Conductive Thread-Based Immunosensor for Pandemic Influenza A (H1N1) Virus Detection.

Infectious agents such as viruses pose significant threats to human health, being transmitted via direct contact as well as airborne transmission without direct contact, thus requiring rapid detection to prevent the spread of infectious diseases. In this study, we developed a conductive thread-based immunosensor (CT-IS), a biosensor to easily detect the presence of airborne viruses. CT-IS utilizes an antibody that specifically recognizes the HA protein of the pandemic influenza A (pH1N1) virus, which is incorporated into the conductive thread. The antigen-antibody interaction results in increased strain on the conductive thread in the presence of the pH1N1 virus, resulting in increased electrical resistance of the CT-IS. We evaluated the performance of this sensor using the HA protein and the pH1N1 virus, in addition to samples from patients infected with the pH1N1 virus. We observed a significant change in resistance in the pH1N1-infected patient samples (positive: n = 11, negative: n = 9), whereas negligible change was observed in the control samples (patients not infected with the pH1N1 virus; negative). Hence, the CT-IS is a lightweight fiber-type sensor that can be used as a wearable biosensor by combining it with textiles, to detect the pH1N1 virus in a person's vicinity.

Droplet digital recombinase polymerase amplification for multiplexed detection of human coronavirus.

Since the outbreak of coronavirus 2019 (COVID-19), detection technologies have been attracting a great deal of attention in molecular diagnosis applications. In particular, the droplet digital PCR (ddPCR) has become a promising tool as it offers absolute quantification of target nucleic acids with high specificity and sensitivity. In recent years, the combination of the isothermal amplification strategies has made ddPCR a popular method for on-site testing by enabling amplification at a constant temperature. However, the current isothermal ddPCR assays are still challenging due to inherent non-specific amplification. In this paper, we present a multiplexed droplet digital recombinase polymerase amplification (MddRPA) with precise initiation of the reaction. First, the reaction temperature and dynamic range of reverse transcription (RT) and RPA were characterized by real-time monitoring of fluorescence intensities. Using a droplet-based microfluidic chip, the master mix and the initiator were fractionated and rapidly mixed within well-confined droplets. Due to the high heat transfer and mass transfer of the droplets, the precise initiation of the amplification was enabled and the entire assay could be conducted within 30 min. The concentrations of target RNA in the range from 5 copies per µL to 2500 copies per µL could be detected with high linearity (R2 > 0.999). Furthermore, the multiplexed detection of three types of human coronaviruses was successfully demonstrated with high specificity (>96%). Finally, we compared the performance of the assay with a commercial RT-qPCR system using COVID-19 clinical samples. The MddRPA assay showed a 100% concordance with the RT-qPCR results, indicating its reliability and accuracy in detecting SARS-CoV-2 nucleic acids in clinical samples. Therefore, our MddRPA assay with rapid detection, precise quantification, and multiplexing capability would be an interesting method for molecular diagnosis of viral infections.

SERS-ELISA using silica-encapsulated Au core-satellite nanotags for sensitive detection of SARS-CoV-2.

The sensitive detection of viruses is key to preventing the spread of infectious diseases. In this study, we develop a silica-encapsulated Au core-satellite (CS@SiO2) nanotag, which produces a strong and reproducible surface-enhanced Raman scattering (SERS) signal. The combination of SERS from the CS@SiO2 nanotags with enzyme-linked immunosorbent assay (ELISA) achieves a highly sensitive detection of SARS-CoV-2. The CS@SiO2 nanotag is constructed by assembling 32 nm Au nanoparticles (AuNPs) on a 75 nm AuNP. Then the core-satellite particles are encapsulated with SiO2 for facile surface modification and stability. The SERS-ELISA technique using the CS@SiO2 nanotags provides a great sensitivity, yielding a detection limit of 8.81 PFU mL-1, which is 10 times better than conventional ELISA and 100 times better than lateral flow assay strip method. SERS-ELISA is applied to 30 SARS-CoV-2 clinical samples and achieved 100% and 55% sensitivities for 15 and 9 positive samples with cycle thresholds < 30 and > 30, respectively. This new CS@SiO2-SERS-ELISA method is an innovative technique that can significantly reduce the false-negative diagnostic rate for SARS-CoV-2 and thereby contribute to overcoming the current pandemic crisis.

