An electrochemical biosensor for SARS-CoV-2 detection via its papain-like cysteine protease and the protease inhibitor screening.

The persistent coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still infecting hundreds of thousands of people every day. Enriching the kits for SARS-CoV-2 detection and developing the drugs for patient treatments are still urgently needed for combating the spreading virus, especially after the emergence of various mutants. Herein, an electrochemical biosensor has been fabricated in this work for the detection of SARS-CoV-2 via its papain-like cysteine protease (PLpro) and the screening of protease inhibitor against SARS-CoV-2 by using our designed chimeric peptide-DNA (pDNA) nanoprobes. Utilizing this biosensor, the sensitive and specific detection of SARS-CoV-2 PLpro can be conducted in complex real environments including blood and saliva. Five positive and five negative patient throat swab samples have also been tested to verify the practical application capability of the biosensor. Moreover, we have obtained a detection limit of 27.18 fM and a linear detection range from 1 pg mL-1 to 10 µg mL-1 (I = 1.63 + 4.44 lgC). Meanwhile, rapid inhibitor screening against SARS-CoV-2 PLpro can be also obtained. Therefore, this electrochemical biosensor has the great potential for COVID-19 combating and drug development.

Single-Nanoparticle-Based Digital SERS Sensing Platform for the Accurate Quantitative Detection of SARS-CoV-2.

To prevent the ongoing spread of the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), accurate and early detection based on a rapid, ultrasensitive, and highly reliable sensing method is crucially important. Here, we present a bumpy core-shell surface-enhanced Raman spectroscopy (SERS) nanoprobe-based sensing platform with single-nanoparticle (SNP)-based digital SERS analysis. The tailorable bumpy core-shell SERS nanoprobe with an internal self-assembled monolayer of 4-nitrobenzenethiol Raman reporters, synthesized using HEPES biological buffer, generates a strong, uniform, and reproducible SERS signal with an SNP-level sensitive and narrowly distributed enhancement factor (2.1 × 108 to 2.2 × 109). We also propose an SNP-based digital SERS analysis method that provides direct visualization of SNP detection at ultralow concentrations and reliable quantification over a wide range of concentrations. The bumpy core-shell SERS nanoprobe-based sensing platform with SNP-based digital SERS analysis achieves the ultrasensitive and quantitative detection of the SARS-CoV-2 spike protein with a limit of detection of 7.1 × 10-16 M over a wide dynamic range from 3.7 × 10-15 to 3.7 × 10-8 M, far outperforming the conventional enzyme-linked immunosorbent assay method for the target protein. Furthermore, it can detect mutated spike proteins from the SARS-CoV-2 variants, representing the key mutations of Alpha, Beta, Gamma, Delta, and Omicron variants. Therefore, this sensing platform can be effectively and efficiently used for the accurate and early detection of SARS-CoV-2 and be adapted for the ultrasensitive and reliable detection of other highly infectious diseases.

Aggregation-Induced Emission Fluorophore-Incorporated Curcumin-Based Ratiometric Nanoprobe for Hypochlorite Detection in Food Matrices.

The development of efficient, economic, reliable, and accurate monitoring of hypochlorite (ClO-) in food matrices is in great demand for food safety assessment, particularly during its massive use against the COVID-19 epidemic. Here, we prepared an aggregation-induced emission (AIE) fluorophore tetraphenylethylene (TPE)-incorporated curcumin-based hybrid ratiometric fluorescence nanoprobe (Curcumin/TPE@HyNPs) through amphiphilic phospholipid polymer-powered nanoprecipitation, which exhibited a fast, highly sensitive, and selective response to the residual ClO- in real food matrices. Because of the inner filter effect (IFE) from curcumin toward TPE inside the nanoprobe, the bright fluorescence of TPE aggregation at ∼437 nm was effectively quenched, along with an enhanced fluorescence of curcumin at ∼478 nm. Once there was a ClO- residue in food matrices, ClO- triggered the oxidation of o-methoxyphenol inside curcumin and led to the almost complete absorption collapse, thereby terminating curcumin fluorescence at ∼478 nm and the IFE process. Accordingly, the fluorescence of TPE at ∼437 nm was recovered. In this case, a ratiometric fluorescent response of Curcumin/TPE@HyNPs toward the residual ClO- in food matrices (e.g., milk) was proposed with a low detection limit of 0.353 µM and a rapid response time of 140.0 s. Notably, the phospholipid polymer as the protection layer effectively reduced/evaded the nonspecific binding of signal reporters inside the nanoprobe, facilitating it to directly monitor the residual ClO- in real food matrices. This work provided a novel approach to utilize the unconventional AIE luminophors for constructing the efficient and reliable early warning mechanisms toward various food contaminants.

Non-PCR Ultrasensitive Detection of Viral RNA by a Nanoprobe-Coupling Strategy: SARS-CoV-2 as an Example.

Developing efficient and highly sensitive diagnostic techniques for early detections of pathogenic viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is vitally important for preventing its widespread. However, the conventional polymerase chain reaction (PCR)-based detection features high complexity, excessive time-consumption, and labor-intensiveness, while viral protein-based detections suffer from moderate sensitivity and specificity. Here, a non-PCR but ultrasensitive viral RNA detection strategy is reported based on a facile nanoprobe-coupling strategy without enzymatic amplification, wherein PCR-induced bias and other shortcomings are successfully circumvented. This approach endows the viral RNA detection with ultra-low background to maximum signal ratio in the linear signal amplification by using Au nanoparticles as reporters. The present strategy exhibits 100% specificity toward SARS-CoV-2 N gene, and ultrasensitive detection of as low as 52 cp mL-1 of SARS-CoV-2 N gene without pre-PCR amplification. This approach presents a novel ultrasensitive tool for viral RNA detections for fighting against COVID-19 and other types of pathogenic virus-caused diseases.

Nanomolecular Magnetic Probe for Detection of Nucleic Acid Sequence-Based Amplification of Covid-19 RNA with the Naked Eye

Nucleic acid amplification of Covid-19 RNA is the main subject for molecular detection of SARS-COV-2. However, the employment of target amplification methods such as PCR needs a converting step for Covid-19 RNA to DNA template to be amplified. In addition, Covid-19 RNA isolation needs some RNA extraction kits that their providing could increase the time and costs for the molecular detection of the virus. In this study, we introduced a magnetic nanoprobe that could be used to capture and amplify Covid-19 RNA through an isothermal amplification process, so-called nucleic acid sequence-based amplification, without needing to perform a separate step for the viral RNA converting to DNA template. By using engineered sequences appropriate to the target nucleic acid attached to the magnetic nanoparticles, identifying the target RNA from the virus could be possible by clumping the particles that could be seen with naked eyes. According to the isothermal amplification of the viral RNA via nucleic acid sequence-based amplification assisted with the magnetic nanoprobe, the nanomolecular method eliminated the need for special pieces of equipment and the time for detection of Covid-19 in specimens. © 2022 by the authors.