Two Kinds Of Urease-mediated Enzyme-linked Immunosorbent Assays For Highly Sensitive Detection Of Ochratoxin A

Ochratoxin is a toxic secondary metabolite produced by several Penicillium and Aspergillus,which seriously threatens the security of agricultural product on a global scale.Among them,ochratoxin A（OTA）is widely known because of its greatest toxicity,the deepest degree of contamination of crops,the widest distribution,and the closest relationship with human health.Traditional detection methods,such as high-performance liquid chromatography（HPLC）and liquid chromatography-mass spectrometry（LC-MS）are always suffered from its expensive instrumental cost,complex operating procedure and professional trainees,which limit itself promotion and application.Enzyme-linked immunosorbent assay（ELISA）as a classical detection method which has the advantages of simple operation,low cost,and high throughput screening.However,due to the low detection sensitivity,it can not be realized the quantitative detection of trace substances;and tetramethylbenzidine（TMB）has a single color and poor visibility by the naked eye,can not achieve qualitative detection.Therefore,a novel ELISA detection method capable of high-sensitivity and naked-eye interpretation becomes a hot spot in the current research.In this study,OTA was used as the target analyte,anti-OTA monoclonal antibodies（anti-OTA mAbs）and OTA labeled urease were used as bio-recognition elements for the establishment of urease-mediated pH responsed and plasmonic ELISA,respectively.The method is based on the traditional direct competition ELISA,using urease instead of horseradish peroxidase（HRP）and bromcresol purple（BCP）instead of TMB for color development.The competitively conjugates catalyzes urea into ammonia,initiating pH change of the solution.As an acid-base indicator that is extremely sensitive to pH,BCP has a dose-effect relationship between the color change of the solution and the concentration of the analyte.The urease@OTA conjugate was prepared according to the active ester method with the molar ratio of urease and OTA at 1:5.73.The optimal concentrations of anti-OTA mAbs and urease@OTA were optimized at 0.085μg/mL and 1.94μg/mL,respectively,by a checkerboard titration method.In addition,the various parameters that could influenced the immunological and colorimetric reactions were further optimized.The immunoreaction buffer was 10%（v/v）methanol solution containing 0.128 M of NaCl with a pH of 5.96,the concentration of urea was 0.1 M,and the catalysis reaction of urease and urea was performed at 37℃ for 30 min.Under the optimal reaction conditions,the proposed method exhibited a high sensitivity for OTA naked-eye detection with a cutoff limit of 50 pg/m L,and a good linear range of 12.5¹00 pg/mL for OTA quantitative detection（r²=0.9895）,with a half maximal inhibitory concentration of 31.4 pg/mL,which is 12.5-and 11.3-flods lower than that of conventional HRP-based ELISA.Recovery studies of the proposed method were evaluated by analyzing OTA-spiked samples in corn extracts.The average recoveries of the intra-assay ranged from 83.7%to 108.8%with a coefficient of variation（CV）ranging from 3.71%to 13.5%,and those for the inter-assay ranged from 86.2%to111%and CV value from 7.13%to 14.5%,respectively.The reliability of the proposed pH responsed ELISA for OTA quantitative detection by naked eye were evaluated by analyzing 70 OTA-spiked real corn samples,and 95.71%of OTA spiked samples showed the correct results,indicating an acceptable accuracy and precision for the quantitative detection of OTA in real corn samples.（2）The principle of urease-mediated metallization plasmonic enzyme–linked immunosorbent assay（pELISA）is as follow:urease hydrolyzes urea to produce ammonia.Silver nitrate is reduced to silver nanoparticles in the presence of ammonia and glucose,then depositing on the surface of gold nanoflowers（AuNFs）to form the core-shell structure of Au@Ag.With the different deposition thickness of silver particles,the plasmon resonance peaks are blue-shifted and the solution color changes from blue to yellow.Therefore,we can monitor the plasma resonance peaks and color changes of AuNFs to achieve the quantitative and qualitative detection of analytes.The optimal concentrations of anti-OTA mAb and urease@OTA at 0.16μg/mL and7.55μg/mL,respectively,were optimized by a checkerboard titration method.Various parameters which affected the metallization reaction of AuNFs were further optimized.The optimal concentrations of Glu,AgNO₃ and urea were 200 m M,10 mM and 100mM,respectively.The metallization reaction time was 40 min,while enzymatic reaction time was also 40 min.Under the optimal reaction conditions,the proposed method exhibited a favorable linear range of 5.0 pg/mL⁶40 pg/mL for OTA quantitative detection with a limit of detection（LOD）at 8.205 pg/mL.The regression equation could be represented as y=-0.0565ln（x）+1.091 with a reliable correlation coefficient（R²=0.9975）.The proposed method also presented a high sensitivity for OTA qualitative detection by the naked eye with a cutoff limit of 40 pg/mL.The LOD and cut-off limit values are 15.6-and 14.3-folds lower than those of horseradish peroxidase（HRP）-based ELISA.Meanwhile,the method also showed excellent specificity against four other mycotoxins with no obvious cross-reaction.Moreover,the recoveries for OTA-spiked rice,corn,wheat,and white wine samples ranged from 81.5%to 106%,with coefficient of variation ranging from6.13%to 18.7%.These results showed a good agreement with those obtained by an ultra-performance liquid chromatography（UPLC）methodand no false positive and false negative results appeared by analyzing the 72 actual samplesIn conclusion,two kinds of urease-mediated enzyme-linked immunosorbent assays were established for OTA highly sensitive detection by naked eye or microplate reader.