Hybrid CRISPR/Cas protein for one-pot detection of DNA and RNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as next-generation molecular diagnostics. In CRISPR-based diagnostics, Cas12 and Cas13 proteins have been widely employed to detect DNA and RNA, respectively. Herein, we developed a novel hybrid Cas protein capable of detecting universal nucleic acids (DNA and RNA). The CRISPR/hybrid Cas system simultaneously recognizes both DNA and RNA, enabling the dual detection of pathogenic viruses in a single tube. Using wild-type (WT) and N501Y mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as detection models, we successfully detected both virus strains with a detection limit of 10 viral copies per reaction without cross-reactivity. Furthermore, it is demonstrated the detection of WT SARS-CoV-2 and N501Y mutant variants in clinical samples by using the CRISPR/hybrid Cas system. The hybrid Cas protein is expected to be utilized in a molecular diagnostic method for infectious diseases, tissue and liquid biopsies, and other nucleic acid biomarkers.

Evaluation of the T cell and B cell response following the administration of COVID-19 vaccines in Korea.

BACKGROUND: The coronavirus disease (COVID-19) has been a worldwide concern since 2019. Vaccines are predicted to be crucial in preventing further outbreaks. The development and kinetics of immune responses determine the efficacy of COVID-19 vaccines. METHODS: We measured interferon-gamma (IFN-γ) levels upon administering homologous adenovirus vector-based (ChAdOx1-S [AZ], Ad26.COV2.S [JAN]), mRNA-based (BNT162b2 [PF]; mRNA-1273 [MO]), and heterologous (AZ/PF) vaccines in healthy Korean individuals using two IFN-γ release assays: the Covi-FERON ELISA and T-SPOT Discovery SARS-CoV-2 assay. B cell responses were evaluated by assessing the production of neutralizing antibodies by surrogate virus neutralization assay. The immune response among the vaccine groups was compared after adjusting the vaccination dose and interactions between each group. RESULTS: AZ triggered the highest T cell response after the first dose but showed instability after the second. PF and MO yielded stable and higher increments of T and B cell responses compared to AZ. MO yielded a higher immune response than PF. JAN yielded T and B cell responses at lower levels than the other vaccines. The booster dose triggered significant increases in the T and B cell responses and is therefore needed to protect against SARS-CoV-2 given the possibility of waning immune responses. CONCLUSION: Administering two doses of mRNA vaccines provides the most effective results among the administered vaccines in triggering the immune response specific to SARS-CoV-2 in healthy Korean individuals. Administration of booster doses demonstrated a significant increase in the immune response and may provide longer protection against SARS-CoV-2.

Ultrasensitive Isothermal Detection of SARS-CoV-2 Based on Self-Priming Hairpin-Utilized Amplification of the G-Rich Sequence.

The outbreak of the novel coronavirus disease 2019 (COVID-19) pandemic induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of fatalities all over the world. Unquestionably, the effective and timely testing for infected individuals is the most imperative for the prevention of the ongoing pandemic. Herein, a new method was established for detecting SARS-CoV-2 based on the self-priming hairpin-utilized isothermal amplification of the G-rich sequence (SHIAG). In this strategy, the target RNA binding to the hairpin probe (HP) was uniquely devised to lead to the self-priming-mediated extension followed by the continuously repeated nicking and extension reactions, consequently generating abundant G-rich sequences from the intended reaction capable of producing fluorescence signals upon specifically interacting with thioflavin T (ThT). Based on the unique isothermal design concept, we successfully identified SARS-CoV-2 genomic RNA (gRNA) as low as 0.19 fM with excellent selectivity by applying only a single HP and further verified its practical diagnostic capability by reliably testing a total of 100 clinical specimens for COVID-19 with 100% clinical sensitivity and specificity. This study would provide notable insights into the design and evolution of new isothermal strategies for the sensitive and facile detection of SARS-CoV-2 under resource constraints.

Highly Adsorptive Au-TiO2 Nanocomposites for the SERS Face Mask Allow the Machine-Learning-Based Quantitative Assay of SARS-CoV-2 in Artificial Breath Aerosols.

