B-358 Application of Surface-Enhanced Raman Spectroscopy to Improve Sensitivity of Clinical Assays
Authors: Sheik D , Ummadisetti C , Byers K , Suresh V , Pate K , Matthews M , Davisson V J 
Published: September 2023

Abstract

Background
Current efforts to expand the pool of available markers that can be analyzed in clinical settings is hampered by limited volumes of biomaterial-rich fluids and low concentrations of certain analytes, coupled with poor sensitivity of existing detection techniques. Although progress has been made using advanced sample preparation or assay techniques such as miRNeasy or ELISA for genomic and protein assays, respectively, little research has focused on adapting new optical methods as detection modes in the clinic. Amplified Sciences has developed an enzymatic activity assay platform using SERS detection to improve diagnostic sensitivities in multiple disease indications. Along with the experts at Wasatch Photonics, research focused on adaptation of this assay platform has enabled investigation of multiple markers of disease that had previously been undervalued or overlooked, as well as stacking of multiple markers in precious, low-volume samples.

Methods
Building upon previously published data using gastricsin as a marker to identify benign, non-mucinous pancreatic cysts, Amplified Sciences has added several SERS laser wavelengths, and different sample preparation methods to the existing platform. To examine the sensitivities of laser and dye combinations, solutions representative of assay samples containing rhodamine-dimer dye-labeled peptide were tested using Wasatch Photonics Raman spectrometers at 532, 638, 785, and 830 nm laser wavelengths. Samples at different pH were analyzed after addition of colloidal Au or Ag nanoparticles to determine the impacts of sample preparation on SERS spectral features. After drying in a desiccator overnight, the samples were reanalyzed to assess the stability of the SERS signals. As a pertinent application of this technology, an activity assay was developed for the known COVID protease 3ClPro. Release of rhodamine dimer dye as a result of 3ClPro cleavage of the substrate was quantified using SERS. The data were analyzed using Bruker OPUS Software package, and plotted using Graphpad Prism 9.

Results
Each laser, buffer, and metal used for SERS provided a unique spectral signature, suggesting that different transitions are available and potentially tunable depending on the application under investigation. Sensitivity was, as expected, best with the 532 nm SERS system purportedly due to enhancement factors, and the resonance impacts of rhodamine dyes at 532 nm. It was found that the Limit of Detection of Rhodamine-dimer dye was 100-fold lower with SERS compared to fluorescence, even after a 3-fold dilution with colloidal Ag. However, the remaining lasers could be more finely tuned, and, thus, provide alternate spectral properties, and different sensitivities which has implications when new dye classes are introduced into the platform. Furthermore, when SERS was applied to the 3ClPro assay, it was determined that activity could be detected below the dimerization concentration of 3ClPro, described in the literature as ∼25 nM. Also, a monomeric version of 3ClPro that is expected to have no reactivity with the substrate was found to display activity above background at concentrations as low as 1 µM.

Conclusion
SERS is an appropriate method for measuring activity in clinically relevant markers of disease, with tunable characteristics based upon the dye composition and the spectrometer in use.