1 research outputs found
Quantitative and Label-Free Detection of Protein Kinase A Activity Based on Surface-Enhanced Raman Spectroscopy with Gold Nanostars
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<sup>–1</sup>, 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