Study On Biotoxin Detection By Electrochemiluminescence Sensor Based On Screen Printed Electrode

Characterized by high toxicity,relatively stable,difficult to detect and verify and so on,biotoxins is urgent to be detected by new methods with high sensitivity,good specificity,and great anti-interference ability.Electrochemiluminescence(ECL)sensor has the advantages of high sensitivity,wide detection range,good controllability,fast response and strong anti-interference,so it is especially suitable for the detection of trace and ultra-trace of complex substances and has excellent application prospects for the detection of biotoxins.However,the conventional ECL sensor requires an integrated detection cell,and the apparatus has a large frame and great weight which limit its application in a laboratory or a hospital,and make it difficult to be applied to in-situ detection.Furthermore,the conventional ECL sensor requires careful cleaning before and after used,which means a lot of time and reagents are needed to be excessively consumed.And the possible uncleaning may bring interference to the test.In this study,the electrochemical reaction center was transferred to the surface of a screen-printed electrode(SPE).Combining with magnetic separation technology and phage display technology,and fully utilizing the advantages of screen-printed electrodes and magnetic separation immunoassay,a new method of ECL detection of biotoxins with high portability,high sensitivity and simplified operation requirements was established.And this study has obtained the following results.1、Construction and response analysis of the electrochemiluminescence sensor based on screen printed electrodes(SPE-ECL sensor).A portable ECL sensing platform based on screen printed electrodes was constructed,and the SPE chip was characterized by optical and electrochemical methods while the main parameters of instruments were optimized.Using screen-printed electrode as the micro reaction pool to replace the traditional discrete three-electrode system,the sensor had the characteristics of small size,portability and simplified operation requirements.The characterization results showed that the gold electrode had excellent electrochemical performance far beyond the graphite electrode and the platinum electrode,and the gold clusters on the electrode surface are distributed uniformly,which was favorable for the enrichment and immobilization of magnetic microspheres and the electrode surface modification.Simultaneously,the optimized sensor has good detection stability and reproducibility.2、Study on the detection of biotoxins based on SPE-ECL sensor and the labeled monoclonal antibody(mcAb).Immobilizing mcAb on the surface of magnetic microspheres as the capture probe,using Ru(bpy)32+ labeled mcAb as the special signal probe,a ECL sensor based on screen printed electrode was constructed.The linear detection range of the sensor for abrin was 0.1 ng/mL-1000 ng/mL,the relationship between ECL intensity(Y)and abrin concentration(X)conformed to the linear regression equation Y=83.6241gX+92.285(R=0.9943,n=8,P<0.0001),and the limit of detection(LOD)was 0.1 ng/mL.With high sensitivity and wide linear range,this sensor also showed good reproducibility indicated by the relative standard deviation(RSD)of different samples ranging from 2.5%to 16.5%.The sensor can overcome the influence of non-target proteins and complex environmental interferences on biotoxins detection,and has good specificity and great anti-interference ability.It can meet the needs of environmental analysis for milk,soil and honey samples with good recovery(83.6%～107.0%).3.Study on the detection of biotoxins based on SPE-ECL sensor and the labeled phage display antibody.Using phage display antibody as the ECL labeled probe and combining with magnetic separation technology,a biotoxins detection method based on SPE-ECL sensor was constructed.The phage display antibody could provide more Ru(bpy)32+ modification sites,which not only ensured the specificity but also increased the detection sensitivity of the sensor.The linear detection range for abrin was 5 pg/mL-500 ng/mL,the linear regression equation was Y= 1122.590 lgX+ 3235.284(R=0.9969,n=9,P<0.0001),and the LOD was 5 pg/mL which meant the detection sensitivity was 20 times higher than that with mcAb as the labeled probe.This sensor showed good reproducibility indicated by the the RSD of different samples ranging from 1.8%to 10.8%,and can overcome the influence of non-target proteins and complex environmental interferences on biotoxins detection with high specificity and great anti-interference ability.It can meet the needs of environmental analysis for milk,soil and honey samples with good recovery(84.3%～104.4%).4.Study on the detection of biotoxins based on SPE-ECL sensor and the labeled affibody.For the first time,the affibody was used as the specific recognition ligand in the ECL detection.Combining with the phage display technology to amplify the sensing signal,a new detection method for biotoxins based on SPE-ECL sensor was constructed.The linear detection range of the sensor for abrin was 5 pg/mL～500 ng/mL,the linear regression equation was Y=1683.838lgX+4495.319(R=0.9963,n=9,P<0.0001),and the LOD was 5 pg/mL.The sensitivity is similar to that with phage display antibody as the labeled probe and about 20 times higher than that with the antibody as the labeled probe.This sensor showed good reproducibility indicated by the the RSD of different samples ranging from 2.9%to 10.4%,and can overcome the influence of non-target proteins and complex environmental interferences on biotoxins detection with high specificity and great anti-interference ability.It can meet the needs of environmental analysis for milk,soil and honey samples with good recovery(85.6%～125.5%).And this method is a successful attempt to expand the application area of the affibody and enrich the ECL sensing system.Combineing the advantages of screen-printed electrodes such as high degree of integration,simplified operation requirements with the characteristics of high sensitivity,good controllability,fast response and great anti-interference ability of ECL detection,this study established a new sensor-based system for detecting biological toxins based on SPE-ECL sensor respectively with mcAb,phage display antibody and affibody as the specific signal probe,which can provide technical basis and guidance for outdoor field environmental monitoring and food hygiene inspection.