Department of Medical Devices, Shanghai University of Medicine & Health Sciences, Shanghai, China.
Department of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai, China.
Biotechnol Appl Biochem. 2024 Feb;71(1):193-201. doi: 10.1002/bab.2531. Epub 2023 Oct 30.
In this study, an electrochemical biosensor was constructed for the detection of fibrin, specifically by a simple two-step approach, with a novel artificial enzyme (Tetrazyme) based on the DNA tetrahedral framework as signal probe. The multichannel screen-printed electrode with the activated surface cannot only remove some biological impurities, but also serve as a carrier to immobilize a large number of antigen proteins. The DNA tetrahedral nanostructure was employed to ensure the high sensitivity of the probe for biological analysis. The hemin was chimeric into the G-quadruplex to constitute the complex with peroxidase catalytic activity (hemin/G4-DNAzyme), subsequently, Tetrazyme was formed through combining of this complex and DNA tetrahedral nucleic acid framework. The artificial enzyme signal probe formed by the covalent combination of the homing peptide (Cys-Arg-Glu-Lys-Ala, CREKA), which is the aptamer of fibrin and the new artificial enzyme is fixed on the surface of the multichannel carbon electrode by CREKA-specific recognition, so as to realize the sensitive detection of fibrin. The feasibility of sensing platform was validated by cyclic voltammetry (CV) and amperometric i-t curve (IT) methods. Effects of Tetrazyme concentration, CREKA concentrations and hybridization time on the sensor were explored. Under the best optimal conditions of 0.6 μmol/L Tetrazyme, 80 μmol/L CREKA, and 2.5 h reaction time, the immunosensor had two linear detection ranges, 10-40 nmol/L, with linear regression equation Y = 0.01487X - 0.011 (R = 0.992), and 50-100 nmol/L, with linear regression equation Y = 0.00137X + 0.6405 (R = 0.998), the detection limit was 9.4 nmol/L, S/N ≥ 3. The biosensor could provide a new method with great potential for the detection of fibrin with good selectivity, stability, and reproducibility.
在这项研究中，构建了一种用于检测纤维蛋白的电化学生物传感器，具体方法是通过一种简单的两步法，利用基于 DNA 四面体型框架的新型人工酶（四酶）作为信号探针。具有活化表面的多通道丝网印刷电极不仅可以去除一些生物杂质，还可以作为载体固定大量抗原蛋白。DNA 四面体型纳米结构用于确保探针对生物分析的高灵敏度。血红素被嵌合到 G-四链体中，构成具有过氧化物酶催化活性的复合物（血红素/G4-DNAzyme），随后，通过将该复合物与 DNA 四面体型核酸框架结合，形成四酶。通过将纤维蛋白的适体（Cys-Arg-Glu-Lys-Ala，CREKA）与新的人工酶共价结合形成的人工酶信号探针固定在多通道碳电极表面上，通过 CREKA 特异性识别，实现了纤维蛋白的灵敏检测。通过循环伏安法（CV）和安培电流-时间曲线（IT）方法验证了传感平台的可行性。探讨了四酶浓度、CREKA 浓度和杂交时间对传感器的影响。在最佳条件下，Tetrazyme 浓度为 0.6 μmol/L，CREKA 浓度为 80 μmol/L，反应时间为 2.5 h 时，该传感器具有两个线性检测范围，10-40 nmol/L，线性回归方程为 Y = 0.01487X-0.011（R = 0.992），50-100 nmol/L，线性回归方程为 Y = 0.00137X + 0.6405（R = 0.998），检测限为 9.4 nmol/L，S/N≥3。该生物传感器为检测纤维蛋白提供了一种具有良好选择性、稳定性和重现性的新方法，具有很大的潜力。
