B-055 A Multiplexed SERS Assay for Quantitative Protease Turnover Detection at Picomolar Levels

Abstract

Surface-Enhanced Raman Spectroscopy (SERS) allows for ultrasensitive detection of analytes in complex biofluids. This capability is particularly important when measuring analytes at low concentrations or when clinical sample volumes are limited. SERS improves workflows by eliminating complex sample-preparation steps and instead employs dilution to reduce matrix interference in analyte measurements. Amplified Sciences has previously used its proprietary dyes and automated BioMatra™ hardware platform to develop a low-volume assay panel (PanCystPro™) which is offered as a Lab Developed Test in a CLIA-certified lab. This multi-analyte test includes an ultra-low-volume SERS-based proteolytic activity measurement of the digestive enzyme gastricsin to differentiate mucinous and non-mucinous pancreatic cysts. Beyond this initial use, significant opportunities exist to apply protease activity biomarkers to other clinical conditions. However, highly accurate diagnostics often demand panels of biomarkers, not single markers, regardless of technology or analyte type. Advancing BioMatra’s multiplexing capabilities will enable additional efficiencies and cost-effective testing. A recently developed multiplexed protease assay panel featuring isotopically-labeled SERS-active dyes demonstrates the platform's potential to analyze complex biological matrices utilizing small sample volumes. A variant of the CLIA laboratory-installed BioMatra was developed, integrating a Wasatch Photonics 785nm Raman spectrometer into a fully automated multi-axis plate reader system (Fig. 1A). The platform software enables high-throughput analysis of 384-well plates with ∼10 second/well acquisition to ensure reproducible readout. A molecular reporter system uses deuterated (d0/d8) isotope-labeled R19 dimer-conjugated SERS-active peptide substrates for neutrophil elastase and MMP9, producing partially overlapping but resolvable spectral signatures centered at 1327 cm-1 (d8, hNE) and 1353 cm-1 (d0, MMP9) (Fig. 1B). Commercial preparations of recombinant neutrophil elastase and MMP9 were mixed with their respective magnetic bead-bound peptide substrates and cleavage reactions terminated via bead removal. Samples for multiplex data acquisition were generated by mixing a portion of the collected supernatants, and analysis included a ratiometric correction factor based on Area Under the Curve of characteristic peaks within overlapping spectra. Increasing concentrations of recombinant enzymes were used to generate standard curves, and both activity assays demonstrated excellent linearity (R2 > 0.99), with coefficients of variation ranging from 1.5-9.7%. The limit of detection was 0.07 ng/mL (2.5 pM) for neutrophil elastase and 0.89 ng/mL (13.4 pM) for MMP9 (Fig. 1C and 1D). Dynamic range spanned approximately two orders of magnitude (0.2-25 ng/mL for neutrophil elastase; 0.7-125 ng/mL for MMP9). Multiplex measurements showed strong concordance with singleplex values (R2 > 0.99, slope near unity), validating the ratiometric deconvolution approach. Despite overlapping d0 and d8 isotopologue features in the 1313-1370 cm-1 region, the d8 peak at 1327 cm-1 remains spectrally distinct, enabling ratiometric correction to resolve individual analyte contributions at 1353 cm-1. These results demonstrate that isotopically-labeled SERS-active dyes enable multiplexed protease activity measurement at picomolar sensitivity from sub-microliter sample volumes using 785 nm laser excitation. The ratiometric correction approach achieves singleplex-equivalent accuracy in a multiplexed format. Combined with the automated BioMatra platform's throughput, this establishes a scalable framework for multi-biomarker protease panels.