Department of Laboratory Medicine, The Third Xiangya Hospital, Central South University, Changsha 410013, China.
School of Life Sciences, Central South University, Changsha 410013, China.
Spectrochim Acta A Mol Biomol Spectrosc. 2023 Feb 5;286:121939. doi: 10.1016/j.saa.2022.121939. Epub 2022 Oct 2.
As a representative biochemical indicator, alkaline phosphatase (ALP) is of great importance in indicating and diagnosing clinical diseases. Herein, we developed a signal-on fluorescence sensing method for sensitive ALP activity detection based on the enzyme-assisted target recycling (EATR) technique. In this method, a two-step signal amplification process is designed. In the presence of ALP, the 3' phosphate group of an ss-DNA is removed explicitly by ALP, thus releasing free 3'-OH. Terminal deoxynucleotidyl transferase (TdT) can subsequently extend this substrate to generate poly(A) tails, converting the trace-level ALP information into multiple sequences and achieving the first-time amplification. A poly(T) Taqman probe labeled with FAM and BHQ1 provides the second one under the assistance of T7 exonuclease (T7 Exo) through alternate hybridization and degradation of ds-DNA regions. The previously quenched fluorescence is recovered due to the departure of FAM/BHQ1 during the cleavage of T7 Exo. Thus, taking advantage of template-free TdT-mediated polymerization and T7 Exo-based EATR, this strategy shows a sensitive LOD at 0.0074 U/L (S/N = 3) and a linear range of 0.01-8 U/L between ALP concentration and fluorescence intensity. To further verify the specificity and accuracy in practical application, we challenged it in a set of co-existing interference and biological environments and have gained satisfying results. The proposed method successfully quantified the ALP levels in clinical human serum samples, suggesting its applicability in practical application. Moreover, we have used this method to investigate the inhibition effects of NaVO. Above all, the proposed assay is sensitive, facile, and cost-effective for ALP determining, holding a promising perspective and excellent potential in clinical diagnosis and drug screening.
作为一种有代表性的生化指标，碱性磷酸酶（ALP）在指示和诊断临床疾病方面非常重要。在此，我们开发了一种基于酶辅助靶标循环（EATR）技术的灵敏 ALP 活性检测的信号开启荧光传感方法。在该方法中，设计了两步信号放大过程。在 ALP 的存在下，ALP 明确去除 ss-DNA 的 3' 磷酸基团，从而释放游离的 3'-OH。末端脱氧核苷酸转移酶（TdT）随后可以扩展该底物以生成 poly(A)尾巴，将痕量水平的 ALP 信息转换为多个序列，并实现第一次放大。在 T7 外切酶（T7 Exo）的辅助下，带有 FAM 和 BHQ1 的 poly(T) Taqman 探针通过交替杂交和 ds-DNA 区域的降解提供第二个信号。由于 T7 Exo 切割过程中 FAM/BHQ1 的离去，先前被猝灭的荧光得到恢复。因此，利用无模板 TdT 介导的聚合和基于 T7 Exo 的 EATR，该策略在 0.0074 U/L（S/N = 3）的检测限下表现出灵敏的 LOD 和 0.01-8 U/L 之间的线性范围，与 ALP 浓度和荧光强度呈线性关系。为了进一步验证实际应用中的特异性和准确性，我们在一组共存的干扰和生物环境中对其进行了挑战，并获得了令人满意的结果。该方法成功地定量了临床人血清样本中的 ALP 水平，表明其在实际应用中的适用性。此外，我们还使用该方法研究了 NaVO 的抑制作用。总之，该方法用于 ALP 测定具有灵敏度高、简便、经济的特点，在临床诊断和药物筛选方面具有广阔的前景和巨大的潜力。
