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The disastrous spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has induced severe public healthcare issues and weakened the global economy significantly. Although SARS-CoV-2 infection is not as fatal as the initial outbreak, many infected victims suffer from long COVID. Therefore, rapid and large-scale testing is critical in managing patients and alleviating its transmission. Herein, we review the recent advances in techniques to detect SARS-CoV-2. The sensing principles are detailed together with their application domains and analytical performances. In addition, the advantages and limits of each method are discussed and analyzed. Besides molecular diagnostics and antigen and antibody tests, we also review neutralizing antibodies and emerging SARS-CoV-2 variants. Further, the characteristics of the mutational locations in the different variants with epidemiological features are summarized. Finally, the challenges and possible strategies are prospected to develop new assays to meet different diagnostic needs. Thus, this comprehensive and systematic review of SARS-CoV-2 detection technologies may provide insightful guidance and direction for developing tools for the diagnosis and analysis of SARS-CoV-2 to support public healthcare and effective long-term pandemic management and control.

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The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide which triggered serious public health issues. The search for rapid and accurate diagnosis, effective prevention, and treatment is urgent. The nucleocapsid protein (NP) of SARS-CoV-2 is one of the main structural proteins expressed and most abundant in the virus, and is considered a diagnostic marker for the accurate and sensitive detection of SARS-CoV-2. Herein, we report the screening of specific peptides from the pIII phage library that bind to SARS-CoV-2 NP. The phage monoclone expressing cyclic peptide N1 (peptide sequence, ACGTKPTKFC, with C&C bridged by disulfide bonding) specifically recognizes SARS-CoV-2 NP. Molecular docking studies reveal that the identified peptide is bound to the "pocket" region on the SARS-CoV-2 NP N-terminal domain mainly by forming a hydrogen bonding network and through hydrophobic interaction. Peptide N1 with the C-terminal linker was synthesized as the capture probe for SARS-CoV-2 NP in ELISA. The peptide-based ELISA was capable of assaying SARS-CoV-2 NP at concentrations as low as 61 pg/mL (∼1.2 pM). Furthermore, the as-proposed method could detect the SARS-CoV-2 virus at limits as low as 50 TCID50 (median tissue culture infective dose)/mL. This study demonstrates that selected peptides are powerful biomolecular tools for SARS-CoV-2 detection, providing a new and inexpensive method of rapidly screening infections as well as rapidly diagnosing coronavirus disease 2019 patients.

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The SARS-CoV-2 spreading rapidly has aroused catastrophic public healthcare issues and economy crisis worldwide. It plays predominant role to rapidly and accurately diagnose the virus for effective prevention and treatment. As an abundant transmembrane protein, spike protein (SP) is one of the most valuable antigenic biomarkers for diagnosis of COVID-19. Herein a phage expression of WNLDLSQWLPPM peptide specific to SARS-CoV-2 SP was screened. Molecular docking revealed that the isolated peptide binds to major antigenic epitope locating at S2 subunit with hydrogen bonding. Taking the specific peptide as antigen sensing probe and tyramine signal amplification (TSA), an ultrasensitive "peptide-antigen-antibody" ELISA (p-ELISA) was explored, by which the limit of detection (LOD) was 14 fM and 2.8 fM SARS-CoV-2 SP antigen for first TSA and secondary TSA, respectively. Compared with the LOD by the p-ELISA by direct mode, the sensitivity with 2nd TSA enhanced 100 times. Further, the proposed p-ELISA method can detect SARS-CoV-2 pseudoviruses down to 10 and 3 TCID50/mL spiked in healthy nasal swab sample with 1st TSA and 2nd TSA, separately. Thus, the proposed p-ELISA method with TSA is expected to be a promising ultrasensitive tool for rapidly detecting SARS-CoV-2 antigen to help control the infectious disease.

