Fast Detection Of Atrazine Using Calorimetric Biosensor

The rapid development of modern agriculture is inseparable from the use of pesticides, veterinary drugs and other agro-chemical products. Triazine, for example, was widely used in agricultural practice in China and even around the world. The use of these chemicals has pros and cons, on the one hand the quantity and quality of agricultural products can be improved, on the other hand irrational use will lead to residues in the soil, water and corresponding products, evoking a range of issues, like environmental pollution and food safety incidents. Developing rapid detection method of high stability, and specificity with simple sample preparation, is an integral part of effective food safety monitoring system.This project targeted typical pesticide residues, Atrazine, for investigating the feasibility of constructing novel thermometric sensor to circumvent drawbacks like tedious sample pretreatment, multi-step signal amplication and/or costly apparatus in traditional immunoassay or instrumental analysing. Conditions like flow injection and control, thermal signal acquisition and regeneration would be carefully optimized and the mechanisms of signal conversion and control be elucidated and finally the targeted pesticide residues be specificly and quantitatively analyzed. All the work will theoretically and technically pave the way for the calorimetric analysis in the field of pesticide residues detection.Calorimeter contains a thermostat unit(including reaction column), a flow pump, an injection valve, a sample loop, a Wheatstone bridge equipped with an amplifier, and a data recorder N2000 chromatography data system and connecting duct. Based on different experimental ideas, we successfully constructed calorimetric detection system against typical triazine pesticides, Atrazine through the preparation of appropriate materials and optimization of a series of experimental conditions.1、Direct competitive calorimetric assay using immobilized m Ab columnATZ coupled with β-lactamase was prepared by using active ester method. The purified anti-ATZ monoclonal antibody was covalently immobilized onto a solid support, controlled porous glass(CPG) by glutaraldehyde method. The immobilized m Ab would be filled into reactor, using as recognition element. Based on the principle of direct competition, ATZ and a constant amount of labeled ATZ derivative in mobile phase competed for the binding sites on the immobilized Ab, followed by the injection of enzyme-specific substrate. The amount of labeled antigens bound to the Ab is proportional to the enthalpy change recorded as peak height and is negatively related to the analyte concentration in the sample. Finally, the immunosorbent is regenerated for new use through the application of a desorbent solution that dissociates the Ab from the antigen–antibody(Ag–Ab) complexes without affecting the supporting characteristics. Several parameters that affected the performance of the dc TELISA were evaluated, such as concentration and nature of the enzyme substrate, ATZ-E concentration, efficiency of the regeneration liquid, effect of temperature on the sensor response, effect of organic solvent DMSO in the buffer solution, flow rate, and interval between two immediate steps during detection. ATZ determination was conducted in spiked corn stalk fresh cut and silage samples. The results were calculating through calibration plot and compared with that by HPLC for validation.The ATZ-E conjugate was successfully prepared by active ester method. The coupling ratio was approximately 2:1, determined by MALDI-TOF/MS. The linear range of new assay was 0.7-3.9 μg m L-1, with detection limit(LOD) of 0.4 μg m L-1. The reproducibility of the biosensor response in terms of the relative standard deviation for repeated injections of 16 mmol L-1 of penicillin was 5%. The cross-reactivity was most pronounced in the other s-triazine, SIM. The high cross-reactivity exhibited by SIM could be attributed to the structural similarity of the ATZ and the SIM, except for an extra methyl group in the SIM. The novel assay is immune to interference induced by complex matrix. The spiked corn stalk fresh cut and silage samples could be injected only after filtration and p H adjustment, with recoveries between 88%-107%, comparable with that of high-performance liquid chromatography(HPLC). This thermometric signal response of the novel assay is fast, reproducible, with simple sample pretreatment. The immobilized antibody column can be used continuously for about four months, facilitating application in daily work.Fast thermometric detection of ATZ with simple sample pretreatment was realized. The reactor filled with antibody-immobilized CPG was rubust enough to meet the on-site rapid detection