Study On DNA-based Immunoassay For The Quantitative Detection Of Cardiac Troponin I

Cardiac troponin I(cTnI)is a part of the troponin protein complex,which involved in the process of myocardial contraction.It plays an important role in the diagnose of myocardial injury,myocardial infarction and other cardiovascular disease.The commonly used detection methods for cTnI are enzyme-linked immunosorbent assay(ELISA),chemiluminescence immunoassay(CLIA),gold standard immunoassay and so on.Most of these methods are based on the specificity of antibody identification.But it is difficult to detect trace substances.At present,cTnI as myocardial infarction "gold standard",has been widely used in clinical diagnosis.At the same time,many of the literature also reported the relativity between a low concentration levels of cTnI changes and other cardiovascular diseases.This requires a higher sensitivity,better accuracy to detect cTnI.Aptamer can be used as a kind of nucleic acid molecule which can specifically bind to target molecule,and has the characteristics of high affinity,easy to modification and preparation.In recent years,in order to improve the detection sensitivity,nucleic acid molecules are increasingly being used in analytical testing,these methods was converted protein detection to nucleic acid detection.Amplification reaction was used to increase the amount of detectable nucleic acid target,to achieve the purpose of detection of trace substances.These methods were immune polymerase chain reaction(IPCR),immuno-rolling circle amplification(IRCA),proximity Ligation Assay(PLA)and so on.The sensitivity and specificity of these assays are significantly higher than conventional ELISA methods.In this study,we selected the rolling circle amplification(RCA)as the signal amplification method,using cTnI specificity of nucleic acid aptamers,to achieve high sensitivity detection of cTnI.Firstly,the RCA reaction system was established,and the type,dosage and reaction time of the enzyme were optimized.The agarose gel electrophoresis was used to characterize the reaction.The results showed that the best condition for the cyclization reaction was 10 U T4 DNA ligase with 2 h incubation.The optimal conditions of the amplification reaction were 10 U phi 29 DNA polymerase with overnight incubation.Secondly,two aptamer sequences were selected and the corresponding aptamer primer complexes were designed and synthesized.The effects of aptamer primer complexes and cyclization products on the affinity and specificity of cTnI were investigated.The results showed that the cyclization product did not affect the specific recognition of cTnI.Thirdly,we designed and synthesized a molecular beacon probe that binds to RCA products.On the basis of its validity,a method based on specific recognition of nucleic acid aptamers,RCA signal amplification and molecular beacon detection was developed for cTnI detection.The linear range,detection limit and limit of quantification were obtained by quantitative detection and optimization.The interference and recoveries of the related substances were also investigated.The linear range was 50-500 pg / mL,the detection limit was 9.03 pg / mL,and the limit of quantification was 51.41 pg / mL.Four interfering substances,myoglobin,human serum albumin,EDTA and glucose,did not interfere with the detection.The recovery of standard cTnI in human blood samples ranged from 99% to 101%.Then,a detection system based on oxidized graphene sensor was established.Combined with the identification of nucleic acid aptamer and the amplification of RCA signal,the quantitative detection method of cTnI was established.The linear range,detection limit and limit of quantification were optimized,and the interference and recovery of related substances were also investigated.The linear range was 25-500 pg/mL,the detection limit was 11.96 pg/mL,and the limit of quantification was 23.88 pg/mL.The recoveries of standard cTnI in human blood samples were between 95% and 105%.Finally,we compared the two sensitive cTnI quantitative detection methods established in this study with the most commonly used enzyme-linked immunosorbent assay(ELISA).The results show that the sensitivity of the two methods is better than The current commercial ELISA kits.The detection accuracy and sensitivity of both methods are high.The molecular beacon method is simple,but the requirement of designing probe is high.The heterogeneous structure and electronic properties of the graphene oxide molecules make it possible to combine reversibly with biomolecules to provide a platform for the construction of sensors,which is flexible and inexpensive.