Detection Of MG And AT Residues By Signal Amplified SPR Biosensor

Abuse of pesticide and veterinary drug causes residues in soil, water and agriculturalproducts, resulting in environmental pollution and entering the food through the foodchain. Pesticide and veterinary drug residue in food is a serious threat to human health,and makes our food and agricultural exports suffer a "green barrier" in international trade,which retards the development of food and agricultural industry. Currently, commondetecting methods of pesticide and veterinary drug residues, such as gas chromatography(GC), high performance liquid chromatography (HPLC), gas chromatography-massspectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), requirecomplicated and time-consuming pre-treatment process and can’t achieve rapidassay.Surface plasmon resonance (SPR) biosensor technology is widely used in the fieldof environmental science and food and drug detection because of its remarkableadvantages such as real-time, fast, label-free and non-destructive. However, when SPRsensor is used to directly detect samples with complex matrix or low level analytes, theresponse signal is weak with low sensitivity. This work, combined with molecularimprinting and immune technique, developed some signal amplification SPR sensingsystems for the detection of pesticide and veterinary drug residue using some signalamplification materials.1. Preparation and application of MIP embeded with gold nanoparticles on thechip surface of SPR sensorThe molecular imprinting technique (MIT) is a newly developed approach that cancreate specific molecular recognition sites for templates. Molecularly imprinted polymers(MIP) are stable, robust and suitable as a recognition element of SPR sensor, which cancomplete separation, enrichment and detection in a single step. Gold nanoparticle (GNP),as signal amplification materials, was introduced in MIP and the GNP signalamplification MIP-SPR sensing system was constructed as follows: the initiator (AIBN)was pasted on the SPR gold chip by spin-coating with PVC. Then the SPR chip wasimmersed in the pre-polymerization solution, which containing MUA-modified GNP toprepare malachite green (MG) MIP film and NIP film. Atomic force microscope (AFM) results indicated that the thickness of the MIP film embedded with GNP was56nm,which was proper as a SPR recognition element. Adsorption results of different polymerfilms to different concentrations of MG indicated that the SPR response of MIP filmembedded with GNP is twice more than that of MIP film without GNP. The SPR signalwas successfully amplified. The calibration curve was Y=0.03X+6.15(r=0.9971). Therecoveries of spiked waters samples were84-110%.2. Construction of signal amplified SPR immunoassay using goldnanoparticles-labeled secondary antibodyAntibody, presenting advantages such as high affinity and specificity, can improvethe detection sensitivity and specificity. Therefore, we constructed a SPR immunosensorfor the detection of atrazine (AT), and employed GNP-labeled secondary antibody(GN-Ab2) to enhance the SPR signal. Detailed works were carried out to obtain theoptimum condition of detection including AT-OVA amount, AT-mAb concentration,regeneration buffer, regeneration time, and amount of second antibody. Under the optimalconditions, the working range of the biosensor was2.18-629.06ng/mL with asignificantly low detection limit at0.72ng/mL. Compared with SPR immunoassaymethod without signal amplification, the detection limit of which was2.34ng/mL and thedetection range was6.4-188.0ng/mL, the signal amplified SPR immunoassay presentedlower detection limit and a wider detection range. Results of specificity experimentsshowed that melamine, chlorpyrifos, and2,4-D were not react with AT-mAb exceptprometryn, which indicated a well specificity. Water samples spiked with AT could bedetermined directly without further treatment and the recoveries were between83~95%with relative standard deviation (RSD) below5%. Compared to HPLC method, the signalamplified SPR immunoassay had higher sensitivity and lower detection limit.Furthermore the developed method was scarcely interfered by color or ionic strength ofsample solution, and all reagents could be added to the detection system in a single step,thereby removing the need for time-consuming incubations.3. Construction of signal amplified SPR immunoassay using magneticnanoparticlesIn order to simplify the tedious pretreatment of complex samples, magneticnanomaterials were employed in SPR detection system, which was used as a separationand enrichment material, and also a signal amplified material of SPR sensor to improvethe detection sensitivity. The AT-mAb was coupled to the carboxyl-modified magneticnanoparticles (MNP) by EDC/NHS activation. After optimizing conditions, such as the amount of AT-mAb and MNP, combination time and the concentration of AT-mAb-MNP,the SPR immunosensor for indirect competitive detection of AT was developed. Thecalibration curve was Y=-0.0743+0.2397X(r=0.9924), IC50was74.77ng/mL. Theworking range was0.89-6309.57ng/mL. The results indicated that MNP couldsuccessfully amplify the SPR signal and improve the detection sensitivity.In this work, based on the MIT and immunoassay, the signal amplified effect of SPRby GNP, gold-labeled secondary antibody and MNP were investigated. Thenano-gold-MIP-SPR sensor, gold nanoparticle-labeled secondary antibody–SPR sensorand antibody-MNP-SPR sensor were respectively established for detecting pesticide andveterinary drug residue. The results showed that the signal amplification materials couldeffectively improve the detection sensitivity and the working range. After furtheroptimization and improvement, the developed SPR sensor platforms are expected toprovide highly sensitive detection methods for other fields, such as environmentalmonitoring, biological engineering, clinical medicine and drug analysis. It presents apromising practical value and application prospect.