We developed a colorimetric method for assay of protease activity through the growth of gold nanoparticles (AuNPs) with ascorbic acid (AA) as the reducing agent. The method is based on the difference in the catalytic activity of various Cu²⁺ species toward AA oxidation. A mutational peptide is employed as the protease substrate, in which only one amino acid residue is replaced by the histidine residue. Specifically, HAuCl₄ can be reduced into AuNPs by AA; Cu²⁺ ion promotes the oxidation of AA under oxygen atmosphere by a redox cycling, thus preventing the formation of AuNPs. Cleavage of the substrate peptide results in the exposure of an amino terminal Cu²⁺ and Ni²⁺-binding (ATCUN) motif in the NH₂-terminus of one fragment. The resultant ATCUN peptide can sequestrate Cu²⁺ and thus depress the catalytic oxidation of AA. The consumption of AA is monitored by UV–vis spectra and differential pulse voltammetry. The high extinction coefficient of the generated AuNPs enables the quantitative and sensitive colorimetric analysis of protease. To demonstrate the analytical performances, β-secretase was tested as a model protease. By employing peptide-functionalized magnetic beads, β-secretase in serum was determined with a satisfactory result. This work provides valuable information for designing of novel protease biosensors.

我们开发了一种比色法，通过以抗坏血酸（AA）为还原剂的金纳米颗粒（AuNPs）的生长来测定蛋白酶活性。该方法基于各种Cu ²⁺物种对AA氧化的催化活性的差异。突变肽被用作蛋白酶底物，其中仅一个氨基酸残基被组氨酸残基代替。具体而言，HAuCl ₄可以通过AA还原为AuNPs。Cu ²⁺离子在氧气氛下通过氧化还原循环促进AA的氧化，从而阻止了AuNPs的形成。底物肽的切割导致氨基端暴露于氨基末端的Cu ²⁺和Ni ²⁺结合（ATCUN）基序一个片段的_2-末端。所得的ATCUN肽可以螯合Cu ²⁺并因此抑制AA的催化氧化。AA的消耗量通过紫外可见光谱和差分脉冲伏安法进行监测。生成的AuNPs的高消光系数能够对蛋白酶进行定量和灵敏的比色分析。为了证明其分析性能，测试了β-分泌酶作为模型蛋白酶。通过使用肽功能化的磁珠，血清中的β-分泌酶被测定，结果令人满意。这项工作为新型蛋白酶生物传感器的设计提供了有价值的信息。