Human respiratory aerosols contain diverse potential biomarkers for early disease diagnosis. Here, we report the direct and label-free detection of SARS-CoV-2 in respiratory aerosols using a highly adsorptive Au-TiO2 nanocomposite SERS face mask and an ablation-assisted autoencoder. The Au-TiO2 SERS face mask continuously preconcentrates and efficiently captures the oronasal aerosols, which substantially enhances the SERS signal intensities by 47% compared to simple Au nanoislands. The ultrasensitive Au-TiO2 nanocomposites also demonstrate the successful detection of SARS-CoV-2 spike proteins in artificial respiratory aerosols at a 100 pM concentration level. The deep learning-based autoencoder, followed by the partial ablation of nondiscriminant SERS features of spike proteins, allows a quantitative assay of the 101-104 pfu/mL SARS-CoV-2 lysates (comparable to 19-29 PCR cyclic threshold from COVID-19 patients) in aerosols with an accuracy of over 98%. The Au-TiO2 SERS face mask provides a platform for breath biopsy for the detection of various biomarkers in respiratory aerosols.

In-situ fabrication of 3D interior hotspots templated with a protein@Au core–shell structure for label-free and on-site SERS detection of viral diseases

Nanoscale plasmonic hotspots play a critical role in the enhancement of molecular Raman signals, enabling the sensitive and reliable trace analysis of biomedical molecules via surface-enhanced Raman spectroscopy (SERS). However, effective and label-free SERS diagnoses in practical fields remain challenging because of clinical samples' random adsorption and size mismatch with the nanoscale hotspots. Herein, we suggest a novel SERS strategy for interior hotspots templated with protein@Au core–shell nanostructures prepared via electrochemical one-pot Au deposition. The cytochrome c and lysates of SARS-CoV-2 (SLs) embedded in the interior hotspots were successfully functionalized to confine the electric fields and generate their optical fingerprint signals, respectively. Highly linear quantitative sensitivity was observed with the limit-of-detection value of 10−1 PFU/mL. The feasibility of detecting the targets in a bodily fluidic environment was also confirmed using the proposed templates with SLs in human saliva and nasopharyngeal swabs. These interior hotspots templated with the target analytes are highly desirable for early and on-site SERS diagnoses of infectious diseases without any labeling processes.

Rapid and Accurate On-Site Immunodiagnostics of Highly Contagious Severe Acute Respiratory Syndrome Coronavirus 2 Using Portable Surface-Enhanced Raman Scattering-Lateral Flow Assay Reader.

In early 2022, the number of people infected with the highly contagious mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), called Omicron, was increasing worldwide. Therefore, several countries approved the lateral flow assay (LFA) strip as a diagnostic method for confirming SARS-CoV-2 instead of reverse transcription-polymerase chain reaction (RT-PCR), which takes a long time to generate the results. However, owing to the limitation of detection sensitivity, commercial LFA strips have high false-negative diagnosis rates for patients with low virus concentrations. Therefore, in this study, we developed a portable surface-enhanced Raman scattering (SERS)-LFA reader based on localized surface plasmon effects to solve the sensitivity problem of the commercial LFA strip. We tested 54 clinical samples using this portable SERS-LFA reader, which generated 49 positive and 5 negative results. Out of the 49 positive results, SERS-LFA classified only 2 as false negative, while the commercial LFA classified 21 as false negative. This confirmed that the false-negative rate had significantly improved compared to that of commercial LFA strips. We believe that the proposed SERS-LFA system can be utilized as a point-of-care diagnostic system to quickly and accurately determine a virus infection that could spread significantly within a short period.

3D interior hotspots embedded with viral lysates for rapid and label-free identification of infectious diseases

In recent decades, biomedical sensors based on surface-enhanced Raman spectroscopy (SERS), which reveals unique spectral features corresponding to individual molecular vibrational states, have attracted intensive attention. However, the lack of a system for precisely guiding biomolecules to active hotspot regions has impeded the broad application of SERS techniques. Herein, we demonstrate the irreversible active engineering of three-dimensional (3D) interior organo-hotspots via electrochemical (EC) deposition onto metal nanodimple (ECOMD) platforms with viral lysates. This approach enables organic seed-programmable Au growth and the spontaneous bottom-up formation of 3D interior organo-hotspots simultaneously. Because of the net charge effect on the participation rate of viral lysates, the number of interior organo-hotspots in the ECOMDs increases with increasingly positive polarity. The viral lysates embedded in the ECOMDs function as both a dielectric medium for field confinement and an analyte, enabling the highly specific and sensitive detection of SARS-CoV-2 lysates (SLs) at concentrations as low as 10–2 plaque forming unit/mL. The ECOMD platform was used to trace and detect the SLs in human saliva and diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2);the results indicate that the proposed platform can provide point-of-care diagnoses of infectious diseases.

