Signal Amplification For Elisa Based On Color Reaction Catalyzed By 5-Nitro-8-Hydroxyquinoline Iron Complex

Enzyme-linked immunosorbent assay(ELISA)has been widely used in clinical diagnosis,food safety and biotechnology due to its high specificity and sensitivity.However,conventional ELISA often has the disadvantage of low sensitivity due to the limited enzyme captured on the adsorption carrier and the poor molar absorption coefficient of enzymatic products.In order to solve this problem,it is very important to develop a series of signal enhancement techniques to improve the detection sensitivity.In this paper,we found that 5-nitro-8-hydroxyquinoline-Fe(Ⅲ)(5-NQ-Fe)complex is a mimetic peroxide that can accelerate the reaction between H2O2 and TMB at certain conditions.Based on this found,we described a colorimetric method with high sensitivity for determination of 5-NQ.By introducing this method to ELISA,a highly sensitive ELISA was established.The main research contents are as follows:(1)By preliminary screening of 8-hydroxyquinoline and some of its derivatives.we found that the organic small molecule 5-NQ had the catalytic performance of HRP in the presence of Fe(Ⅲ).It can accelerate the rate of the oxidation TMB by hydrogen peroxide which resulted in blue TMBox.By optimizing the conditions,the sensitivity detection by naked eyes was improved.Additionally,we found the concentration of 5-NQ in the range from 0.01 to 0.7 μM is linear to the absorbance of TMBox(A=0.00635+0.042C5-NQ,r=0.987).The detection limit is 0.01 μM.And the color change caused by 0.03 μM is easy to be observed.The innovation of this method is to realize the detection 5-NQ with high sensitivity by using nake eyes.The method for determination of 5-NQ offers a methodology foundation for development of highly sensitive ELISA.(2)The corresponding substrate of β-galactosidase 5-nitro-8-quinolinyl-β-Dgalactopryanoside was synthesized.By adopting the method for determination of 5-NQ mentioned above,we established a highly sensitive method for monitoring β-galactosidase.Comparing to the traditional method based on direct detection of the absorbance of 5-NQ,the catalytic method improve the detection limit to 0.0007 U/mL,which has amplified the signal about 114 timed.The further introduce the method for determination β-galactosidase to ELISA result in a detection limit lower than 1000 times of traditional ELISA.When we used the established method for determination the concentration of human IgG,the detection limit was improved to 0.001 ng/mL,which is more sensitive than conventional ELISA.(3)The catalytic mechanism of 5-NQ-Fe(Ⅲ)complex was also investigated.By investigating the ratio of 5-NQ and Fe(Ⅲ),we concluded that the complex with a ratio of 2:1(5-NQ to Fe(Ⅲ))had high catalytic effect on the oxidation of TMB by hydrogen peroxide.The steady-state dynamics,ESR,UV-Vis absorption spectra and Raman spectra experiment verified that ·OH and Fe(Ⅳ)were generated in catalytic process.The generation of Fe(IV)is the key factor for explaining the efficient catalysis of 5-NQ.It is also the reason for highly sensitive determination of 5-NQ.