Chemiluminescent Immunosensors For The Detection Of Pesticide Residues And Heavy Metals In Chinese Herbal Medicines

As a significant integrant of Chinese Medical Culture,Chinese herbal medicines(CHMs)are increasingly drawing attention from numerous people all around the world due to their various functions for treatment and health care.However,there exist many restricts in the process of internationalization and modernization of CHMs,among which the instability of CHMs quality is one of the most remarkable aspects.Moreover,the overproof pesticide residues and heavy metals dramatically affect the quality of CHMs.Therefore,the development of a variety of protocols of high efficiency and low cost is gradually becoming vital for promoting the modernization of CHMs.Recently,there are numerous chromatographic and mass spectrometric methods reported for the detection of pesticide residues,such as gas chromatography,high performance liquid chromatography,gas chromatography-mass spectrometry and high performance liquid chromatography-mass spectrometry.Meanwhile,some spectroscopic protocols are developed for monitoring heavy metals,including ultraviolet spectrophotometry,atomic absorption spectroscopy,atomic fluorescence spectroscopy and inductively coupled plasma atomic emission spectrometry.Due to high accuracy and high sensitivity,it is appropriate to utilize these methods as confirmatory assays in a laboratory.However,such disadvantages as high lost and sophisticated operation restrict the application of these methods for field assay and rapid testing.Without the need of external light source,chemiluminescence(CL)is an ideal protocol for the detection of pesticide residues and heavy metals due to the advantages of easy operation,cost efficiency,high sensitivity and wide linear range.In the study,several CL immunosensors were designed for the detection of pesticide residues in CHMs based on several high-efficient CL probes such as enzymes,metal nanoparticles,metal oxide nanoflowers and carbon-metal nanocomposites.Meanwhile a label-free CL immunosensor was also proposed for the detection of heavy metals in CHMs.The detailed information is showed as following:1.CL kinetics-resolved bienzyme immunoassay for the multiplexed detection of methyl parathion and imidacloprid.There are remarkable differences between the reaction kinetics characteristics of horseradish peroxidase(HRP)-luminol-H2O2 and alkaline phosphatase(ALP)-di sodium 3-(4-methoxy-spiro {1,2-dioxetane-3,2'-(5'-chloro)tricyclo(3.3.1.13,7)decan}-4-yl)phenyl phosphate CL systems.After triggering these two systems,CL emission of the former system could reach the maximum at about 0.6 s,while the CL signal of the latter system increased slowly in a long period.After 1000 s,the signal interference between two CL reactions became negligible.And thus,HRP and ALP were utilized as the CL probes to tag the haptens of methyl parathion and imidacloprid,respectively,and 0.6 s and 1000 s were adopted as the time windows for collecting two CL responses.Due to the capability of simultaneous binding of methyl parathion and imidacloprid,bispecific monoclonal antibodies against methyl parathion and imidacloprid were used as the unique recognition reagent for the development of CL kinetics-resolved bienzyme immunosensor for pesticide residues.After two complete competitive immunoreactions,the two analytes were simultaneously detected,and their linear ranges were both 1.0-500 ng/mL with both limits of detection of 0.33 ng/mL(S/N = 3).The CL-resolved immunosensor was successfully applied for the detection of two pesticide residues in Ginseng and American Ginseng with an acceptable recovery of 80-118%.2.A dual-sensing immunochromatographic test strip for detecting chlorpyrifos utilizing MnO2 nanoflowers(MnO2 NFs)as a visual/CL probe.Based on the reduction of KMnO4 by poly(diallyldimethylammonium chloride),MnO2 NFs with a uniform size were successfully synthesized.Compared with Pt-Au bimetal nanoparticles,Pt-Pd bimetal nanoparticles,Prussian blue nanorods and graphene oxide,the MnO2 NFs remarkably enhanced the CL signal of luminol-H2O2 system.After a careful investigation,the enhancing effect of MnO2NFs on CL emission was based on the oxidant activity instead of the peroxidase-like catalytical effect.Due to the dark brown and the enhancing effect of MnO2 NFs,a dual-sensing immunochromatographic test strip utilizing MnO2 NFs as a visual/CL probe was designed for detecting pesticide residues.Chlorpyrifos-bovine serum albumin was deposited as the coating antigen on the NC membrane of a test strip for forming a test line.The chlorpyrifos antibody was tagged to MnO2 NFs as the tracer antibody,after a complete immunoreaction,the tracer antibody was captured by the coating antigen.And then a brown color appearing on the test strip was observed by naked eyes as a visual/semi-quantitative response.After triggering the MnO2 NFs-luminol-H2O2 CL system on the test line,CL signal was collected as a sensitive and quantitative response for detecting chlorpyrifos.The linear range of chlorpyrifos