Evaluation of the T cell and B cell response following the administration of COVID-19 vaccines in Korea

Background The coronavirus disease (COVID-19) has been a worldwide concern since 2019. Vaccines are predicted to be crucial in preventing further outbreaks. The development and kinetics of immune responses determine the efficacy of COVID-19 vaccines. Methods We measured interferon-gamma (IFN-γ) levels upon administering homologous adenovirus vector-based (ChAdOx1-S [AZ], Ad26.COV2.S [JAN]), mRNA-based (BNT162b2 [PF];mRNA-1273 [MO]), and heterologous (AZ/PF) vaccines in healthy Korean individuals using two IFN-γ release assays: the Covi-FERON ELISA and T-SPOT Discovery SARS-CoV-2 assay. B cell responses were evaluated by assessing the production of neutralizing antibodies by surrogate virus neutralization assay. The immune response among the vaccine groups was compared after adjusting the vaccination dose and interactions between each group. Results AZ triggered the highest T cell response after the first dose but showed instability after the second. PF and MO yielded stable and higher increments of T and B cell responses compared to AZ. MO yielded a higher immune response than PF. JAN yielded T and B cell responses at lower levels than the other vaccines. The booster dose triggered significant increases in the T and B cell responses and is therefore needed to protect against SARS-CoV-2 given the possibility of waning immune responses. Conclusion Administering two doses of mRNA vaccines provides the most effective results among the administered vaccines in triggering the immune response specific to SARS-CoV-2 in healthy Korean individuals. Administration of booster doses demonstrated a significant increase in the immune response and may provide longer protection against SARS-CoV-2.

Dual-mode SERS-based lateral flow assay strips for simultaneous diagnosis of SARS-CoV-2 and influenza a virus.

Since COVID-19 and flu have similar symptoms, they are difficult to distinguish without an accurate diagnosis. Therefore, it is critical to quickly and accurately determine which virus was infected and take appropriate treatments when a person has an infection. This study developed a dual-mode surface-enhanced Raman scattering (SERS)-based LFA strip that can diagnose SARS-CoV-2 and influenza A virus with high accuracy to reduce the false-negative problem of the commercial colorimetric LFA strip. Furthermore, using a single strip, it is feasible to detect SARS-CoV-2 and influenza A virus simultaneously. A clinical test was performed on 39 patient samples (28 SARS-CoV-2 positives, 6 influenza A virus positives, and 5 negatives), evaluating the clinical efficacy of the proposed dual-mode SERS-LFA strip. Our assay results for clinical samples show that the dual-mode LFA strip significantly reduced the false-negative rate for both SARS-CoV-2 and influenza A virus.

Smartphone-Based SARS-CoV-2 and Variants Detection System using Colorimetric DNAzyme Reaction Triggered by Loop-Mediated Isothermal Amplification (LAMP) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

Coronavirus disease (COVID-19) has affected people for over two years. Moreover, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns regarding its accurate diagnosis. Here, we report a colorimetric DNAzyme reaction triggered by loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR), referred to as DAMPR assay for detecting SARS-CoV-2 and variants genes with attomolar sensitivity within an hour. The CRISPR-associated protein 9 (Cas9) system eliminated false-positive signals of LAMP products, improving the accuracy of DAMPR assay. Further, we fabricated a portable DAMPR assay system using a three-dimensional printing technique and developed a machine learning (ML)-based smartphone application to routinely check diagnostic results of SARS-CoV-2 and variants. Among blind tests of 136 clinical samples, the proposed system successfully diagnosed COVID-19 patients with a clinical sensitivity and specificity of 100% each. More importantly, the D614G (variant-common), T478K (delta-specific), and A67V (omicron-specific) mutations of the SARS-CoV-2 S gene were detected selectively, enabling the diagnosis of 70 SARS-CoV-2 delta or omicron variant patients. The DAMPR assay system is expected to be employed for on-site, rapid, accurate detection of SARS-CoV-2 and its variants gene and employed in the diagnosis of various infectious diseases.

Sensitive and reproducible detection of SARS-CoV-2 using SERS-based microdroplet sensor.

