College of Chemistry and Life Science, Zhejiang Normal University , Jinhua 321004, China.
Beijing National laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences , Bejing 100190, China.
ACS Appl Mater Interfaces. 2015 Oct 28;7(42):23564-74. doi: 10.1021/acsami.5b06711. Epub 2015 Oct 13.
Sensitive assay of tyrosinase (TYR) activity is in urgent demand for both fundamental research and practical application, but the exploration of functional materials with good biocompatibility for its activity evaluation at the intracellular level is still challenging until now. In this work, we develop a convenient and real-time assay with high sensitivity for TYR activity/level monitoring and its inhibitor screening based on biocompatible dopamine functionalized carbon quantum dots (Dopa-CQDs). Dopamine with redox property was functionalized on the surface of carbon quantum dots to construct a Dopa-CQDs conjugate with strong bluish green fluorescence. When the dopamine moiety in Dopa-CQDs conjugate was oxidized to a dopaquinone derivative under specific catalysis of TYR, an intraparticle photoinduced electron transfer (PET) process between CQDs and dopaquinone moiety took place, and then the fluorescence of the conjugate could be quenched simultaneously. Quantitative evaluation of TYR activity was established in terms of the relationship between fluorescence quenching efficiency and TYR activity. The assay covered a broad linear range of up to 800 U/L with a low detection limit of 7.0 U/L. Arbutin, a typical inhibitor of TYR, was chosen as an example to assess its function of inhibitor screening, and positive results were observed that fluorescence quenching extent of the probe was reduced in the presence of arbutin. It is also demonstrated that Dopa-CQD conjugate possesses excellent biocompatibility, and can sensitively monitor intracellular tyrosinase level in melanoma cells and intracellular pH changes in living cells, which provides great potential in application of TYR/pH-associated disease monitoring and medical diagnostics.
酶(TYR)活性的灵敏检测在基础研究和实际应用中都有迫切的需求,但直到现在,对于其在细胞内水平的活性评估,探索具有良好生物相容性的功能材料仍然具有挑战性。在这项工作中,我们开发了一种方便且实时的测定方法,具有高灵敏度,可用于 TYR 活性/水平监测及其抑制剂筛选,该方法基于生物相容性的多巴胺功能化碳量子点(Dopa-CQDs)。具有氧化还原性质的多巴胺被功能化在碳量子点的表面上,构建了具有强蓝绿色荧光的 Dopa-CQDs 缀合物。当 Dopa-CQDs 缀合物中的多巴胺部分在 TYR 的特定催化下被氧化成多巴醌衍生物时,会发生 CQDs 和多巴醌部分之间的粒子内光诱导电子转移(PET)过程,随后缀合物的荧光会同时被猝灭。通过荧光猝灭效率与 TYR 活性之间的关系,建立了定量评估 TYR 活性的方法。该测定方法的线性范围很宽,可达 800 U/L,检测限低至 7.0 U/L。熊果苷,一种典型的 TYR 抑制剂,被选为评估其抑制剂筛选功能的一个例子,观察到阳性结果,即探针的荧光猝灭程度在存在熊果苷的情况下降低。还证明了 Dopa-CQD 缀合物具有优异的生物相容性,可以灵敏地监测黑素瘤细胞内的酪氨酸酶水平和活细胞内的细胞内 pH 值变化,这为 TYR/pH 相关疾病监测和医学诊断的应用提供了巨大的潜力。