| Cardiovascular disease is the number one cause of death in patients worldwide,it is a serious threat to human health and can cause sudden cardiac death.Acute myocardial infarction(AMI)is myocardial necrosis caused by acute and persistent,ischemia and hypoxia in the coronary arteries.Early detection,diagnosis and treatment are the key to reducing mortality and disability.Cardiac troponin I(cTnI)is the preferred marker for the diagnosis of myocardial injury,and its high sensitive and specific detection are very important for the timely diagnosis,efficacy evaluation and recovery monitoring of the disease.Single particle collision electrochemistry(SPCE)is an analytical method with simple operation,high sensitivity and wide application,realizing the detection and study of single nanoparticles in recent years.Due to its ultra-high sensitivity at the single particle level,SPCE also shows great application prospects in the highly sensitive detection of biomolecules.However,there are few reports on the analysis and detection of SPCE at present.The challenges may be:(1)Low efficiency of signal conversion,one or more targets are converted to a signal marker,affecting the detection sensitivity;(2)In the actual sample,there are many complex matrix interferers,leading to microelectrode poisoning and agglomeration of collision particles.Based on the above challenges,this work combines chain replacement,rolling circle amplification,CRISPR/Cas12a-mediated signal amplification strategy and the efficient separation and enrichment ability of magnetic beads.Constructing the electrocatalytic amplification SPCE biosensor achieves highly specific and sensitive detection of cTnI.The detailed work is as follows:(1)Antibody-modified magnetic beads(m Ab-MBs)could capture and separate cTnI in complex samples with a high capture efficiency of 99%within 120 s,endowing the immunosensor with high specificity and anti-interference ability.Target cTnI could drive the rolling circle amplification reaction to generate magnetic immune complexes.ss DNA-modified Pt NPs as signal probe,one cTnI molecule could trigger the release of 4.5×10~4 Pt NPs,greatly improving the signal conversion efficiency and detection sensitivity.After magnetic separation,the released Pt NPs collided with gold ultramicroelectrode to catalyze the oxidation of hydrazine hydrate and generate transient current.According to the relationship between collision frequency(number of transient currents per unit time)and the concentration of cTnI,the sensor realized sensitive detection of cTnI.The linear range was 1 fg/m L～50ng/m L,and the detection limit was as low as 0.57 fg/m L.The SPCE sensor was successfully used to detect clinical samples of AMI patients,indicating great application prospect of the sensor in clinical disease diagnosis.(2)To simplify the operation process and realize the rapid and sensitive detection of cTnI,an SPCE aptamer sensor was constructed based on CRISPR/Cas12a.Due to its low cost,simple design and ultra-high sensitivity as low as a M,CRISPR/Cas12a is a gene editing technology developed in recent years,and has attracted wide attention in biological detection.In the presence of only target cTnI,the trans-cleavage capability of CRISPR/Cas12a was activated to efficiently shear ss DNA on the surface of Au@Pt NPs within 30 min,restoring Au@Pt NPs activity and participating in the collision reaction.Compared with Pt NPs,Au@Pt NPs had higher catalytic activity for hydrazine hydrate,effectively improving the signal-to-background ratio and detection sensitivity.Due to the excellent shearing activity of CRISPR/Cas12a,each cTnI molecule could induce the release of 4.68×10~4 Au@Pt NPs.The detection limit of the constructed SPCE sensor for cTnI was as low as 0.35 fg/m L,and it had 2～6orders of magnitude higher than other cTnI detection methods,providing a new idea for the application of SPCE in analysis and detection. |
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