A microfluidic biosensor architecture for the rapid detection of COVID-19

Abstract

The lack of enough diagnostic capacity to detect severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has been one of the major challenges in the control the 2019 COVID pandemic; this led to significant delay in prompt treatment of COVID-19 patients or accurately estimate disease situation. Current methods for the diagnosis of SARS-COV-2 infection on clinical specimens (e.g. nasal swabs) include polymerase chain reaction (PCR) based methods, such as real-time reverse transcription (rRT) PCR, real-time reverse transcription loop-mediated isothermal amplification (rRT-LAMP), and immunoassay based methods, such as rapid antigen test (RAT). These conventional PCR methods excel in sensitivity and specificity but require a laboratory setting and typically take up to 6 h to obtain the results whereas RAT has a low sensitivity (typically at least 3000 TCID50/ml) although with the results with 15 min. We have developed a robust micro-electro-mechanical system (MEMS) based impedance biosensor fit for rapid and accurate detection of SARS-COV-2 of clinical samples in the field with minimal training. The biosensor consisted of three regions that enabled concentrating, trapping, and sensing the virus present in low quantities with high selectivity and sensitivity in 40 min using an electrode coated with a specific SARS-COV-2 antibody cross-linker mixture. Changes in the impedance value due to the binding of the SARS-COV-2 antigen to the antibody will indicate positive or negative result. The testing results showed that the biosensor's limit of detection (LoD) for detection of inactivated SARS-COV-2 antigen in phosphate buffer saline (PBS) was as low as 50 TCID50/ml. The biosensor specificity was confirmed using the influenza virus while the selectivity was confirmed using influenza polyclonal sera. Overall, the results showed that the biosensor is able to detect SARS-COV-2 in clinical samples (swabs) in 40 min with a sensitivity of 26 TCID50/ml.

Keywords: Biosensor; Dielectrophoresis; Impedance measurement; Microfluidic; Polymerase chain reaction (PCR); SARS-COV-2; Trapping and focusing electrodes.

Graphical abstract

Fig. 1

(a) An electric field (E-Field) modeling and simulation using COMSOL Multiphysics software of the three regions making the biosensor, i.e., (b) focusing, (c) detection regions and (d) tapping (e) An equivalent electrical circuit of the biosensor was also included in the simulation. (f) Finally, experimental and simulation results were obtained after the SARA- Covid-19 antibody-antigen binding occurred in the microchannel.

Fig. 2

(a) Three-dimensional (3D) Schematics of the biosensor, presenting the focusing, trapping and detection regions, (b) A side view of biosensor showing the materials used in device fabrication, (c) Scanning electron microscope (SEMs) micrographs of the fabricated biosensor.

Fig. 3

(a) A schematic of the device testing setup showing. Top view schematics of the biosensor showing the solution flow direction during (b) antibody coating where the antibody-cross linker mixture was first placed at the antibody inlet and suction was applied to the antibody outlet while all other inlets were closed, (c) SARS-CoV-2 virus antigen sample was placed at the sample inlet while suction was applied to the sample outlet directing the flow toward the focusing region. The flow subsequently continued toward the detection region. (d) Process flow for antibody immobilization, and the antibody/antigen binding on the IDE array, (d-1) sideview of the device, (d-2) the antibody was loaded from the antibody inlet while suction was applied from the antibody outlet. All other inlets and outlets are closed, (d-3) the microchannel was washed after adhesion of antibody to the IDE array, (d-4) the virus sample was loaded into the sample inlet while suction was applied to the sample outlet, (d-5) the microchannel was washed again after antibody antigen binding was completed.

Fig. 4

(a) fluorescent images before focusing the nanobeads into the centerline of the focusing region, (b) fluorescent images after focusing the nanobeads into the centerline of the focusing region. (c) fluorescent images before trapping the nanobeads onto the surface of the detection electrode array. (d) fluorescent images after trapping the nanobeads onto the surface of the detection electrode array.

Fig. 5

(a) The optimal antibody concentration against SARS-CoV-2 virus was determined using multiple antibody dilutions from 0.0166 mg/ml to 1.66 mg/ml mixed with a cross linker (Sulfo-LC-SPDP). Each antibody dilution was tested using a fixed virus concentration at 105 TCID50/ml in PBS and a fixed antibody coating time, i.e., 60 minutes. (b) The impedance change was plotted versus antibody concentration at 1 kHz. (c) The optimal antibody concentration against SARS-CoV-2 virus was determined using wastewater samples spiked with fixed concentration of SARS-CoV-2, i.e., 10³ TCID50/ml. (d) Testing of SARS-CoV-2 in PBS at various titers, (e) The specificity was tested by immobilizing antibody (Ab) SWZ/13 against influenza virus (1.3μg/ml) on the sensing electrode while SARS-CoV-2 samples with a fixed concentration of 10⁵ TCID50/ml in PBS were used. The result was compared with detection of the SARS-CoV-2 antigen using SARS-CoV-2 antibody for the same antigen concentration. (f) The selectivity was measured using influenza virus samples with a concentration of 10⁶ TCID50/ml with the sensing electrode coated with SARS-CoV-2 antibody (0.083 mg/ml).

Fig. 6

Testing of Four inactivated clinical human samples with final various titrations between 0.40 - 6.49′10⁵TCID50/ml after 10-fold dilution.

Fig. 7

(a) wastewater samples were spiked with various concentration (50-10³ TCID50/ml) of SARS-CoV-2, (b) wastewater samples that were collected in April 2020. They were tested negative for SARS-CoV-2. (c) comparison between the results of testing a low concentration of SARS-CoV-2 in PBS (100TCID50/ml), and in wastewater (100 TCID50/ml), and non-specific binding of SARS-CoV-2 with high concentrations (10⁵ TCID50/ml) to influenza antibody, and non-specific binding of Influenza with high concentrations (10⁵ TCID50/ml) to SARS-CoV-2 antibody.