Electrochemically generated paper SERS substrate for detection of exosome in urine samples

Here, a single-drop paper-based surface-enhanced Raman spectroscopy (SERS) immunoassay was developed to pave the way for monitoring exosome numbers for the early diagnosis of prostate cancer. Exosomes are nanosized (40-150 nm) membrane vesicles that provide intercellular communication. In our work, we offer a new paper SERS substrate for exosome detection in urine. We initially electrochemically deposited nanostar-shaped gold nanoparticles (AuNPs) on the working electrode to crate the paper SERS substrates. Then we functionalized them with 11-mercaptoundecanoic acid (11-MUA) and conjugated them with anti-CD9 antibodies. After capturing exosomes, the sandwich immunoassay structure was created by using gold nanorods (AuNRs) modified with 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) as a Raman tag. The SERS signal intensities of DTNB molecules at 1330 cm-1 were monitored to determine the exosome concentration. Each step occurred in only one drop of solution or sample. The developed single-drop paper-based SERS immunoassay exhibited a linear range from 1.0 x 103 to 1.0 x 109 exosome particles/mL with correlation coefficients (R2) of 0.9903. The limit of detection (LOD) was found as 9.9 x 101 exosome particles/mL. The developed system was tested with clinical urine samples from patients with benign prostatic hyperplasia, prostatitis, prostate cancer, and healthy individuals. The obtained results were compared with the exosome particle numbers in these samples determined by an enzyme-linked immunosorbent assay (ELISA) method and the accuracy of the system was evaluated with an average recovery value of 96.7 %. The developed biosensor system enables highly sensitive detection of exosomes in low-volume urine samples. The usage of a paper membrane as a SERS substrate, combined with the electrochemical deposition of gold nanoparticles, provides an eco-friendly and cost-effective solution, enabling wider use and applications.