Surface-enhanced Raman scattering (SERS)-based assays have been recently developed to overcome the low detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SERS-based assays using magnetic beads in microtubes slightly improved the limit of detection (LoD) for SARS-CoV-2. However, the sensitivity and reproducibility of the method are still insufficient for reliable SARS-CoV-2 detection. In this study, we developed a SERS-based microdroplet sensor to dramatically improve the LoD and reproducibility of SARS-CoV-2 detection. Raman signals were measured for SERS nanotags in 140 droplets passing through a laser focal volume fixed at the center of the channel for 15 s. A comparison of the Raman signals of SERS nanotags measured in a microtube with those measured for multiple droplets in the microfluidic channel revealed that the LoD and coefficient of variation significantly improved from 36 to 0.22 PFU/mL and 21.2% to 1.79%, respectively. This improvement resulted from the ensemble average effects because the signals were measured for SERS nanotags in multiple droplets. Moreover, the total assay time decreased from 30 to 10 min. A clinical test was performed on patient samples to evaluate the clinical efficacy of the SERS-based microdroplet sensor. The assay results agreed well with those measured by the reverse transcription-polymerase chain reaction (RT-PCR) method. The proposed SERS-based microdroplet sensor is expected to be used as a new point-of-care diagnostic platform for quick and accurate detection of SARS-CoV-2 in the field.

FRET-based hACE2 receptor mimic peptide conjugated nanoprobe for simple detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a pandemic of acute respiratory disease, namely coronavirus disease (COVID-19). This disease threatens human health and public safety. Early diagnosis, isolation, and prevention are important to suppress the outbreak of COVID 19 given the lack of specific antiviral drugs to treat this disease and the emergence of various variants of the virus that cause breakthrough infections even after vaccine administration. Simple and prompt testing is paramount to preventing further spread of the virus. However, current testing methods, namely RT-PCR, is time-consuming. Binding of the SARS-CoV-2 spike (S) glycoprotein to human angiotensin-converting enzyme 2 (hACE2) receptor plays a pivotal role in host cell entry. In the present study, we developed a hACE2 mimic peptide beacon (COVID19-PEB) for simple detection of SARS-CoV-2 using a fluorescence resonance energy transfer system. COVID19-PEB exhibits minimal fluorescence in its ''closed'' hairpin structure; however, in the presence of SARS-CoV-2, the specific recognition of the S protein receptor-binding domain by COVID19-PEB causes the beacon to assume an ''open'' structure that emits strong fluorescence. COVID19-PEB can detect SARS-CoV-2 within 3 h or even 50 min and exhibits strong fluorescence even at low viral concentrations, with a detection limit of 4 × 103 plaque-forming unit/test. Furthermore, in SARS-CoV-2-infected patient samples confirmed using polymerase chain reaction, COVID19-PEB accurately detected the virus. COVID19-PEB could be developed as a rapid and accurate diagnostic tool for COVID-19.

Evaluation of the Diagnostic Accuracy of Nasal Cavity and Nasopharyngeal Swab Specimens for SARS-CoV-2 Detection via Rapid Antigen Test According to Specimen Collection Timing and Viral Load.

The rapid diagnosis of SARS-CoV-2 is an essential aspect in the detection and control of the spread of COVID-19. We evaluated the accuracy of the rapid antigen test (RAT) using samples from the nasal cavity and nasopharynx based on sample collection timing and viral load. We enrolled 175 patients, of which 71 patients and 104 patients had tested positive and negative, respectively, based on real time-PCR. Nasal cavity and nasopharyngeal swab samples were tested using STANDARD Q COVID-19 Ag tests (Q Ag, SD Biosensor, Korea). The sensitivity of the Q Ag test was 77.5% (95% confidence interval [CI], 67.8-87.2%) for the nasal cavity and 81.7% (95% [CI, 72.7-90.7%) for the nasopharyngeal specimens. The RAT results showed a substantial agreement between the nasal cavity and nasopharyngeal specimens (Cohen's kappa index = 0.78). The sensitivity of the RAT for nasal cavity specimens exceeded 89% for <5 days after symptom onset (DSO) and 86% for Ct of E and RdRp < 25. The Q Ag test performed fairly well, especially in the early DSO when a high viral load was present, and the nasal cavity swab can be considered an alternative site for the rapid diagnosis of COVID-19.

SERS-based dual-mode DNA aptasensors for rapid classification of SARS-CoV-2 and influenza A/H1N1 infection.

