A mask-based diagnostic platform for point-of-care screening of Covid-19

Abstract

Diagnostics of SARS-CoV-2 infection using real-time reverse-transcription polymerase chain reaction (RT-PCR) on nasopharyngeal swabs is now well-established, with saliva-based testing being lately more widely implemented for being more adapted for self-testing approaches. In this study, we introduce a different concept based on exhaled breath condensate (EBC), readily collected by a mask-based sampling device, and detection with an electrochemical biosensor with a modular architecture that enables fast and specific detection and quantification of COVID-19. The face mask forms an exhaled breath vapor containment volume to hold the exhaled breath vapor in proximity to the EBC collector to enable a condensate-forming surface, cooled by a thermal mass, to coalesce the exhaled breath into a 200-500 μL fluid sample in 2 min. EBC RT-PCR for SARS-CoV-2 genes (E, ORF1ab) on samples collected from 7 SARS-CoV-2 positive and 7 SARS-CoV-2 negative patients were performed. The presence of SARS-CoV-2 could be detected in 5 out of 7 SARS-CoV-2 positive patients. Furthermore, the EBC samples were screened on an electrochemical aptamer biosensor, which detects SARS-CoV-2 viral particles down to 10 pfu mL^-1 in cultured SARS-CoV-2 suspensions. Using a "turn off" assay via ferrocenemethanol redox mediator, results about the infectivity state of the patient are obtained in 10 min.

Keywords: Aptamers; Detection; Electrochemistry; Exhaled breath condensate (EBC); Real-time reverse-transcription polymerase chain reaction (RT-PCR); SARS-CoV-2.

Fig. 1

Exhaled breath condensate (EBC)-based diagnostic strategy for SARS-CoV-2 infectivity: Laboratory engineered mask allows collection of EBC by first cooling the mask for 30 min in the freezer, putting on the cooled mask and breathing into it for 5 min. EBS formed in the Teflon-lining of the inside of the mask is collected and directly deposited onto an electrochemical sensing modified with SARS-CoV-2 specific aptamer targeting the receptor-binding domain (RBD) region of the S1 spike protein as surface receptor. Using ferrocenemethanol as a redox meditator before and after viral interaction allows discrimination between positive and negative EBC samples.

Fig. 2

Mask-based EBC collector: (a) Inside of the laboratory engineered mask showing an exhaled breath condensate (EBC) collector (cold trap, indicated with orange line) for converting breath vapor into a fluid sampe. The EBC collector is made of a Teflon-based condensate-forming surface. (b) Image of EBC formed on the Teflon collector after 5 min breathing into the mask. (c) EBC volume collected in 5 min using the EBC mask (n = 14). (d) EBC volume collected in 5 min using commercial RTube condensers (n = 14). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Electrochemical Aptamer Sensor: (a) 2D structure of DNA aptamer with sequence redacted, (b) Biolayer interferometry (BLI) measurements of biotinylated aptamer linked onto streptavidin-activated BLI sensors with different concentrations of SARS-CoV-2 S protein (3.13 nM, 6.25 nM, 12.5 nM and 25 nM): running buffer: 1x PBS, 1 mM MgCl₂, (c) Surface attachment strategy of SARS-Cov-2 aptamer on the gold working electrode of the screen-printed electrode using maleimide-thiol-aptamer linkage. (d) Cyclic voltammograms (CV) of a gold electrode before (black) and after (green) functionalization with SARS-CoV-2 aptamer (10 μg mL⁻¹ for 2 h) using ferrocenemethanol as a redox mediator (1 mM in 0.1 M PBS, pH 7.4, Scan rate = 100 mV s⁻¹). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Electrochemical SARS-CoV-2 aptasensor: (a) Differential pulse voltammogram (DPV) of aptamer modified electrode using ferrocenemethanol (1 mM in 0.1 M PBS, pH 7.4) as a redox meditor. Initial signal (black) and after addition 50 nM RBD for 10 min (grey line), washing and recording a new DPV in ferrocenemethanol. The decrease in current is due to RBD binding to the aptamer. DPV conditions: t_aquis = 3s, E_step = 0.01V, E_pulse = 0.06 V, t_pulse = 0.02 V, scan rate = 0.06 V s⁻¹. (b) Current response to increasing RBD concentrations using ferrocenemethanol (1 mM PBS, pH 7.4) as a redox probe. (c) Langmuir adsorption isotherm as extracted from Fig. 4b. (d) Dose-dependent response curve toward SARS-CoV-2 virus clade 20A.EU2 (black) as well as clade 20I/501Y.V1, “British variant” (green) and the clade 20H/501Y.V2, “South African variant” (blue) on aptamer-modified electrodes. (e) Selectivity of the aptamer sensors towards other real patient virus samples by comparison to SARS-CoV-2 positive samples. All the virus samples have Ct values between 22–25 and are nasal swab samples. All the values are displayed as means ± SEM (n = 5). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)