was 0.1-50 ng/mL with a limit of detection of 0.033 ng/mL(S/N =3).The dual-sensing immunochromatographic test strip was successfully applied to detect chlorpyrifos in Astragalus and Poria cocos with an acceptable recovery of 90-120%.3.Spatially-resolved immunochromatographic test strip based on luminol-reduced gold nanoparticles(LuReGNPs)as a dual-sensing probe for simultaneously detecting methyl parathion and fenpropathrin.LuReGNPs were successfully synthesized by reducing HAuC14 with luminol.The LuReGNPs with red color could emit strong CL signal.Methyl parathion antibody and fenpropathrin antibody were tagged to LuReGNPs as spatially-resoled CL tracers.Methyl parathion-bovine serum albumin and fenpropathrin-bovine serum albumin were deposited on the NC membrane of a test strip to form two test zones.After two competitive immunoreactions,the CL probes were captured by their corresponding coating antigens on test zones,respectively.After that,red colors appearing on two test zones were observed as colorimetric signals for semi-quantitatively detecting the pesticide residues.After triggering the luminiol-HRP-H2O2 CL reaction on the two test zones,the CL signals were collected for sensitively detecting methyl parathion and fenpropathrin.Their linear range were 0.5-200 ng/mL and 0.3-200 ng/mL with the limits of detection of 0.17 and 0.10 ng/mL(S/N = 3),respectively.A recovery test of methyl parathion and fenpropathrin was successfully performed to detect pesticides in Gardenia and Folium Mori,and an acceptable recovery of 95-111%demonstrated the application potential of the novel immunosensor.4.Dual-readout immunochromatographic test strip based on peroxidase-like g-C3N4-BiFeO3 nanocomposites(NCs)for the detection of chlorpyrifos and carbaryl.g-C3N4-BiFeO3 NCs were synthesized through a one-step sol-gel combustion method.The nanomaterials showed high peroxidase-like catalytical activity for enhancing the CL emission of luminol-H2O2 system.Chlorpyrifos antibody and carbaryl antibody were tagged with the g-C3N4-BiFeO3 NCs as two tracer antibodies.The coating antigens of chlorpyrifos and carbaryl were deposited on a NC membrane for forming two test lines.After two competitive immunoreactions,the two tracer antibodies were captured on the test lines by their corresponding immunogens.Brown color of the g-C3N4-BiFeO3 NCs was recorded as a visual/qualitative readout and the CL signal from g-C3N4-BiFeO3 NCs-luminol-H2O2 system was collected as a sensitive/quantitative readout for the detection of chlorpyrifos and carbaryl.The linear range of the two analytes were 0.1-60 ng/mL and 0.1-40 ng/mL,respectively,with both limits of detection of 0.033 ng/mL(S/N = 3).An acceptable recovery value of 81-119%were obtained in the spiked Salvia miltiorrhiza and Codonopsis pilosula sample test,implying the application potential of the dual-readout multiplexed immunochromatographic test strip.5.A label-free immunoassay based on self-enhancement of Cu-EDTA on luminol-H2O2 CL system for the detection of Cu(?).An ultra-facile,rapid,sensitive,low-cost immunoassay was constructed for the detection of heavy metal residues,utilizing Cu(?)as a model analyte.Cu(?)reacted previously with ethylenediaminetetraacetate(EDTA),and then the chelate was captured by immobilized monoclonal antibody for Cu(?)-EDTA chelate.After the immunoreaction,urea was adopted as a chemical denaturant of the monoclonal antibody to release the captured Cu(?)-EDTA chelate.Then,a very strong CL signal was obtained utilizing its remarkable enhancement to the CL reaction of luminol-H2O2 system.The CL intensity is linear relative with the Cu(?)concentration in a very wide range of 1.0-1000 ng/mL,with a detection limit of 0.33 ng/mL(S/N = 3).Based on dual selectivity of the antibody and the luminol-H2O2 system,the facile label-free immunoassay showed a great specificity of the detection of Cu(?).The proposed method was successfully used to detect Cu(?)in Ginseng and Salvia Miltiorrhiza with an acceptable recovery value of 82-113%.In conclusion,based on several novel CL probes including enzymes,metal oxide nanoflowers,luminophore-modified metal nanoparticles and carbon-metal nanocomposites,several CL immunosensors were developed for the detection of pesticide residues in CHMs.Furthermore,three multiplexed immunoassays were obtained through the usage of the multi-tag model and the spatially-resolved model.The proposed immunosensors showed such advantages as high throughout,high sensitivity,miniaturization and portability,implying their great application potential in field screening and rapid testing.Meanwhile,by utilizing the self-enhancing effect of heavy metal(Cu(?)as a model analyte)on luminol-H2O2 system,an ultra-facile label-free immunosensor was designed for detecting heavy metals in CHMs.Superior to traditional indirect immunoassay for heavy metals,the immunosensor showed high sensitivity and high analytical efficiency.Compared with traditional spectroscopic methods,the proposed immunoassay showed such merits as low cost,good portability for rapid assay and field testing.