Department of Biomedical Engineering , National University of Singapore , Singapore 117583.
TechnoSpex Private Limited , Singapore 169203.
Anal Chem. 2018 May 15;90(10):6071-6080. doi: 10.1021/acs.analchem.7b05417. Epub 2018 Apr 26.
The activity of extracellular protein kinase A (PKA) is known to be a biomarker for cancer. However, conventional PKA assays based on colorimetric, radioactive, and fluorometric techniques suffer from intensive labeling-related preparations, background interference, photobleaching, and safety concerns. While surface-enhanced Raman spectroscopy (SERS)-based assays have been developed for various enzymes to address these limitations, their use in probing PKA activity is limited due to subtle changes in the Raman spectrum with phosphorylation. Here, we developed a robust colloidal SERS-based scheme for label-free quantitative measurement of PKA activity using gold nanostars (AuNS) as a SERS substrate functionalized with bovine serum albumin (BSA)-kemptide (Kem) bioconjugate (AuNS-BSA-Kem), where BSA conferred colloidal stability and Kem is a high-affinity peptide substrate for PKA. By performing principle component analysis (PCA) on the SERS spectrum, we identified two Raman peaks at 725 and 1395 cm, whose ratiometric intensity change provided a quantitative measure of Kem phosphorylation by PKA in vitro and allowed us to distinguish MDA-MB-231 and MCF-7 breast cancer cells known to overexpress extracellular PKA catalytic subunits from noncancerous human umbilical vein endothelial cells (HUVEC) based on their PKA activity in cell culture supernatant. The outcome demonstrated potential application of AuNS-BSA-Kem as a SERS probe for cancer screening based on PKA activity.
细胞外蛋白激酶 A（PKA）的活性是癌症的一个生物标志物。然而，基于比色法、放射性和荧光法的传统 PKA 测定法在标记相关制备、背景干扰、光漂白和安全问题方面存在局限性。尽管基于表面增强拉曼光谱（SERS）的测定法已被开发用于各种酶来解决这些局限性，但由于磷酸化引起的拉曼光谱的细微变化，它们在探测 PKA 活性方面的应用受到限制。在这里，我们开发了一种使用金纳米星（AuNS）作为 SERS 基底的稳健胶体 SERS 方案，该基底通过牛血清白蛋白（BSA）-kemptide（Kem）生物缀合物（AuNS-BSA-Kem）进行功能化，用于无标记定量测量 PKA 活性，其中 BSA 赋予胶体稳定性，而 Kem 是 PKA 的高亲和力肽底物。通过对 SERS 光谱进行主成分分析（PCA），我们在 725 和 1395 cm 处鉴定出两个拉曼峰，其相对强度变化提供了 Kem 磷酸化的定量测量，PKA 在体外和允许我们根据细胞培养上清液中的 PKA 活性，区分已知过表达细胞外 PKA 催化亚基的 MDA-MB-231 和 MCF-7 乳腺癌细胞与非癌细胞人脐静脉内皮细胞（HUVEC）。结果表明，AuNS-BSA-Kem 作为基于 PKA 活性的癌症筛查的 SERS 探针具有潜在应用。
