The COVID-19 pandemic, caused by SARS-CoV-2, resulted in a global health and economic crisis. The need for rapid, sensitive, and accessible diagnostic tools is paramount. While RT-PCR is the gold standard, it requires specialized equipment and trained personnel, limiting its widespread applicability, especially in resource-limited settings. This necessitates the development of alternative diagnostic platforms, particularly point-of-care (POC) technologies. Several reviews have addressed various SARS-CoV-2 detection strategies targeting RNA, proteins, or host antibodies. However, this review specifically focuses on biosensors designed for the detection and quantification of SARS-CoV-2 RNA. The emphasis will be on the critical components of these biosensors: the biorecognition layer responsible for selectively binding to the viral RNA, and the transduction mechanism that converts the biorecognition event into a measurable signal. This detailed analysis will provide a comprehensive understanding of the current state-of-the-art in SARS-CoV-2 RNA biosensors and highlight potential avenues for future improvement.

The literature extensively covers various SARS-CoV-2 detection methods. Rubio-Monterde et al. [19] critically discussed lateral flow biosensors and nucleic acid amplification techniques (NAATs). Tessaro et al. [20] summarized colorimetric biosensors, highlighting the advantages of nanomaterials and POC applications. Filchakova et al. [21] reviewed commercialized RT-PCR methods, while Oishee et al. [22] compared FDA-approved RT-PCR and immunoassay kits. Mostafa et al. [23] provided a comprehensive overview of SARS-CoV-2 detection strategies based on the target biomolecule and quantification technique, addressing the impact of mutations. This review builds upon this existing knowledge by focusing specifically on biosensors for SARS-CoV-2 RNA detection, providing a detailed analysis of the biorecognition and transduction elements employed in these systems.

This review compiles and analyzes research published between 2020 and 2022 on biosensing strategies for SARS-CoV-2 nucleic acid detection. The review categorizes biosensors based on their transduction mechanism: electrochemical, electrochemiluminescent, and optical. Each category is discussed in detail, examining the specific biorecognition layers and transduction schemes employed in various studies. For electrochemical biosensors, examples include genosensors utilizing sandwich hybridization schemes with magnetic beads and electrochemical detection of the oxidized product of TMB, impedimetric transduction using modified carbon nanotubes, and non-amplified detection using carbon dots and gold nanoparticles. Electrochemiluminescent biosensors are described, emphasizing enzyme-free approaches, dual-wavelength ratiometric detection with nanomaterials, and the use of DNA walker amplification strategies. Optical transduction methods are analyzed, covering CRISPR-based systems with nanocluster reporters, colorimetric and fluorescent assays utilizing CRISPR-Cas12a or Cas13a, and label-free methods using molecular beacons. Lateral flow biosensors, combining amplification techniques like RT-LAMP and CRISPR detection, are also discussed, along with examples of commercially available tests. The analytical performance parameters, such as linear range and detection limit, are compared across different biosensor types, and their applications in various sample matrices (e.g., clinical samples, saliva, serum) are highlighted. The figures illustrate the working principles of several key biosensors.

The review identifies a diverse range of biosensing strategies for SARS-CoV-2 RNA detection with varying analytical performance characteristics. Electrochemical biosensors, particularly those using modified electrodes with nanomaterials, demonstrate high sensitivity, often reaching attomolar detection limits. Several studies showcase the successful detection of SARS-CoV-2 RNA in diluted clinical samples, even after significant dilution, indicating the potential for direct detection without amplification. Electrochemiluminescent biosensors, while requiring specialized equipment, also achieve high sensitivity by incorporating amplification strategies like DNA walkers and resonance energy transfer. Optical transduction methods, including CRISPR-based approaches, offer advantages in terms of simplicity and visual readout, suitable for point-of-care diagnostics. Lateral flow biosensors, integrating isothermal amplification and CRISPR detection, provide a portable and cost-effective solution, with several commercially available tests demonstrating the clinical utility of this approach. Amplification-free methods, though less sensitive, offer the potential for faster turnaround times and simplified workflows. The detection limits achieved by different methods vary widely, from a few copies per microliter to attomolar concentrations, depending on the technique and amplification strategy. The selection of a particular biosensing strategy depends on the desired sensitivity, cost, complexity, and required level of portability.

This review demonstrates the significant progress made in developing biosensors for SARS-CoV-2 RNA detection. While RT-PCR remains the gold standard, the limitations in accessibility and cost have driven the exploration of alternative approaches. The biosensors reviewed here offer compelling advantages in terms of sensitivity, speed, and portability, addressing the limitations of RT-PCR. The high sensitivity achieved by several electrochemical and electrochemiluminescent methods suggests their potential for early detection of SARS-CoV-2 infections, even at low viral loads. The simplicity and portability of lateral flow and optical biosensors are particularly relevant for point-of-care diagnostics, enabling rapid and widespread testing in diverse settings. The findings highlight the value of integrating amplification strategies with various transduction mechanisms to improve sensitivity and detection limits. The diverse array of biosensors presented reflects the multifaceted nature of the problem and the innovative solutions being explored to combat the pandemic. However, further research is needed to address the challenges in ensuring consistent performance and minimizing false-positive or false-negative results across various sample types and clinical contexts.

Biosensors represent a promising alternative to RT-PCR for SARS-CoV-2 RNA detection. Electrochemical, electrochemiluminescent, and optical methods, particularly when coupled with amplification strategies, demonstrate high sensitivity and selectivity. Lateral flow biosensors offer an attractive option for point-of-care diagnostics due to their ease of use and portability. Future research should focus on developing amplification-free, multiplex platforms with high sensitivity and robustness, capable of detecting low viral loads and emerging variants. The goal is to create affordable, readily deployable, and accurate biosensors for widespread and efficient SARS-CoV-2 detection.

The review primarily focuses on research published between 2020 and 2022, potentially excluding more recent advancements in the field. The evaluation of biosensors is based on reported data from individual studies, and direct comparisons across different platforms may be limited by variations in experimental conditions and sample types. While the review highlights the potential of various biosensors, it does not include a comprehensive evaluation of their clinical performance or cost-effectiveness in real-world settings. The focus is primarily on the detection of SARS-CoV-2 RNA, and other aspects of COVID-19 diagnostics, such as antigen detection or antibody-based assays, are not covered in detail. Finally, data availability was limited by the confidential nature of some of the studies' data.