requirement, and the novel method could be an alternative for large-scale pesticide-residue screening.2、Universally competitive calorimetric assay using Protein G sepharoseThe established method was improved by introducing the incubation step and universal recognition element. ATZ and β-lactamase-labeled ATZ were employed in a competitive immunoassay using a monoclonal antibody(m Ab). After the off-column liquid phase competition, the formed antigen-antibody complex and enzyme-labeled antigen-antibody complex were captured on the Protein G Sepharose TM 4 Fast Flow(PGSFF) column support material. Injected β-lactamase substrate ampicillin was degraded by the column bound ATZ-β-lactamase generating detectable heat signal. The signal strength is inversely proportional to the analyte amount. Several assay parameters were optimized, including: substrate concentration, regeneration conditions, as well as the mAb and ATZ-β dilution ratios and concentrations. ATZ determination was conducted in spiked tap water samples. The results were calculating through calibration plot and compared with that by HPLC-MS/MS for validation.A highly sensitive thermal enzyme linked immunoassay, TELISA, was developed for the rapid detection of ATZ in tap water,with linear range of 0.7-4.8 μg m L-1, detection limit(LOD) of 0.7 μg m L-1. The reproducibility of the biosensor response in terms of coefficient of variation for repeated detection of sample in the presence of 6.25 μg m L-1 ATZ was no more than 8%. Sample with certain amount of ATZ mixed with ATZ-β-lactamase and m Ab, and then the competitive reaction was carried out at 37 oC for 30 min.The target was subjected to recovery experiments of spiked tap water, with recovery of 103%-116%, comparable with that of liquid chromatography-mass spectrometry/mass spectrometry(UHPLC-MS/MS). We attributed this dramatic increase in sensitivity over our previously published work to: 1) capture of already formed immune-complexes on the column via immobilized Protein G, which eliminates chemical immobilization of the antibody, 2) off-column pre-incubation allows immune complexes to form under nearly ideal conditions, which speeds-up and enhances complex formation, as well as allowing control over incubation times, 3) multiple buffers can be used to, in one case enhance immune-complex formation, and in the other maximize enzyme activity. Furthermore, the scheme creates an universal assay platform in which sensing is performed in the off-column incubation and detection after capture in the ET detector, opening up the possibility of detecting any antibodies-available substance.Compared with our previously published work, a highly sensitive and universal assay platform was constructed by introducing pre-incubation step and Fc-region-specific immunosorbent. Separating the sensing phase from the detection phase is help of finishing the competitive reaction, which was thought to be crucial to sensitivity. The immunosorbent capturing immune-complexes by combining with Fc region of Ig G make the assay target change in the off-column incubation without switching columns possible, which provided the basis for universal detection.Sensing phase, off-column pre-incubation in 37℃ water bath, like that of traditional ELISA, wasn`t conditioned by detection phase. Thus, the reaction could be carried out under nearly ideal conditions, such as optimized dilution ratio of m Ab, buffer additive, full immune-complex formation time, etc. While on-column sensing in previous work took place simultaneously with detection phase. The analyte, ATZ competed with ATZ-β-lactamase conjugate for binding sites on the immobilized m Ab in flowing buffer, which obviously restricted optimization. The Protein G capture column was less stable than the antibody column used in our previous work. We assume the main cause is the much harsher regeneration condition, which could result in more severe activity loss of PGSFF. However, the stability was more than adequate for routine use with dozens determinations being able to be performed with each column. Approaches for extending column life are under investigation.3、Biofunctional surface-imprinting thermometric-biomimetic sensor for fast detection of atrazineA thermometric biomimetic sensor, using surface-imprinted micron-sized porous polymers(S-MIPs) as antibody-mimicing recognition element and enthalpy-generating element, was established to circumvent activity-reducing induced by organic solvent and coloration-interfering by complex food matrix in traditional immunoassay. The influence of functional monomer was investigated and sodium p-styrenesulfonate(SSS) was choosen. S-MIPs were synthesized using synchronously graft-polymerizing and imprinting method. S-MIPs were filled into reaction column and directly used to