Biosensing strategies for the detection of SARS-CoV-2 nucleic acids

SARS-CoV-2, first reported in late 2019, led to a global pandemic with hundreds of millions of cases and millions of deaths. The virus is a positive-sense single-stranded RNA coronavirus (~30 kb) with key structural and non-structural genes (including S, E, M, N, ORF1a/1b). Diagnosis has largely relied on detection of viral genetic material, with RT-PCR as the gold standard, but it requires thermal cycling, trained personnel, and laboratory infrastructure. Isothermal amplification methods (e.g., LAMP/RT-LAMP, RCA, RPA, SDA, NASBA, CRISPR-based assays) have emerged as attractive alternatives for point-of-care (POC) use. This review focuses on biosensing strategies specifically for SARS-CoV-2 RNA, discussing biorecognition layers and transduction mechanisms, and situating them within the broader diagnostic landscape to address the need for rapid, sensitive, and decentralized testing.

The paper synthesizes advances (primarily 2020–2022) in SARS-CoV-2 RNA biosensing covering electrochemical, electrochemiluminescent (ECL), optical, and lateral flow biosensors. It contextualizes these within prior reviews on nucleic acid amplification techniques (NAATs), colorimetric and lateral flow methods, and commercial RT-PCR/immunoassay kits. It contrasts the strengths and limitations of RT-PCR with isothermal amplification approaches and highlights CRISPR-based detection. The review compiles platforms, target genes (ORF1ab, N, E, RdRp), analytical performances (linear ranges, detection limits), selectivity against related viruses, and validation in clinical matrices (swabs, saliva, serum). It also references commercialization and regulatory approvals for integrated LF-NAAT devices.

As a narrative review, the authors collected and analyzed recent reports of SARS-CoV-2 RNA biosensors, organizing them by transduction mechanism. For each platform, they describe the biorecognition design (e.g., probes, nanomaterials, CRISPR enzymes), amplification strategies when used (CHA, TdT, DNA walkers, HCR, LAMP/RAA/RPA), the transduction/readout (electrochemical, ECL, optical, lateral flow), target gene region, analytical figures of merit (linear range, limit of detection), selectivity, and performance in clinical or spiked samples. They also summarize commercially available LF-integrated NAAT devices and note regulatory status.

- Electrochemical biosensors: - Sandwich hybridization with magnetic beads and HRP/TMB readout achieved 0.6 pM LOD and discrimination against homologous viruses (Cajigas et al.). - Impedimetric/amperometric sensors on MWCNTs–avidin platforms reached aM-level LODs and detected RT-PCR–amplified RNA diluted up to 10^10-fold (López Mujica et al.). - BNQDs/flower-like Au nanostructures with methylene blue (MB) indicators provided turn-on/off DPV detection down to 0.27 aM and validated in clinical nasopharyngeal swabs (Hatamluyi et al.; Pina-Coronado et al.). - Graphene-based paper chip with AuNP-capped antisense probes enabled amplification-free detection at 6.9 copies/μL with no cross-reactivity (Alafeef et al.). - EDL-gated BioFET detected N-gene RNA in saliva from 1 fM to 1 pM (Paulose et al.). - CHA+TdT cascade amplified signal for RdRp/ORF1ab targets with LODs of 45 fM and 26 fM and successful swab testing (Deng et al.; Peng et al.). - Self-actuated molecular-electrochemical system (MECS) detected as few as 4 copies in 80 μL artificial saliva and enabled direct testing without amplification (Ji et al.). - Electrochemiluminescent (ECL) biosensors: - DNA tetrahedron with entropy-driven amplification enabled fM detection in serum (Fan et al.). - Dual-wavelength ratiometric ECL via RET between g-C3N4 and Ru-SiO2 with 3D DNA walker achieved sub-fM LOD (0.18 fM) and recoveries ~92–102% in throat swabs (Yin et al.). - Combined entropy-driven and bipedal DNA walker strategies yielded 7.8 aM LOD in serum (Fan et al.). - HCR-based ECL reached 59 aM LOD in human pharyngeal swabs (Zhang et al.). - 3D DNA walker + CRISPR-Cas12a on MXene ECL gave 12.8 aM LOD (Zhang et al.). - Optical/CRISPR-based biosensing: - Label-free DNA-Cu nanocluster + exonuclease I reporter for Cas12a delivered 20 copies LOD (Xie et al.). - Smartphone-compatible CRISPR-Cas12a colorimetry and FRET reporters detected down to 1 copy/μL across vectors, pseudoviruses, and clinical samples (Ma et al.). - PGM-CRISPR assay converted Cas12a activity to glucose readout with dynamic range 10^1–10^4 copies/μL and 10 copies/μL LOD; validated in clinical samples (Huang et al.). - Amplification-free SERS-CRISPR detected 1.0 fM in clinical swabs (Liang et al.). - CASCADE (Cas13a + AuNPs) enabled naked-eye detection at 100 pM; with RPA/NASBA, LOD improved to fM/aM; validated with swabs (López-Valls et al.). - Molecular beacon using telomeric G-triplex and ThT reached 0.01 nM LOD with DSN recycling (Qin et al.). - ASOs templating in situ anisotropic AuNPs allowed extraction-free colorimetric detection directly from throat samples, concordant with RT-qPCR (Borghei et al.). - Lateral flow biosensing (LF): - RT-MCDA + Cas12a LF detected 7 copies/test for ORF1ab and N; validated on clinical RNA (Zhu et al.). - RT-LAMP + Cas12a LF (iSCAN) detected 10 copies/reaction in nasopharyngeal swabs (Ali et al.). - Multiplex RT-LAMP-LFB targeting ORF1ab and N detected 12 copies/reaction with 100% sensitivity/specificity (Zhu et al.). - Amplification-free hybrid capture fluorescence immunoassay (HC-FIA) LF detected 500 copies/mL with 100% sensitivity and 99% specificity for throat swabs and sputum; portable reader suitable for POC and approved in China/EU (Wang et al.). - Wearable face-mask FDCF integrating lysis, RT-RPA, Cas12a, and LF achieved 500 copies LOD in ~1.5 h at room temperature (Nguyen et al.).

