Optical Immunoassays For The Detection Of Exogenous Harmful Substances In Traditional Chinese Medicines

Traditional Chinese medicines?TCMs?with thousands of years history have attracted increasing attention around the world due to their good therapeutic effect and low toxicity.However,they still face many obstacles in global marketing because of their unstable quality.Excessive pesticide residues,heavy metals and aflatoxins play key roles in the quality instability of TCMs.In order to ensure the safety and promote the internationalization of TCMs,the development of detection protocols for pesticide residues,heavy metals and aflatoxins has gradually attracted increasing interest of researchers.In decades,various chromatographic and mass spectrometric methods such as gas chromatography-flame photometric detection,high performance liquid chromatography?HPLC?,desorption electrospray ionization mass spectrometry and gas chromatography-mass spectrometry have been well developed for detecting pesticide residues in TCMs.In the meantime,some instrumental methods such as atomic absorption spectrometry,inductively coupled plasma mass spectroscopy,inductively coupled plasma atomic emission spectrometry and X-ray fluorescence spectrometry have been well developed for the detection of heavy metal ions.Additionally,the detection methods of aflatoxin are mainly based on chromatographic techniques including immunoaffinity column-based HPLC.Usually these protocols are utilized as confirmatory assays due to their ideal reliability.Nevertheless,they always require complicated manipulation,skilled personnel,sophisticated instrumentation and time-consuming sample pretreatment.Thus,they are not fit for rapid screening and field assay.Chemiluminescence?CL?and fluorescence?FL?detection have been widely used in immunoassay,and showed their remarkable properties such as high sensitivity,wide linear range and simple instrumentation.In this study,we designed a CL immunochromatographic test strip to detect pesticide residues in TCMs.Meanwhile,two label-free FL immunosensors were proposed to detect heavy metals and aflatoxin in TCMs,respectively.The detailed information is showed as following:1.Multiplexed immunochromatographic test strip for time-resolved CL detection of methyl parathion and imidaclopridA multiplexed immunochromatographic test strip based on a time-resolved chemiluminescence?CL?strategy was developed for quantitative detection of pesticide residues.We used a novel bifunctional antibody as the sole recognition reagent that could recognize methyl parathion and imidacloprid simultaneously.Horseradish peroxidase?HRP?and alkaline phosphatase?ALP?were used as the CL probes to label the haptens of methyl parathion and imidacloprid,respectively.After the labeled haptens competed with methyl parathion and imidacloprid to bind with the BfAb immobilized on the test strip,the two CL reactions catalyzed by the enzymes were triggered simultaneously by coreactants injection.Due to the distinct CL kinetics characteristics of HRP and ALP,the signals for methyl parathion and imidacloprid detections were collected at 2.5 s and 300s,respectively.The linear ranges for methyl parathion and imidacloprid were both 0.1-250 ng/mL,with detection limits of 0.06 ng/mL?3??.The whole assay process could be accomplished within 22 min.The detection results for spiked in Angelica and Liquorice samples demonstrated its application potential,with the recovery of 95.6-117.0%.The proposed method provided a low-cost,facile and rapid tool for multiplexed screening of pesticide residues using single antibody.2.Label-free fluorescent immunoassay for Cu²⁺ion detection based on UV degradation of immunocomplex and metal ion chelatesMouse anti Cu²⁺-ethylenediaminetetraacetate?EDTA?monoclonal antibody?McAb?was used to capture EDTA chelated Cu²⁺ions.Then UV radiation was adopted to degrade the formed immunocomplex and Cu²⁺-EDTA chelates to release free Cu²⁺ions.The released Cu²⁺ions could be quantified based on their quenching effect to the fluorescent emission of CdSe/ZnS quantum dots.This protocol showed high sensitivity although it was free of probe labeling of antibody.It also showed many merits such as simple manipulation,low cost and high throughput.Importantly,this protocol avoided the interference from most common cations which could also quench CdSe/ZnS quantum dots FL.The linear range for Cu²⁺detection was 10-1000 ng/mL with a detection limit of0.33 ng/mL?3??.The proposed method has been successfully used to detect Cu²⁺in American ginseng and Ganoderma,with acceptable recovery values of 81.9-128.6%.We believe that the proof-of-concept work can be extended to detect other heavy metals by using their specific antibodies in future studies.3.A label-free fluorescent immunoassay based on fluorescence self-enhancement of aflatoxin B₁ by UV degradationMouse anti aflatoxin B₁?AFB₁?McAb was modified on carboxylic-magnetic beads to achieve efficient enrichment and separation of AFB₁.Thus,we obtained the immunocomplex of AFB₁-McAb by magnetic separation.Then a UV lamp with an emission at 365 nm was utilized to enhance the FL emission of AFB₁ according to published research.Finally,the FL signal was collected to quantify AFB₁.The linear range for detection of AFB₁ was 1.0-1000 ng/mL,with a detection limit of 0.21 ng/mL?3??.The proposed method has been successfully used for real samples including Astragalus membranaceus and Salvia Miltiorrhiza,with recovery values of 95.4-107.7%.This developed method showed many advantages such as low cost,easy fabrication,short assay time,and user-friendliness.It has good development prospect in practical application.In conclusion,CL and FL techniques were combined with immunoassay to detect pesticide residues,heavy metals and aflatoxins in TCMs.Compared with the traditional ELISA and other immunoassays,the present protocols show many advantages including higher sensitivity,simpler manipulation and shorter assay time,which are expected to provide a new pathway for rapid screening and field detection.