Schematic illustration of the working principle of detecting PSA protease activity using peptide-conjugated NPR SERS nanosensors. (a) NPRs exhibit a tunable plasmon resonance and highly enhanced local electromagnetic field through coupled plasmonic resonance. NPRs with a short axis of 150 nm and long axis of 200 nm were made of multi-stacks of silver and SiO₂ layers with thicknesses of 25 nm and 5 nm, respectively. (b) The molecular structure of the biomarker that consists of Raman dye R19, PSA specific peptide sequence HSSKLQLAAAC, and cysteine. The peptide can be cleaved by PSA enzyme between HSSKLQ and LAAAC. (c) The detection scheme of NPR functionalized with peptide sequence HSSKLQLAAAC and the Raman dye R19 (star). The presence of the PSA enzyme will cleave the peptide sequence. After cleavage, the diffusion of the R19 (star) away from the surface will be monitored by the loss of the SERS signatures of the R19 moiety. Packing molecule octanethiol (HS-(CH₂)₂-CH₃) was used to reduce the packing density of the reporting peptide on NPR surface, and thus, allows PSA enzyme to access the reporting peptide.

Real-time kinetic measurement of PSA protease activity. (a) SERS spectra for 6 nM PSA incubation taken over 30 minutes with an integration time of 30 seconds. (b) SERS spectra for the negative control, 6 nM granzyme B, taken over 30 minutes. (c) Time-resolved measurements of relative change in Raman peak intensity at 1316 cm^-1, 1456 cm^-1, 1526 cm^-1, and 1597 cm^-1 before and after addition of PSA protease. Negative time represents time before protease addition.

(a) Optical microscopic image of NPRs arrays fabricated using standard e-beam lithography and thin film deposition process. Fabricated NPRS arrays consists of 30 × 30 NPRs with 500 nm spacing. Multiple NPRs arrays and alignment mark can be conveniently fabricated on the same substrate. (b) Magnified image of NPR array measured by Scanning Electron Microscope. Using precision lithography methods, the NPR can be prepared in a controlled manner. (c) Image of NPRs measured at higher magnification using Atomic Force Microscope (AFM). (d) Measured extinction spectrum of an NPR-peptide-R19 conjugate array at a wavelength range of 425 to 650 nm. The resonance peak of the NPR has been tuned to closely match the laser excitation and Raman emission frequencies, and thus, maximize the overall enhancement of the Raman signal. (e) Reproducible Raman spectra of NPR-peptide-R19 conjugates measured from six different NPRs arrays. Integration time is 30 seconds.

Time-resolved measurements of PSA activity by varying the active PSA concentration. (a) Normalized SERS intensity change for 1316 cm^-1 peak at active PSA concentration from 6 pM to 6 nM. The decreasing of SERS intensity can be clearly measured while no significant change can be observed in the control experiment with no active PSA protease. (b) Concentration dependence of normalized SERS intensity change obtained at 30 min. (c) Protease activity measurement obtained from unprocessed extracellular fluid (ECF): Raman spectra obtained at the beginning of exposing LNCaP cells (positive control) ECF to NPR nanosensors (t = 0 min) and after 30 minutes. (d) Normalized change of SERS intensity at 1316 cm^-1 peak indicating PSA proteolytic activity in the fluid extracted from LNCaP and K562 cell lines.