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The COVID-19 pandemic has led to a global crisis with devastating effects on public healthcare and the economy. Sensitive detection of SARS-CoV-2 is the key to diagnose and control its spread. The spike (S) protein is an abundant viral transmembrane protein and a suitable target protein for the selective recognition of SARS-CoV-2. Here, we report that with bovine serum albumin prescreening, a specific phage peptide targeting SARS-CoV-2 S1 protein was biopanned with the pIII phage display library. The identified phage #2 expressing the peptide (amino acid sequence: NFWISPKLAFAL) shows high affinity to the target with a dissociation constant of 3.45 ± 0.58 nM. Furthermore, the identified peptide shows good specificity with a binding site at the N-terminal domain of the S1 subunit through a hydrogen bond network and hydrophobic interaction, supported by molecular docking. Then, a sandwiched phage-based enzyme-linked chemiluminescence immunoassay (ELCLIA) was established by using phage #2 as a bifunctional probe capable of SARS-CoV-2 S1 antigen recognition and signal amplification. After optimizing the conditions, the proposed phage ELCLIA exhibited good sensitivity, and as low as 78 pg/mL SARS-CoV-2 S1 could be detected. This method can be applied to detect as low as 60 transducing units (TU)/mL SARS-CoV-2 pseudovirus in 50% saliva. Therefore, specific phage peptides have good prospects as powerful biological recognition probes for immunoassay detection and biomedical applications.

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As a vascular growth regulator, vascular endothelial growth factor (VEGF) exerts significant biological roles through specific binding to its receptors on the vascular endothelial cells. VEGF165 is generally referenced as a potential therapeutic target of many malignant tumors. In this study, a negative pre-screening strategy with structurally analogous members of VEGF121, VEGFC and VEGFD was first proposed for VEGF165 biopanning, aiming at significantly improving the specificity of the selected phage monoclones. Indirect ELISA experiment showed that the phage monoclone expressing peptide SPFLLRM demonstrates excellent affinity and specificity. Then a VEGF165 electrochemical impedimetric spectroscopy (EIS) immunosensor was constructed by above specific phage modified electrode. After optimizing the experimental conditions, the as-explored EIS immunosensor had a linear range of 0.5-1000 pg/mL with the limit of detection of 0.15 pg/mL VEGF165. In addition, the developed phage-based EIS immunosensor was applied to satisfactorily detect VEGF165 in human serum samples. Considering its ultra-sensitivity, good selectivity, batch reproducibility and stability, the screened selective phage-based EIS sensor is envisioned potential application in diagnosis and therapy.

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The sequential enzyme biosensors hold significant importance in measuring species which are usually hard to process with single-enzyme-based biosensors. However, sequential enzyme electrodes experience critical issues such as low catalytic efficiency, insensitivity and poor reproducibility. In this work, yeast surface co-displaying sequential enzymes of glucoamylase (GA) and glucose oxidase (GOx) with controllable ratios through the specific cohesion-dockerin protein interaction was explored, by which starch hydrolyzing by GA into glucose is the rate-limiting step. The modified electrodes were prepared by immobilizing yeast-GA&GOx whole-cell and reduced graphene oxide (RGO) on glassy carbon electrode (GCE), for which the direct electron transfer between the electrode and recombinant GOx was arrived. Interestingly, the current responses of sensors to starch and glucose are dependent on the displayed enzyme composition, of which the yeast-GA&GOx (2:1) exhibited the highest current. Thereafter, sequential enzyme sensor of yeast-GA&GOx (2:1)/RGO/GCE was developed. Based on reduction detection at negative potential without interference, the sensor is stable and capable of assaying glucose (linear range: 2.0-100 mg/L) or starch (linear range, 50-3500 mg/L), separately. Coupled with yeast-GOx/RGO/GCE glucose sensor, both glucose and starch in real samples can be detected satisfactorily. This work provides new ideas for the development of other sequential enzyme electrodes for potential applications.

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Gold nanoplates (AuNPTs) exhibit outstanding photothermal conversion efficiency (68.5%) and peroxidase-like activity. The combination of the very low H2O2 concentration (0.1 mM) and the low AuNPT dosage (50 µg mL-1) with 808 nm laser irradiation (1 W cm-2, 3 min) shows excellent synergistic antibacterial ability and healing of MRSA-infected wounds in vivo.

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