State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Biosens Bioelectron. 2014 Nov 15;61:321-7. doi: 10.1016/j.bios.2014.05.038. Epub 2014 May 23.
In the present day, oligonucleotide-encapsulated silver clusters (DNA-AgNCs) have been widely applied into bio-analysis as a signal producer. Herein, we developed a novel method to synthesize DNA-AgNCs encapsulated by long-chain cytosine (C)-rich DNA. Such DNA was polymerized in a template-free way by terminal deoxynucleotidyl transferase (TdT). We demonstrated that TdT-polymerized long chain C-rich DNA can serve as an excellent template for AgNCs synthesis. Based on this novel synthesis strategy, we developed a label-free and turn-on fluorescence assay to detect TdT activity with ultralow limit of detection (LOD) of 0.0318 U and ultrahigh signal to background (S/B) of 46.7. Furthermore, our proposed method was extended to a versatile biosensing strategy for turn-on nucleases activity assay based on the enzyme-activated TdT polymerization. Two nucleases, EcoRI and ExoIII as model of endonuclease and exonuclease, respectively, have been detected with high selectivity and competitive low LOD of 0.0629 U and 0.00867 U, respectively. Our work demonstrates the feasibility of TdT polymerization-based DNA-AgNCs synthesis strategy as a versatile and potent biosensing platform to detect the activity of DNA-related enzymes.
在当今社会，寡核苷酸包裹的银纳米簇（DNA-AgNCs）已被广泛应用于生物分析作为信号产生体。在此，我们开发了一种新的方法来合成由长链富含胞嘧啶（C）的 DNA 包裹的 DNA-AgNCs。这种 DNA 可以通过末端脱氧核苷酸转移酶（TdT）在无模板的方式下聚合。我们证明 TdT 聚合的长链富含 C 的 DNA 可以作为 AgNCs 合成的优良模板。基于这种新的合成策略，我们开发了一种无标记和开启型荧光测定法，用于检测 TdT 活性，其检测限（LOD）低至 0.0318 U，信号与背景比（S/B）高达 46.7。此外，我们的方法还扩展为一种基于酶激活 TdT 聚合的用于开启型核酸酶活性测定的通用生物传感策略。两种核酸酶，EcoRI 和 ExoIII，分别作为内切核酸酶和外切核酸酶的模型，具有高选择性和竞争性低 LOD，分别为 0.0629 U 和 0.00867 U。我们的工作证明了基于 TdT 聚合的 DNA-AgNCs 合成策略作为一种通用且强大的生物传感平台，用于检测与 DNA 相关的酶的活性的可行性。
