Computational aptamer design for spike glycoprotein (S) (SARS CoV-2) detection with an electrochemical aptasensor

Abstract

A new bioinformatic platform (APTERION) was used to design in a short time and with high specificity an aptamer for the detection of the spike protein, a structural protein of SARS-CoV-2 virus, responsible for the COVID-19 pandemic. The aptamer concentration on the carbon electrode surface was optimized using static contact angle and fluorescence method, while specificity was tested using differential pulse voltammetry (DPV) associated to carbon screen-printed electrodes. The data obtained demonstrated the good features of the aptamer which could be used to create a rapid method for the detection of SARS-CoV-2 virus. In fact, it is specific for spike also when tested against bovine serum albumin and lysozyme, competitor proteins if saliva is used as sample to test for the virus presence. Spectrofluorometric characterization allowed to measure the amount of aptamer present on the carbon electrode surface, while DPV measurements proved the affinity of the aptamer towards the spike protein and gave quantitative results. The acquired data allowed to conclude that the APTERION bioinformatic platform is a good method for aptamer design for rapidity and specificity. KEY POINTS: • Spike protein detection using an electrochemical biosensor • Aptamer characterization by contact angle and fluorescent measurements on electrode surface • Computational design of specific aptamers to speed up the aptameric sequence time.

Keywords: Aptamer; Bioinformatic platform; Biosensor; Screen-printed electrodes; Spike protein.

Fig. 1

Contact angle analysis after incubation of 0.5 µM up to 5 µM of aptamer-TAMRA on electrode surface. Data are reported as mean value of left and right angle on the sample and the standard deviation

Fig. 2

Quantification of aptamer by spectrofluorimetric evaluation in two different buffers using calibration curves reported in Fig. S1. Aptamer-TAMRA incubation was performed at 0.5 µM concentration in PBS 1X and 1 mM concentration in TRIS buffer

Fig. 3

Quantification of the bounded aptamer in TRIS buffer, subtracting the fluorescence signal of unbounded and washing from stock solutions. Data are reported as mean of at least two different electrodes, and error bars represent the standard deviation. Linear regression was performed considering data up to 1 μM aptamer incubated on surface

Fig. 4

Fluorescent microscopy images of aptamer-TAMRA incubated on carbon electrodes at 0.1 µM (A), 0.5 µM (B), 1 µM (C). Images were acquired at × 10 magnification with 1 s of exposure time

Fig. 5

Results obtained from the differential pulse voltammetry (DPV) tests. A The original curves resulting after the addition of different spike concentrations (0.01 μM—0.05–0.1 µM) on the surface of the WE with the aptamer at 0.5 µM. B The Ipa values (µA) obtained after aptamer adsorption and after the addition of increasing concentrations of the spike protein. C The correspondent ∆Ipa values (µA) obtained after the addition of spike protein concentrations on the screen-printed electrode with the aptamer

Fig. 6

Curves and ΔIpa obtained for the tests performed with bovine serum albumine (BSA) and lysozyme. A Reports the original curves obtained using the modified SPE with the serial addition of Bovine Serum Albumine (BSA) at concentrations of 1.5 – 3 - 6 μM. B Shows the values of ΔIpa obtained for the BSA concentrations tested. C Shows the original curves of the deposition of increasing concentrations of lysozyme (1.5 – 2 – 3 - 10 μM). D Reports the values of ΔIpa obtained for the lysozyme concentrations tested

Fig. 7

Linear range obtained for the carbon SPE used

Fig. 8

Image of the results of the aptamer designed with docking using the in silico design. The structure reported is obtained from the sequence selected by the programs used