detect enthalpy change induced by recognition between ATZ and imprinted cavities. The signal intensity was positively proportional to analyte concentration. ATZ was specificly and quantitatively analyzed after conditions like flow injection and control, thermal signal acquisition and amplification and regeneration were carefully optimized and the mechanisms of signal conversion and control were elucidated. By comparison with the HPLC method, the novel method was assessed.The influence of the functional monomer sodium p-styrenesulfonate(SSS) and methacrylic acid(MAA) on the binding characteristics was analysed. SSS cross-linked S-MIPs showed a much higher binding enthalpies of atrazine compared with MAA cross-linked S-MIPs. Furthermore interactions of a SSS cross-linked S-MIPs against model template atrazine was studied using a thermistor. Several assay parameters were optimized, including: imprinting solution, running buffer and flow rates. Label-free detection of atrazine in spiked water samples were realized in the range of 3.9 – 120 μg m L-1. The detection limit was 1.1 μg m L-1. The reproducibility of the biosensor response in terms of the relative standard deviation for repeated injections of 15 mg L-1 of ATZ was no more than 1%.ATZ-spiked water samples exhibited a recovery rate of 82.8% to 92.5% which correlated well with that of HPLC measurements. The S-MIPs sensor was used to characterize the cross activity and the influence of shape selective molecular recognition is discussed. The novel method can consecutively detect hundreds of samples in a row, without regeneration. After filtration and p H adjustment, the spiked sample can be added, suggesting easy application in fast detection. Isothermal binding experiments showed the S-MIPs contained two kinds of imprinted cavities, but in the calorimetric measurement, a good linear response with increasing concentration of ATZ were obtained.Compared with biological recognition element, biomimetic element is easy-made, time-effective and quality-homogeneous. The novel assay realize label-free, specific and direct determination of enthalpy change caused by recognition, with greatly improved throughput compared with the first two methods. Hundreds of assay could be carried out without frequent regeneration. The assay is robust enough to be applied to in-situ fast detection.4、Highly sensitive thermometric-biomimetic sensor for fast detection of atrazine based on direct competitivenessThe established biomimetic method was improved by introducing competitiveness. Taking methacrylic acid(MAA) as the functional monomer, S-MIPs were synthesized using synchronously graft-polymerizing and imprinting method. ATZ and β-lactamase-labeled ATZ in mobile phase compete for the imprinted cavities on S-MIPs, filled in reactor. Enthalpy change induced by catalysis was inversely proportion to analyte concentration. Conditions like components of imprinting system, mobile phase and regeneration phase and enzyme substrate concentration, ATZ-β dilution ratios should be fully optimized. The contents of ATZ in tap water samples were calculated based on calibration curve. Assessment of the new assay was carried out by comparing with detection results of spiked tap water samples by HPLC-MS / MS.Contrary to SSS cross-linked S-MIPs, MAA cross-linked S-MIPs showed a sensitivity similar to that of the second established method mentioned above. After optimization, the calibration curve was obtained,with experimental linear detection range of 0.6-4.1ng m L-1, LOD of 0.4 μg m L-1. The reproducibility of the biosensor response in terms of the relative standard deviation for repeated injections of 4 mmol L-1 of Ampicillin was about 2%.The novel assay is highly selective and the cross-reactivity with structural analogues were 5.7% and 3.7% against simazine and melamine perspectively, which was much better than that of ATZ-m Ab. The recoveries were between 89.8%-95.3% of spiked tap water, comparable with that of high performance liquid chromatography-mass spectrometry/mass spectrometry(HPLC-MS/MS), and the S-MIPs volumn was stable after dozens of tests. Continuous detection needs regeneration between samples, which restricted high-throughput detection. In competitively calorimetric measurement,a good linear response with was obtained as increasing concentration of ATZ.The improved biomimetic assay, based on thermometric assay and surface-imprinting technique combined, was highly sensitive and specific by introducing competition. Regeneration reduced throughput, but it still worth it while high sensitivity was realized.Compared with the second established assay, the present method was similar in sensitivity and much better in specificity and without off-line incubation, suggesting the excellent recognition property of S-MIP.