to characterize and analyze the different stages of sensor construction,and the relevant parameters affecting the performance of electrochemical biosensor were optimized.Under the optimal conditions of the experiment,DPV method was used to detect the linear relationship between different CRP concentrations and the difference of sensor response current.The linear equation was:I=4.6741-0.4033lg c(I is the current valueμA,c is the CRP concentrationmg×m L^-1),the linear range was 0.0001～100mg×m L^-1,the correlation coefficient was 0.9920,the minimum detection limit was0.03 ng×m L^-1,and the sensitivity was 5.7055μA?mg^-1?m L?cm^-2.In this chapter,the selectivity,stability and reproducibility of the sensor are studied,which proves that the sensor has good selectivity,stability and reproducibility.The recovery rate of CRP in human serum samples was determined by standard addition method.The results show that the recovery rate of the sensor ranges from 98.2%～102.2%,indicating that the performance of the immunosensor is good,and its RSD range is 2.44～5.98%.Therefore,the sensor can be used for the detection of actual samples.（2）BSA/Anti-CRP/Py-Au/GCE electrochemical immunosensor for Detecting CRP was constructed.Py-Au was used as the amplification signal element,and the probe was Fe（CN）₆^3-/4-(0.1 mol×L^-1 KCl).The oxidation potential in the DPV diagram was about 0.3 V,and the current value increased about 4 folds.The different stages of sensor construction and nanocomposites were characterized and analyzed by SEM,EDS,TEM and electrochemical methods.The feasibility of the sensor was studied by DPV method.It was proved that the sensor can be used for the detection of CRP,and the relevant parameters affecting the performance of electrochemical biosensor were optimized.Under the optimal conditions of the experiment,DPV method was used to detect the linear relationship between different CRP concentrations and the difference of sensor response current.The linear equation was:I=49.4684-4.5802lg c(I is the current valueμA,c is the CRP concentrationmg×m L^-1),the linear range is 0.0005～60mg×m L^-1,the correlation coefficient is 0.9959,the minimum detection limit is 0.17ng×m L^-1,and the sensitivity is 0.0105μA?mg^-1?m L?cm^-2.The selectivity,stability and reproducibility of the sensor are studied.By exploring the current response effect of the multiple immunosensors to detect CRP,it shows that the sensor has good selectivity,stability and reproducibility.The recovery of CRP in human serum samples was determined by standard addition method.The results show that the RSD range is 1.73～6.51%,and the recovery is 99.1～102.6%.Therefore,the sensor can be applied to the detection of actual samples.（3）The probe of BSA/Anti-CRP/indole/Au NPs/SPCE electrochemical immunosensor is indole with a potential of about 0.6 V,and the probe of BSA/Anti-CRP/Py-Au/GCE electrochemical immunosensor is Fe（CN）₆^3-/4-,with a potential of about 0.3 V.while the first one has a wide linear range and low minimum detection limit,but the steps used in BSA/Anti-CRP/Py-Au/GCE are less than those in BSA/Anti-CRP/indole/Au NPs/SPCE,The linear range is very close to the minimum detection limit.Both sensors can be used for detection in actual samples.