A DNA biosensors-based microfluidic platform for attomolar real-time detection of unamplified SARS-CoV-2 virus

Abstract

The emergence of the coronavirus 2019 (COVID-19) arose the need for rapid, accurate and massive virus detection methods to control the spread of infectious diseases. In this work, a device, deployable in non-medical environments, has been developed for the detection of non-amplified SARS-CoV-2 RNA. A SARS-CoV-2 specific probe was designed and covalently immobilized at the surface of glass slides to fabricate a DNA biosensor. The resulting system was integrated in a microfluidic platform, in which viral RNA was extracted from non-treated human saliva, before hybridizing at the surface of the sensor. The formed DNA/RNA duplex was detected in presence of SYBR Green I using an opto-electronic system, based on a high-power LED and a photo multiplier tube, which convert the emitted fluorescence into an electrical signal that can be processed in less than 10 min. The limit of detection of the resulting microfluidic platform reached six copies of viral RNA per microliter of sample (equal to 10 aM) and satisfied the safety margin. The absence of non-specific adsorption and the selectivity for SARS-CoV-2 RNA were established. In addition, the designed device could be applicable for the detection of a variety of viruses by simple modification of the immobilized probe.

Keywords: DNA-biosensor; Fluorescence; Microfluidic; RNA extraction; SARS-CoV-2 detection; Silica slide.

Graphical abstract

Fig. 1

Test workflow for the identification of SARS-CoV-2 viral charge in human saliva samples based on the direct detection of viral RNA at the surface of ssDNA-coated sensing SiO₂ slides. From collection to readout, the sample is processed through extraction of the viral genetic material (step ②), sensing on DNA-functionalized silica slides (step ③) and fluorescence detection (step ④).

Scheme 1

a: Schematic illustration of PP1 immobilization at the surface of amino-modified slides. b: Schematic illustration of the procedure for PP1 quantification at the surface of the sensors. A Cy3-labeled complementary reverse probe (Supporting Information, Table S2) was added to the functionalized slides, followed by removal of non-hybridized sequences through washing cycles. The resulting DNA duplexes were denaturated by thermal treatment for quantification of the Cy3-labeled sequences released in the supernatant.

Fig. 2

a: Concept of the SARS-CoV-2 diagnostics sensing device. b: Manufactured test bench. c: Concept of the microfluidic diagnostic circuit. d: Produced disposable microfluidic kit. e: Concept of the opto-mechanical microscopy setup of the SARS-CoV-2 diagnostics sensing device. f: Manufactured fluorimetric detection system. For the main block diagram of the automated SARS-CoV-2 diagnostics sensing device, see Supporting Information, Fig. S6.

Fig. 3

SARS-CoV-2 RNA detection with S-PP1 slides. a: Light intensities emitted by SYBR Green I upon hybridization of SARS-CoV-2 RNA with PP1 probes at the surface of S-PP1 sensing slides, using decreasing viral loads expressed in copies per μL of analyzed saliva samples. Results are expressed as the mean ± SD of two independent experiments b: Difference between S-PP1 samples and baseline at decreasing viral loads represented as logarithmic colorbars of normalized intensities over LED current intensity (mA) and PMT gain (mV).

Fig. 4

a: Immunofluorescence detection of SARS-CoV-2 RNA with S-PP1 slides in saliva samples containing 50, 12, 6 and 3 copies/μL of the virus. Fluorescence was revealed through SYBR Green emission. b: Control experiments; S-PP1 slides with SYBR Green I only, S-PP1 slides with MERS virus at 50 copies/μL, S-PP1 slides with pure saliva, S-Suc slides with SARS-CoV-2 virus at 50 copies/μL. Scale bar: 10 μm.