Development Of Highly Sensitive And Rapid Detection Kits For Cardiac Troponin Ⅰ

Cardiac troponin Ⅰ is a subunit of cardiac troponin,which can inhibit the activity of actin ATPase,so as to relax the muscle and prevent muscle fiber contraction.It is an important substance to maintain the normal relaxation and contraction of the heart,and can be used as an important detection indicator of myocardial injury.At present,cTnⅠ kit has been developed abroad for clinical detection and diagnosis of acute myocardial infarction,but the research and development of this diagnostic technology in China is still in its infancy,with complex diagnostic process,low efficiency and long-term dependence on imported products.In this study,we first used the colloidal gold method to screen and pair four kinds of cardiac troponin monoclonal antibodies that bind different epitopes from hytest,and then selected the 2 + 2 type pair with better sensitivity for subsequent development.Through the optimization and improvement of the colloidal gold method and fluorescence immunochromatography,we developed an immunodetection strip with high sensitivity,strong specificity,simple and fast for early diagnosis of myocardial infarction.As an important marker for the detection of acute myocardial infarction,cTnⅠ cannot reach the high sensitivity of 100 pg/m L with ordinary colloidal gold strip.In addition,cTnⅠ is easy to degrade and difficult to detect,which is the main obstacle to the development of cTnⅠ efficient detection strips.Therefore,through the optimization and improvement of three kinds of flow chromatography detection methods of cardiac troponin I,the detection sensitivity was improved.The main experimental results are as follows:a)A stable colloidal gold solution with good dispersion and uniform particle size was successfully prepared by optimizing the flow chromatography strip for the detection of cardiac troponin by colloidal gold method.During the experiment,antibody pairs with strong specificity and high sensitivity were selected for subsequent fluorescence immunochromatography strip and Raman probe immunoassay strip experiments.Subsequently,the process conditions of colloidal gold strip were optimized,and the sensitivity experiment of the optimized colloidal gold immunochromatographic strip for cardiac troponin was carried out,and the minimum detection limit was calculated to be 0.403 ng/m L,and no false positive was detected in random samples.b)The technological process of fluorescence immunochromatography strip was optimized.The results showed that the concentration of c-line antibody was 0.5 mg/m L,the concentration of T-line antibody was 1 mg/m L,the optimal reaction time was 15 min,the optimal microsphere activation time was 20 min,and the optimal amount of antibody was 10 μg.Under these conditions,the sensitivity of immunofluorescence strip was the highest,the detection limit was0.04 ng/m L,and the correlation coefficient was 0.99582.c)Gel modification was carried out on the flow measurement chromatography strip,and different concentrations of hydrogels were added after the C line of the flow measurement chromatography strip,so that the sample flow rate slowed down during the whole reaction process,increasing the reaction time and improving the sensitivity.The initial detection limit of colloidal gold reagent strip was 0.403 ng/m L,and 8% gelatin had the best effect.The detection limit increased to 0.174 ng/m L,which was 1.3 times higher.The initial detection limit of the fluorescent immunoassay strip was 0.04 ng/m L,and the modified effect of 4% gelatin was the best.The detection limit increased to 0.00613 ng/m L,which was 5.5 times higher.The initial detection limit of the fluorescence immunoassay strip of Wanfu biological Co.,Ltd.was0.023 ng/m L.the modified gelatin with a concentration of 4% had the best effect,and the detection limit increased to 0.00477 ng/m L,which was 3.8 times higher.d)The nanoprobe was prepared by covalent ligation and backfilled with PEG.The detection limit was 0.0895 ng/m L.the sensitivity of covalent ligation was 3.3 times higher than that of physical backfill strip.To sum up,this study combined the research advantages of the three methods to develop a highly sensitive cTnⅠ detection device to achieve rapid qualitative and quantitative detection of cardiac troponin I,which can be used for POCT detection and clinical auxiliary diagnosis,laying the foundation for the construction of a rapid quantitative detection system of cardiovascular markers.