Lab-on-a-chip for the easy and visual detection of SARS-CoV-2 in saliva based on sensory polymers

Abstract

The initial stages of the pandemic caused by SARS-CoV-2 showed that early detection of the virus in a simple way is the best tool until the development of vaccines. Many different tests are invasive or need the patient to cough up or even drag a sample of mucus from the throat area. Besides, the manufacturing time has proven insufficient in pandemic conditions since they were out of stock in many countries. Here we show a new method of manufacturing virus sensors and a proof of concept with SARS-CoV-2. We found that a fluorogenic peptide substrate of the main protease of the virus (Mpro) can be covalently immobilized in a polymer, with which a cellulose-based material can be coated. These sensory labels fluoresce with a single saliva sample of a positive COVID-19 patient. The results matched with that of the antigen tests in 22 of 26 studied cases (85% success rate).

Keywords: COVID-19; Coating; Fluorimetry; Immobilization; Pandemics; Paper-supported; Peptide; Substrate.

Graphical abstract

Fig. 1

Preparation of sensory labels: a) chemical structure and schematic view of the fluorogenic peptide substrate for SARS-CoV-2 main protease; b) graphical abstract of the sensory labels preparation procedure containing covalently anchored substrates; and c) graphical abstract of the sensory labels preparation procedure containing non-covalently anchored substrates.

Fig. 2

Enzyme tests carried out in 96-well plates, including a 6 mm diameter sensory label at the bottom of each well and 20 μL of a 0.5 μM solution of Mpro in 20 mM Tris-HCl pH 7.3, 100 mM NaCl, 1 mM EDTA, 1 mM DTT buffer. Experimental conditions: temperature = 30ºC, λ_ex = 360 nm, λ_em = 460 nm, excitation slit = 40 nm, emission slit = 40 nm. (a) Response time study of sensory labels 1–4, by monitoring the fluorescence at 15, 30, 60, 120, 180, and 240 min; image of a sensory label 1 before and after interaction with Mpro. (b) Storage stability study of sensory labels 1, 3 and free peptide by measuring the fluorescence response at 60 min after 1, 7, 14, 28 and 60 days of storage using zip bags. Data are means ± standard error of 3 independent replicates.

Fig. 3

EpiDermTM tissues were exposed to sensory labels 1 and 2 for 1 h. The viability was analysed by MTT assay, and it is expressed as a percentage of negative control. Data represented the mean ± standard error of 3 independent replicates. Differences were established using a one-way ANOVA followed by a multiple comparisons test (Tukey test) and considered significant when p ≤ 0.05. The same letter indicates no significant differences between treatments.

Fig. 4

Results for Subject #4 testing with sensory label coated with copolymer 1 (sensory label 1), sensory label coated with copolymer 2 (sensory label 2), and antigen test. The image shows the real photograph and the cropped image. Each photo contains 2 negative controls, 2 positive controls, and 2 replicates.