| Glyphosate(GLYP)was developed by Monsanto in the 1960s as a nonselective broad-spectrum herbicide.Since 2005,it has ranked the first in the global pesticide sales list.In addition,Anti-GLYP crops were successfully bred and the GLYP amount is increasing year by year.GLYP has become the world's most widely used and the largest production of herbicides.It is also the largest herbicide used in China.Large-scale use of glyphosate leads to high levels of GLYP residues in crops,which poses a serious threat to human health.In 2015,the World Health Organization's International Agency for Cancer Research classified GLYP as to 2A categories,which can cause cancer to humans.As a result,the development of GLYP residue detection technique in agricultural products and drinking water has attracted more and more attentions.Chromatographic technology is the main analysis method for detecting GLYP.Although it has high sensitivity and accuracy,the disadvantages of complex sample pretreatment,high cost,long detection period and expensive equipment requirement still existed.Hence,it is necessary to develop a rapid detection method with good selectivity,high sensitivity and convenience process for detection of GLYP.Compared with other detection techniques,immunoassay has the advantages of short detection period,low cost and simple operation.Nevertheless,the main immunoassay for GLYP detection is the traditional enzyme-linked immunoassay(ELISA).Compared with chromatography and other instrument analysis methods,the detection sensitivity needed to be improved.In recent years,with the emergence of novel nanomaterials and biological enzymes,immunoassay technology has been rapidly developed.It has become a new multidisciplinary analytical technique integrating nanotechnology,enzyme engineering and genetic engineering,which provides new ideas for the development of more rapid,sensitive and specific immunological analysis methods.Based on bio-functionalized nanoparticle materials can further improve the sensitivity and stability of immunoassays for GLYP detection.Besides,the usage of nanomaterials in immunoassays can also make up for the shortcomings of the existing GLYP immunoassay methods,such as low sensitivity,more numbers of detection steps and dull detection strategy.This topic aims to develop new immunoassays for detection of GLYP based on DNA nanostructure functionalized AuNPs probes,using the advantages of DNA nanostructures and gold nanoparticles(AuNPs)in biosensing,fluorescence analysis and molecular probe labeling.Moreover,the aim is to improving the performance of immunoassay for GLYP detection,achieving higher sensitivity,rapid and economical detection protocols.Meanwhile,we want to verify the potential of oligonucleotide functionalized AuNPs probes for the detection of small molecular substances such as GLYP.The contents and conclusions of this study are as follows:1.Synthesis and identification of complete antigens of GLYP.Immunogen(BSA-GLYP)and detection antigen(OVA-GLYP)of GLYP were synthesized by activated lipid method and mixed anhydride method,respectively.The synthetic complete antigens were analyzed and identified by UV-Vis spectral scanning,agarose electrophoresis and Native-PAGE.After comparing the titer and corresponding inhibition rate of serum from different immune antigens,c BSA-PEA-GLYP exhibiting the highest serum titer and inhibition rate was selected for subsequent preparation of polyclonal antibodies.2.Preparation and identification of anti-GLYP polyclonal antibody.An AKTA protein purification system and a protein antibody purification column were used to purify the GLYP antibody in the rabbit serum with higher serum titer.The concentration,purity,titer and affinity of the antibody were also determined.The titer of antibody was 1:256000,and the affinity constant of antibody was 3.68×109 L mol-1,belonging to high affinity antibody.3.Development of ic-ELISA.The ic-ELISA for GLYP detection was established by using the prepared rabbit anti-GLYP polyclonal antibody.Under the optimized detection condition,a linear detection range of 0.125mg m L-1?4 mg m L-1 was obtained,and the LOD is 139.9?g m L-1.Cross-reactivity of the ic-ELISA for detection of AMPA,GLUF and PMIDA were 0.61%?0.003%and 0.014%,respectively.A recovery of 97.1%?99.9%was obtained.The assay time was 3 h.4.Establishment of indirect-competitive fluorescence immunoassay based on AuNP-TDNs nanoflare probe.The tetrahedral DNA(TDNs)labeled with goat anti-rabbit antibody and fluorescent dye SYBR Green I(SG)were used to modify AuNPs for synthesis of AuNP-TDNs nanoflare probes with controllable fluorescence"turn on"function,the fluorescence of which was initially quenched by AuNPs through FRET action.The prepared probes were characterized by TEM?UV-Vis scanning and fluorescence spectra,respectively.Then the indirect competitive fluorescence immunoassay(ic-FLIA)for detecting GLYP was established based on the probe.The mixture of GLYP antibody and the sample containing GLYP was added to the enzymatic plate coated with OVA-GLYP.At this point,GLYP in the sample competes with the OVA-GLYP to bind GLYP antibody.Then,AuNP-TDNs nanoflare probe was added and incubated in the plate.After the washing