We developed a dual-mode surface-enhanced Raman scattering (SERS)-based aptasensor that can accurately diagnose and distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/H1N1 at the same time. Herein, DNA aptamers that selectively bind to SARS-CoV-2 and influenza A/H1N1 were immobilized together on Au nanopopcorn substrate. Raman reporters (Cy3 and RRX), attached to the terminal of DNA aptamers, could generate strong SERS signals in the nanogap of the Au nanopopcorn substrate. Additionally, the internal standard Raman reporter (4-MBA) was immobilized on the Au nanopopcorn substrate along with aptamer DNAs to reduce errors caused by changes in the measurement environment. When SARS-CoV-2 or influenza A virus approaches the Au nanopopcorn substrate, the corresponding DNA aptamer selectively detaches from the substrate due to the significant binding affinity between the corresponding DNA aptamer and the virus. As a result, the related SERS intensity decreases with increasing target virus concentration. Thus, it is possible to determine whether a suspected patient is infected with SARS-CoV-2 or influenza A using this SERS-based DNA aptasensor. Furthermore, this sensor enables a quantitative evaluation of the target virus concentration with high sensitivity without being affected by cross-reactivity. Therefore, this SERS-based diagnostic platform is considered a conceptually new diagnostic tool that rapidly discriminates against these two respiratory diseases to prevent their spread.

Development of surface‐enhanced Raman scattering‐based immunoassay platforms using hollow Au nanostars for reliable SARS‐CoV‐2 diagnosis

We developed a novel surface‐enhanced Raman scattering (SERS)‐based SARS‐CoV‐2 assay platform using hollow Au nanostars to realize high‐sensitivity diagnosis of SARS‐CoV‐2. The assay was performed using SARS‐CoV‐2 lysate as the target in a wide dynamic range with virus concentrations ranging from 0 to 104 PFU/ml and has a limit of detection (LOD) of 5.1 PFU/ml. This LOD value shows 100 times and 10 times better sensitivity compared to the LODs measured on the same sample using a commercially available rapid kit and enzyme‐linked immunosorbent assay, respectively. Therefore, we believe that this SERS‐based SARS‐CoV‐2 assay platform has high diagnostic accuracy for early or asymptomatic infected patients with low virus concentrations. Furthermore, the probability of a false‐negative diagnosis is likely to be very low. A new magnetic bead‐based SERS assay platform was developed to overcome the sensitivity limit of the commercially available SARS‐CoV‐2 immunodiagnostic kits. A novel hollow gold nanostars (HAuNSs), with a high enhancement factor and good stability, was employed for the detection of nucleocapsid protein from SARS‐CoV‐2. The assay could be performed in a wide dynamic range with virus concentrations ranging from 10−2 to 104 PFU/ml and showed an LOD value of 5.1 PFU/ml using this platform.

Development of surface-enhanced Raman scattering-based immunoassay platforms using hollow Au nanostars for reliable SARS-CoV-2 diagnosis.

We developed a novel surface-enhanced Raman scattering (SERS)-based SARS-CoV-2 assay platform using hollow Au nanostars to realize high-sensitivity diagnosis of SARS-CoV-2. The assay was performed using SARS-CoV-2 lysate as the target in a wide dynamic range with virus concentrations ranging from 0 to 104 PFU/ml and has a limit of detection (LOD) of 5.1 PFU/ml. This LOD value shows 100 times and 10 times better sensitivity compared to the LODs measured on the same sample using a commercially available rapid kit and enzyme-linked immunosorbent assay, respectively. Therefore, we believe that this SERS-based SARS-CoV-2 assay platform has high diagnostic accuracy for early or asymptomatic infected patients with low virus concentrations. Furthermore, the probability of a false-negative diagnosis is likely to be very low.

Detection of Infectious Viruses Using CRISPR-Cas12-Based Assay.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has severely influenced public health and economics. For the detection of SARS-CoV-2, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)-based assays have been emerged because of their simplicity, sensitivity, specificity, and wide applicability. Herein, we have developed a CRISPR-Cas12-based assay for the detection of SARS-CoV-2. In the assay, the target amplicons are produced by isothermal reverse transcription recombinase polymerase amplification (RT-RPA) and recognized by a CRISPR-Cas12a/guide RNA (gRNA) complex that is coupled with the collateral cleavage activity of fluorophore-tagged probes, allowing either a fluorescent measurement or naked-eye detection on a lateral flow paper strip. This assay enables the sensitive detection of SARS-CoV-2 at a low concentration of 10 copies per sample. Moreover, the reliability of the method is verified by using nasal swabs and sputum of COVID-19 patients. We also proved that the current assay can be applied to other viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), with no major changes to the basic scheme of testing. It is anticipated that the CRISPR-Cas12-based assay has the potential to serve as a point-of-care testing (POCT) tool for a wide range of infectious viruses.