The compiled evidence shows that biosensors can sensitively and selectively detect SARS-CoV-2 RNA with simplified workflows suitable for decentralized and point-of-care testing. Electrochemical platforms, often without target amplification, reach aM-level or few-copies sensitivity and work with diverse clinical matrices (swabs, saliva), leveraging nanomaterials and innovative probe architectures (e.g., MECS). ECL approaches provide ultralow detection limits via signal amplification circuits (DNA walkers, HCR, entropy-driven reactions) and ratiometric readouts that enhance robustness. Optical and CRISPR-based sensors translate nuclease activity into visual, fluorescent, SERS, or even glucose meter signals, enabling instrument-light diagnostics and smartphone readouts. Lateral flow integrations with isothermal amplification deliver rapid, user-friendly tests with clinically relevant sensitivity and specificity, several already commercialized or approved. Collectively, these findings address the need for rapid, decentralized diagnostics that complement RT-PCR, particularly in low-resource or high-throughput screening settings. Remaining challenges include achieving robust amplification-free early-stage detection, multiplexing for variants, and large-scale manufacturing with rigorous clinical validation.

Biosensors have emerged as powerful tools for SARS-CoV-2 RNA detection, offering fast, user-friendly, and sensitive assays that can be miniaturized and adapted for POC use. Electrochemical biosensors, in particular, demonstrate high sensitivity and selectivity, sometimes without nucleic acid amplification, while LF-based readouts coupled to extraction/amplification enable sensitive and selective diagnostics suited for mass production and home use. The widespread deployment of antigen LF tests demonstrated the feasibility and impact of such platforms; extending similar success to RNA-targeting LF or hybrid systems could enable earlier diagnosis. Future work should focus on fast, accurate, amplification-free, multiplex POC devices capable of detecting very low copy numbers and new viral mutations, along with robust clinical validation and scalable manufacturing.

As a review, this work compiles reported performances and validations but does not generate new experimental data. The authors highlight that despite significant progress, developing mass-producible, amplification-free biosensors with sufficient sensitivity for very early-stage SARS-CoV-2 RNA detection remains challenging. Some approaches still require specialized reagents, controlled temperatures, or instrumentation (e.g., ECL, certain CRISPR formats), and broad clinical validation across variants and sample types is needed.