steps,flare DNAs on the probe were released by adding dithiothreitol(DTT),leading to the fluorescence recovery and the amplification of the detection signal.Under optimized detection conditions,the detection range of the method is from 0.5?g m L-1 to 32?g m L-1 and the LOD was 0.18?g m L-1.Compared with the ic-ELISA established in this study,the sensitivity of the AuNP-TDNs-ic-FLIA was increased by about 700 times.Cross-reactivity of the method for detection of AMPA,GLUF and PMIDA were below 0.01%.A recovery of 95.5%?100.0%was obtained.The assay time was 3.5 h.5.Establishment of competitive fluorescence immunoassay based on AuNP-dsDNA nanoflare probe.SG stained dsDNA and anti-GLYP antibody were simultaneously modified on AuNPs for the preparation of AuNP-dsDNA nanoflare probe with controllable fluorescence"turn on"function,the fluorescence of which was initially quenched by AuNPs through FRET action.The prepared probe was characterized by TEM?UV-Vis scanning and fluorescence spectra,respectively.Then,a competitive fluorescence immunoassay(c-FLIA)for detecting GLYP was established based on the prepared nanoflare probe.The microtiter plate was coated with OVA-GLYP and a mixture of AuNP-dsDNA probe and samples containing GLYP was added followed with an incubation step.At this point,GLYP in the sample directly competes with the OVA-GLYP to bind the anti-GLYP antibody modified on the probe.After several washing steps to remove unconjugated components,DTT was added to release the flare DNA(dsDNA-SG)on the probe.Then the fluorescence recovery and the amplification of the detection signal are realized.Under optimized detection conditions,the detection range of the method is from 62.5 ng m L-1 to 4?g m L-1 with a LOD of28.6 ng m L-1.Cross-reactivity of the method for detection of AMPA,GLUF and PMIDA were below 0.01%.A recovery of 97.5%?101%was obtained.The method established in this study has a detection sensitivity 4800 times and 6 times higher than previous developed ic-ELISA and AuNP-TDNs-ic-FLIA based on the same anti-GLYP antibody.This method adopts direct competitive detection mode.Thus,the detection process is simpler and time-saving.The assay time was reduced to 1.5 h.Additionally,the usage of dsDNA-SG as flare DNA to replace TDNs-SG,further improve the signal amplification performance of the probe.6.Establishment of bio-barcode immuno-PCR based on dual-functionalized gold nanoparticles probe.Dual-functionalized AuNPs probes were prepared by conjugating AuNPs with both anti-GLYP antibodies and dsDNA,which simultaneously possessed the functions of recognition capture and signal amplification.The dsDNA consisted with a thiol-modified capture DNA(SH-capture DNA)and a bar-code DNA(signal DNA)which is complementary pairing bound to the capture DNA.Based on this probe,the bio-barcode immuno-PCR was established(AuNP-BB-iPCR)for detecting GLYP.Firstly,the mixture of AuNP probe and samples were added to the PCR tube pre-coated with OVA-GLYP.During this process of immune competition response,the GLYP in the sample competes with OVA-GLYP to bind the anti-GLYP antibody on the AuNP probe in the PCR tube.After washing to remove the unconjugated components,real-time PCR was directly performed by adding PCR reaction system to the tube.The amount of GLYP id determined by detecting the amount of signal DNA in the tube.The prepared probe was characterized by TEM,UV-Vis scanning spectroscopy,real-time PCR and SDS-PAGE,respectively.Under optimized detection conditions,a LOD of 8.9 pg m L-1 was obtained,and the linear range is from 122.1 pg m L-1 to 62.5 ng m L-1.Cross-reactivity of the method for detection of AMPA,GLUF and PMIDA were 2.36%?0.02%and 0.34%,respectively.A recovery of 98.3%?102.9%was obtained.In this study,real-time PCR was used for the cycle amplification of the biocode DNA resulting in the exponential amplification of the detection signal.Comparing with the previously established ic-ELISA,AuNP-TDNs-ic-FLIA and AuNP-dsDNA-c-FLIA based on the same anti-GLYP antibody,the detection sensitivity was increased by 7 orders of magnitude,4orders of magnitude(20000 times)and 3 orders of magnitude(3200 times),respectively.The assay time was 3.5 h.7.Conclusions.This paper focused on the interdisciplinary research between nanotechnology,fluorescence labeling techniques and immunoassay for the preparation of nanomaterial biosensing probes based on AuNPs and oligonucleotides.After the probe characterization,condition optimization and performance evaluation tests,effectiveness of the prepared probes in improving the sensitivity of GLYP immunoassays was demonstrated.Furthermore,an ic-ELISA method and three novel immunoassays were developed.Besides ic-ELISA method,the sensitivity of the other three methods meets the requirements of maximum residual limit of GLYP in food stipulated in the national standard.Compared with the GC-MS standard method for the determination of GLYP residues in food stipulated in the national standard,these methods does not require special testing conditions and expensive instruments.Only simple incubation steps were needed to complete the analysis within 1.5 h?3.5 h,with low cost and simple operation. |
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