To improve long-term graft patient outcomes and develop more effective antirejection therapies, noninvasive monitoring of acute cellular rejection (ACR) after organ transplantation is urgently needed. As a biomarker of ACR, Granzyme B (GrB) is expected to be applied in the noninvasive monitoring of ACR. Herein, we have developed a method for detecting the GrB activity based on the target-initiated great change in electrochemical steric hindrance by designing a nanoprobe. The nanoprobe is prepared by conjugating a specific peptide, which is responsive to GrB cleavage activity, to gold nanoparticles (AuNPs). Meanwhile, a piece of DNA sequence with G-quadruplex (G4) is attached at the distal end of the peptide. Upon exposure to GrB, the peptide substrate is cleaved to eliminate the steric hindrance between inter-nanoprobes as well as nanoprobe and DNA tetrahedron (TDN), allowing the released DNA strand to hybridize with TDN, giving sensitive signal output. The method can also be used to detect GrB activity in complex biological settings, so it has a great potential for monitoring GrB activity in the blood or urine of graft patients.

中文翻译：

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用于测定粒酶 B 活性的电化学空间位阻的靶标引发的巨大变化

为了改善长期移植患者的预后并开发更有效的抗排斥疗法，迫切需要对器官移植后的急性细胞排斥 (ACR) 进行无创监测。作为ACR的生物标志物，颗粒酶B（GrB）有望应用于ACR的无创监测。在此，我们通过设计纳米探针开发了一种基于靶引发的电化学空间位阻的巨大变化来检测 GrB 活性的方法。纳米探针是通过将响应 GrB 裂解活性的特定肽与金纳米粒子 (AuNP) 结合来制备的。同时，一段带有G-四链体（G4）的DNA序列附着在肽的远端。接触 GrB 后，肽底物被切割以消除纳米探针之间以及纳米探针和 DNA 四面体 (TDN) 之间的空间位阻，使释放的 DNA 链与 TDN 杂交，从而产生灵敏的信号输出。该方法还可用于检测复杂生物环境中的 GrB 活性，因此在监测移植患者血液或尿液中的 GrB 活性方面​​具有巨大潜力。