The one-pot seedless protocol provides a facile approach in the synthesis of gold nanostars (AuNS) that involves only three reagents, gold (III) chloride (HAuCl 4 ), silver nitrate (AgNO 3 ) and ascorbic acid (C 6 H 8 O 6 ). While studies correlating the synthesis parameters of the seedmediated protocol to surface-enhance Raman scattering (SERS) enhancement is well reported, the same understanding of the one-pot seedless protocol is limited. Here, we aim to elucidate how the synthesis parameters of AuNS from the one-pot seedless protocol, the AuNS concentration, surface passivation and aggregation level affect the colloidal SERS enhancement. Using crystal violet (CV) as a Raman probe molecule, we found that the SERS enhancement increases with Au 3+ /C 6 H 8 O 6 molar ratio up to 0.60 and Au 3+ /Ag + molar ratio up to 18. Although the surfactant, cetyltrimethylammonium bromide (CTAB) maintained colloidal stability, it reduced the SERS enhancement. Interestingly, the SERS enhancement did not increase monotonically with AuNS concentration, but decreased when AuNS concentration was beyond 15 pM. The SERS enhancement also increased with the increasing level of salt-induced aggregation of AuNS, but only within a few minutes. While the concept of SERS with colloidal nanostructures is not new, we have shown for the first time, a detailed systematic study of various parameters that affect the SERS enhancement of AuNS synthesized using a one-pot seedless protocol. This study enables us to optimize the SERS enhancement of AuNS at the synthesis level to make them effective colloid-based SERS substrates for potential use in intracellular biosensing.

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.

Nanoparticle-based phototherapy has evolved to include immunotherapy as an effective treatment combination for cancers through inducing anti-cancer immune activation leading to downstream adaptive responses and immune protection. However, most cancer...