Novel Immunosensing Approaches For The Quantification Of Exogenous Proteins

In recent years,with the increasing issues of genetically modified organisms?GMOs?in illegal planting and food contamination,the strengthening of the detection and supervision of GMOs is in need.Due to the huge amount of GM crops involved in illegal cultivation and international trade,the development of simple,rapid and point-of-care testing?POCT?quantitative approaches for GMOs on site is crucial for the implementation of labeling regulations and safety control of GMOs.There is an increased attention for GMO detection technologies that are protein-based immunoassays targeting of exogenous proteins expressed in GM crops.These protein-based approaches are fit for field analysis and rapid screening of GMO ingredients because they are simple to manipulate,low cost and do not need sophisticated instruments.In some commonly used immunoassays a visible colorimetric readout strategy was used,including enzyme-linked immunosorbent assay?ELISA?,western blotting and immunochromatographic test strip.These approaches usually suffer from insufficient sensitivity.More recently,a number of immunoassay methods have already been developed to achieve high assay sensitivity,such as fluorescent immunoassay,chemiluminescent immunoassay and polymerase chain reaction?PCR?-ELISA.Unfortunately,sensitively quantitative technologies for POCT of exogenous proteins in GM crops have still failed to access.Immunosensors with desirable features of integration and miniaturization provide a promising pathway for POCT of GM crops.In present study,three immunosensing methods have been developed for the quantification of GM proteins with high sensitivity,rapidity and simplicity using nanomaterials as ideal antibody carriers and signal amplification nanoprobes.The main contents are as follow:?1?An ultrasensitive label-free electrochemiluminescent immunosensor for measuring PAT/bar protein level and genetically modified crops contentIn this work,we have tested for the first time a method that uses novel carbon nanospheres?CNPs?label-free electrochemiluminescent?ECL?immunosensor for the ultrasensitive quantification of PAT/bar protein in GM crops.Novel CNPs were prepared from printer toner with a very facile approach,and linked with antibody for PAT/bar protein to modify a glassy carbon working electrode.After an immunoreaction between the PAT/bar protein and its antibody,the immunocomplex formed on the electrode receptor region resulted in an inhibition of electron transfer between the electrode surface and the ECL substance,thus led to a decrease of ECL response.Under the optimal conditions,the ECL responses linearly decreased as the increase of the PAT/bar protein concentration and the GM rapeseed RF3content in the ranges of 0.10-10 ng m L^-1and 0.050-1.0%,with the limits of detection of 0.050ng m L^-1 and 0.020%?S/N=3?,showing significant improvement of sensitivity than that of the most reported immunoassays.The application of novel CNPs and ECL detection greatly improved the detection sensitivity of PAT/bar protein,which was comparable to that of the highly PCR-based approach.The developed label-free ECL immunosensor provided a promising and versatile tool for GMO detection.?2?A portable electrochemical immunosensor for highly sensitive point-of-care testing of CP4-EPSPS proteinTo date the development of sensitively quantitative POCT system has not yet been reported for GMO detection.In presented work,an electrochemical immunosensor towards CP4-EPSPS has been fabricated by integrating a portable bioanalytical device with a disposable screen-printed carbon electrode?SPCE?for POCT of exogenous proteins in GM crops.The dual-functionalized Au NPs were used as nanoprobes and prepared by simultaneously tagging horseradish peroxidase?HRP?and antibody on Au NPs with an exceptionally simple protocol.The sensitivity of the developed nanoprobe-based immunosensor was 62.5-fold higher than that using HRP-labeled antibody.As a result,the proposed immunosensor using SPCE could detect CP4-EPSPS down to 0.050 ng m L^-1 with the linear range of 0.10-10 ng m L^-1 within 65 min.In addition,the developed method has been validated with genuine GM crops and the results show a good correlation coefficient of0.9909 compared with those of a commercial ELISA kit.Therefore,this portable electrochemical immunosensor is suitable for sensitive detection and provides a convenient and reliable platform for POCT assay.?3?Highly sensitive immunosensing platform for one-step quantification of Cry 1AbHerein,A highly sensitive immunosensing platform has been developed for both colorimetric and chemiluminescent?CL?detection of Cry 1Ab using dual-functionalized Au NPs as signal amplification nanoprobes and antibody-labeled magnetic beads as capture probes.Combined with immunomagnetic separation,this immunosensing platform based on colorimetric method could detect Cry 1Ab in one step in a linear range from 1.0 to 40 ng m L^-1 within 1.5 h,with a limit of detection of 0.50 ng m L^-1.The sensitivity of fabricated nanoprobes was 15.3 times higher than that using commercial HRP-conjugated antibody.Meanwhile,the fabricated nanoprobes coupled with CL detection was successfully applied for Cry 1Ab detection with a minimum detection concentration of 0.050 ng m L^-1 within a linear range of 0.10-20 ng m L^-1.The proposed approach was validated with genuine GM crops,and the results showed a good correlation coefficient of 0.9906 compared to those of a commercial ELISA kit.Compared with ELISA,the developed immunosensing platform significantly simplified the assay procedure and shortened the analytical time,thus expanding the applications of ELISA approach in POCT assay.