基于多肽均相裂解电化学发光法检测前列腺特异性抗原

Electrochemiluminescent Detection of Prostate Specific Antigen Based on Homogeneous Polypeptide Cleavage

以前列腺特异性抗原(PSA)为目标分析物,多肽(CHSSKLQK)为分子识别物质,钌联吡啶衍生物作为信号物质,建立了基于金纳米粒子(AuNPs)均相电化学发光法检测PSA的新方法。利用多肽末端上的巯基将肽自组装在AuNPs上,当PSA加入含有多肽结合的AuNPs悬液中时,PSA选择性地切断信号物质标记的多肽。被切断的信号物质多肽片段离开AuNPs表面,离心分离后检测到的电化学发光强度与PSA质量浓度的对数在5. 0×10^(-12)~1. 0×10-9g/m L范围内呈良好的线性关系,检出限为2. 0×10^(-12)g/mL。用于血清样品的测定,该法的测定结果与化学发光免疫分析法基本一致,具有灵敏度高、重现性好等优点,为高灵敏检测蛋白酶提供了新思路。

A novel peptide-based electrochemiluminescent(ECL)method for the determination of prostate-specific antigen(PSA)was developed based on the target-induced cleavage of homogeneous specific polypeptide with gold nanoparticles(AuNPs)to overcome drawbacks from probes directly immobilized on electrodes.A specific peptide CHSSKLQK was employed as a molecular recognition element,and ruthenium(Ⅱ)complexes(Ru1)were used as ECL emitting species.A site-specific enzymatic-cleavage-reaction-based biosensor using Ru1/peptide-conjugated gold nanoparticle complexes(AuNPs-peptide-Ru1)was developed.When PSA was added into the solution containing AuNPs-peptide-Ru1,PSA specifically recognized and cleaved the sequence of the peptides attached to the AuNPs.As a result,Ru1 were separated from the AuNPs and resulted in the increase of ECL intensity in the presence of co-reactant tripropylamine.Results showed that the increased ECL intensity was directly linear to the logarithm of PSA concentration in range of 5.0×10-12-1.0×10-9 g/mL with a detection limit of 2.0×10-12 g/mL.The method was applied in the detection of clinical serum samples,and the results were consistent with those obtained by CL method.With the advantages of high sensitivity and good reproducibility,this method provides a promising strategy for the sensitive detection on protease.