Label-Free Saliva Test for Rapid Detection of Coronavirus Using Nanosensor-Enabled SERS

Abstract

The recent COVID-19 pandemic has highlighted the inadequacies of existing diagnostic techniques and the need for rapid and accurate diagnostic systems. Although molecular tests such as RT-PCR are the gold standard, they cannot be employed as point-of-care testing systems. Hence, a rapid, noninvasive diagnostic technique such as Surface-enhanced Raman scattering (SERS) is a promising analytical technique for rapid molecular or viral diagnosis. Here, we have designed a SERS- based test to rapidly diagnose SARS-CoV-2 from saliva. Physical methods synthesized the nanostructured sensor. It significantly increased the detection specificity and sensitivity by ~ten copies/mL of viral RNA (~femtomolar concentration of nucleic acids). Our technique combines the multiplexing capability of SERS with the sensitivity of novel nanostructures to detect whole virus particles and infection-associated antibodies. We have demonstrated the feasibility of the test with saliva samples from individuals who tested positive for SARS-CoV-2 with a specificity of 95%. The SERS-based test provides a promising breakthrough in detecting potential mutations that may come up with time while also preparing the world to deal with other pandemics in the future with rapid response and very accurate results.

Keywords: COVID-19; SERS; coronavirus; nanosensors.

Figure 1

Schematic representation of the label-free rapid SARS-CoV 2 saliva test.

Figure 2

Synthesis and characterization of nanostructured COVID sensor (a) Schematic representation of the synthesis of nanostructured COVID sensor using multiphoton ionization process (b) Representative TEM image showing the morphology of quantum immunoprobe (Scale 50 nm) (c) Histogram showcasing the ultrasmall size of nanostructured COVID sensor based on TEM imaging, (d) Optical characterization of nanostructured COVID sensor using fluorescent spectroscopy and UV visible spectroscopy (e) Fluorescence mapping of the COVID nanosensor (f) UV-Vis spectra of the nanosensor.

Figure 3

(a) Establishing the unique SERS signature of SARS-CoV-2 viral components. Analytical sensitivity of Label-free SERS assay by determining the limit of detection of the nanostructured sensor (b) SERS spectra of whole virus particles spiked in saliva varying from 10,000 particles to 50 particles per ml of saliva (c) Linear regression analysis based on the limit of detection (d) Hierarchical clustering analysis showing the cluster similarities between whole virus particles and their structural components.

Figure 4

Mechanism of signal enhancement in the label-free SERS assay demonstrated through the fluorescence quenching on adsorption of whole virus particles on the nanostructured sensor.

Figure 5

Cross-reactivity analysis shows the ability of the nanostructured sensor to distinguish different viruses clearly (a) Representative SERS spectra of SARS-CoV-2, Influenza, HCoV OC43, Respiratory syncytial virus, (b) Violin plot showing the F1 scores using partial least square method (c) tSNE analysis showing the clear distinction between different viral species, showing the high specificity of the label-free SERS saliva test using a nanostructured sensor (d) Heatmap showing the significant principal component contributing to the high specificity of the label-free SERS saliva test using the nanostructured sensor.(*—(p-value < 0.01), ***—(p-value < 0.0001)).

Figure 6

Direct diagnosis of SARS-CoV-2 virus from saliva samples (a) diagnostic parameters of COVID detection using Nanosensors (b) Applicability of nanostructured sensor for differentiating similar viruses (c) Clinical validation of the assay with RT-PCR.