Catching COVID: Engineering Peptide-Modified Surface-Enhanced Raman Spectroscopy Sensors for SARS-CoV-2

Abstract

COVID-19 remains an ongoing issue across the globe, highlighting the need for a rapid, selective, and accurate sensor for SARS-CoV-2 and its emerging variants. The chemical specificity and signal amplification of surface-enhanced Raman spectroscopy (SERS) could be advantageous for developing a quantitative assay for SARS-CoV-2 with improved speed and accuracy over current testing methods. Here, we have tackled the challenges associated with SERS detection of viruses. As viruses are large, multicomponent species, they can yield different SERS signals, but also other abundant biomolecules present in the sample can generate undesired signals. To improve selectivity in complex biological environments, we have employed peptides as capture probes for viral proteins and developed an angiotensin-converting enzyme 2 (ACE2) mimetic peptide-based SERS sensor for SARS-CoV-2. The unique vibrational signature of the spike protein bound to the peptide-modified surface is identified and used to construct a multivariate calibration model for quantification. The sensor demonstrates a 300 nM limit of detection and high selectivity in the presence of excess bovine serum albumin. This work provides the basis for designing a SERS-based assay for the detection of SARS-CoV-2 as well as engineering SERS biosensors for other viruses in the future.

Keywords: COVID-19; SARS-CoV-2; SERS; biomimetic; biosensor; peptide; virus.

Figure 1.. Characterization of peptide-modified sensor for detection of SARS-CoV-2.

A) Schematic of peptide-modified SERS substrates before (i) and after (ii) binding the spike protein of SARS-CoV-2. B) Sequence and chemical structure of ACE2 derived peptide used to modify surfaces and bind the spike protein. C) Normalized XPS spectra of SERS substrates showing peptide modification and RBD binding. D) Atomic composition of surfaces used in (c) showing successful modification and RBD binding. E) (i) CLSM 3D reconstructed side-view images of immunolabeled SERS surfaces modified with SBP-PEG₄ before and after RBD binding (scale bar = 2 μm). (ii) Relative fluorescence from quantification of integrated density before and after RBD binding (n=4, area per n = 156 μm²).

Figure 2.. Linker affects binding affinity of ACE2 derived peptides for RBD.

A) Steady-state analysis of BLI data to determine Kd values. B) CD spectra of cysteine-modified peptides with and without linker comparing the ability of each to form alpha-helical structures. C) BLI response of SBP-PEG₄ showing specific binding to RBD from SARS-CoV-2 compared to SARS-CoV-1 and MERS.

Figure 3.. SERS detection of SARS-CoV-2 spike protein and RBD on peptide-modified substrates.

A) SERS spectra of unmodified substrate and of both peptide-modified substrates before and after addition of 2 μM spike protein. B) Comparison of SERS signal from the SBP-PEG₄-modified surface and in the presence of 2 μM RBD and 2 μM full spike, with highlighted regions indicating important spectral similarities associated with the spike/RBD (maroon shading) and SBP-PEG₄ (teal shading). The spectra are offset for clarity.

Figure 4.. Specificity of SBP-PEG₄ SERS sensor for SARS-CoV-2 RBD (blue) versus SARS-CoV-1 RBD (red) and MERS-CoV RBD (orange).

The peptide surfaces (teal) prior to treatment with each RBD (5 μM) are shown below each spectrum, respectively. The spectra are offset for clarity.

Figure 5.. Selectivity and quantitative capabilities of SBP-PEG₄ SERS sensor.

A) Average spectra from SBP-PEG₄-modified substrate treated with: no protein, 15 μM BSA, 2 μM spike, and 8 μM BSA plus 1 μM spike (8:1 mixture). B) MCR component 2, representing SERS signature of spike protein. C) MCR component 1, representing SERS signature of peptide. D) MCR scores on component 2 for SBP-PEG₄-modified substrate with: no protein, 15 μM BSA, 2 μM spike, and 8 μM BSA plus 1 μM spike (8:1 mixture). *p =0.05. ****p <0.0001. E) MCR scores on component 1 for SPB-PEG4-modified substrate with: no protein, 8 μM BSA, 2 μM spike, and 8 μM BSA plus 1 μM spike (8:1 mixture). F) Average spectra (normalized to 1002 cm⁻¹) from SBP-PEG₄-modified substrate treated with varying concentrations of spike protein. G) SERS-based calibration curve for spike using MCR scores on the spike component, showing a limit of detection of 300 nM. The spectra are offset for clarity.