Small form factor flow virometer for SARS-CoV-2

Abstract

Current diagnostics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection heavily rely on reverse transcription-polymerase chain reaction (RT-PCR) or on rapid antigen detection tests. The former suffers from long time-to-result and high cost while the latter from poor sensitivity. Therefore, it is crucial to develop rapid, sensitive, robust, and inexpensive methods for SARS-CoV-2 testing. Herein, we report a novel optofluidic technology, a flow-virometry reader (FVR), for fast and reliable SARS-CoV-2 detection in saliva samples. A small microfluidic chip together with a laser-pumped optical head detects the presence of viruses tagged with fluorescent antibodies directly from saliva samples. The technology has been validated using clinical samples with high sensitivity (91.2%) and specificity (90%). Thanks also to its short time-to-result (<30 min) and small size (25 × 30 × 13 cm), which can be further reduced in the future, it is a strong alternative to existing tests, especially for point-of-care (POC) and low resource settings.

Fig. 1.

A comprehensive overview of the developed diagnostic method for detection of SARS-CoV-2 antigen, from sample collection to SARS-CoV-2 antigen measurement. 1) Saliva is collected by spit, heat inactivated at 56°C for 1 hour, diluted in PBS and filtered with a 0.2 µm syringe filter. 2) The sample is then labelled with Alexa 488-anti-SARS-CoV spike antibodies and incubated for 20 minutes at room temperature. 3) The labelled sample is circulated inside the FVR and a fluorescence signal is detected every time antibody-virus complexes pass through the interrogation point.

Fig. 2.

FVR characterization. (a) A schematic diagram of the FVR detection system. (b) A lab built FVR prototype with the tablet. (c) Standard curve of the polystyrene beads concentration vs. total counts, R² = 0.99. (d) Standard curve of the fluorescent (Alexa 488) anti-SARS-CoV spike antibody concentration vs. counts per minute, R² = 0.94. For visual representation purposes the y-axis is broken into two regions [0:10], [60:100] and the x-axis into three [0:6], [49:52] and [498:501]. The error bar represents the standard deviation.

Fig. 3.

Determination of FVR cut-off value and standard curve for SARS-CoV-2 detection (a) Counted events per minute of the Alexa 488-anti-SARS-CoV spike antibody (blue) and of filtered saliva samples (orange). Mean values (dotted lines) and standard deviations (shaded area) are indicated. Cut-off, according to the antibody signal (blue), was calculated as mean plus two times its standard deviation and indicated as solid line. (b) Standard curve of the SARS-CoV-2 viral particles concentration vs. normalised counts; R² = 0.94.

Fig. 4.

Detection of SARS-CoV-2 in saliva samples from SARS-CoV-2 infected and uninfected individuals. SARS-CoV-2 was determined in saliva samples from both SARS-CoV-2 infected (n = 34) and uninfected (n = 20) individuals in a blind test. (a) Sample identification by color code (red: SARS-CoV-2 positive samples; green: SARS-CoV-2 uninfected samples) according to RT-qPCR. (b) Distribution of saliva samples showing the normalized count obtained with the FVR. (c) Correlation between normalized counts and RT-qPCR viral load [copies mL^-1] of blind test samples. The dashed line indicates a linear fit (R² = 0.43) to the data from positive samples that fall within our analytical range excluding the outliers marked with a circle. Here outliers are defined as data points whose residual values are greater than two times the standard deviation of all the data points.