Rapid, Ultrasensitive, and Quantitative Detection of SARS-CoV-2 Using Antisense Oligonucleotides Directed Electrochemical Biosensor Chip

Abstract

A large-scale diagnosis of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is essential to downregulate its spread within as well as across communities and mitigate the current outbreak of the pandemic novel coronavirus disease 2019 (COVID-19). Herein, we report the development of a rapid (less than 5 min), low-cost, easy-to-implement, and quantitative paper-based electrochemical sensor chip to enable the digital detection of SARS-CoV-2 genetic material. The biosensor uses gold nanoparticles (AuNPs), capped with highly specific antisense oligonucleotides (ssDNA) targeting viral nucleocapsid phosphoprotein (N-gene). The sensing probes are immobilized on a paper-based electrochemical platform to yield a nucleic-acid-testing device with a readout that can be recorded with a simple hand-held reader. The biosensor chip has been tested using samples collected from Vero cells infected with SARS-CoV-2 virus and clinical samples. The sensor provides a significant improvement in output signal only in the presence of its target—SARS-CoV-2 RNA—within less than 5 min of incubation time, with a sensitivity of 231 (copies μL –1 ) −1 and limit of detection of 6.9 copies/μL without the need for any further amplification. The sensor chip performance has been tested using clinical samples from 22 COVID-19 positive patients and 26 healthy asymptomatic subjects confirmed using the FDA-approved RT-PCR COVID-19 diagnostic kit. The sensor successfully distinguishes the positive COVID-19 samples from the negative ones with almost 100% accuracy, sensitivity, and specificity and exhibits an insignificant change in output signal for the samples lacking a SARS-CoV-2 viral target segment ( e . g ., SARS-CoV, MERS-CoV, or negative COVID-19 samples collected from healthy subjects). The feasibility of the sensor even during the genomic mutation of the virus is also ensured from the design of the ssDNA-conjugated AuNPs that simultaneously target two separate regions of the same SARS-CoV-2 N-gene.

Detection of the SARS-CoV-2 spike protein in saliva with Shrinky-Dink© electrodes

“…The biosensor still needs to be validated to detect the whole virus but given the location of the S1 protein at the outermost layer of the virus, we expect that this should not be an issue. 26 In future studies, the varying performance at different frequencies as well as different probe densities should be investigated to better understand aptamer behaviour on the molecular level. Additionally, we expect using aptamers specifically designed for binding-induced conformational changes would produce even more sensitive results than with the aptamer used in this study, which has no confirmed conformational change upon binding with the S1 protein.…”

Electrochemical sensing: A prognostic tool in the fight against COVID-19

“… Analyte Electrode/modification Receptor Matrix/Solvent LOD (copies/mL)1 Linear range (copies/mL)1 Ref. Viral nucleocapsid phosphoprotein (N-gene) Calixarene functionalized graphene oxide electrodes, super sandwich type sensor Custom designed capture, signal, and auxillary probes Pharmaceutical preparation 200 n/a [ 192 ] Spike Receptor Binding Domain Cobalt-functionalized TiO2 nanotubes (Co-TNTs) Cobalt SARS-CoV-2 Wuhan-Hu-1 sRBD with C-terminal hexa-histidine tag pharmaceutical preparation 0.7 nM 14-1.4*10ˆ3 nM [ 189 ] COVID-specific viral RNA or the corresponding c-DNA Electrode made of Gold nanoparticles on Ti surface Complementary thiolated probes n/a n/a n/a [ 193 ] Viral nucleocapsid phosphoprotein (N-gene) Electrode made of AuNPs on graphene film Highly specific antisense oligonucleotides (ssDNA) Pharmaceutical Preparation 6.9 5.85*10ˆ7- 1*10ˆ9 [ 194 ] +Spike protein Graphene FET-based sensor with Au/Cr electrodes SARS-CoV-2 spike antibody Nasopharyngeal secretions from COVID-19 infected individuals 242 n/a [ 190 ] Multiplex sensor for SARS-CoV-2 nucleocapsid protein (NP), S1-IgM, S1 IgG, and CRP Laser-engraved graphene electrodes, sandwich/double-sandwich type assay (depending on the analyte) Capture antigens and antibodies for NP, S1-IgM, S1-IgG and CRP immobilized onto the graphene electrode surface Serum and saliva samples from RT PCR-confirmed COVID-19 positive patients and healthy patients n/a n/a [ 191 ] 1 The units used are copies/mL unless otherwise noted. …”

Trends in biosensing platforms for SARS-CoV-2 detection: A critical appraisal against standard detection tools

“…Recently, Pan’s research group fabricated Au NPs capped with specific antisense oligonucleotides (ssDNA) based electrochemical biosensor chip which enabled digital detection of SARS-CoV-2 nucleocapsid phosphoprotein (N-gene) [ 99 ]. The paper based electrochemical system was used to immobilize sensing probes in order to construct nucleic acid testing device.…”

“… Biosensor Pathogen Detection target Limit of detection Linear range Ref. EC SARS-CoV-2 S or N protein 19 ng/mL and 8 ng/mL - [ 103 ] EC SARS-CoV-2 Antibodies - - [ 101 ] EC SARS-CoV-2 N-gene 6.9 copies/μL 585.4 to 5.854 × 10 7 copies/μL [ 99 ] Cell-based SARS-CoV-2 Antigen 1 fgmL 1- 10 fg and 1 μg mL 1- [ 105 ] Optofluidic SARS-CoV-2 Antibody 0.5 pM 1- [ 83 ] Nanoplasmonic SARS-CoV-2 Virus particles 370 vp/mL 0 to 10 7 vp/mL [ 80 ] SPR SARS-CoV-2 Antibody 1.02 pM 2-1000 pM [ 82 ] LSPR SARS-CoV-2 RNA 1 pM 1 nM to 1 μM [ 106 ] Lateral flow optical/chemiluminescence SARS-CoV-2 Serum IgA - - [ 78 ] SERS-LFIA SARS-CoV-2 Antibody 1.28×10 7 -fold dilution - [ 79 ] Lateral flow SARS-CoV-2 RNA 12 copies...…”