Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 36
Filter
1.
Analyst ; 145(12): 4173-4180, 2020 Jun 21.
Article in English | MEDLINE | ID: covidwho-1721601

ABSTRACT

Studies have shown that microRNAs, which are small noncoding RNAs, hold tremendous promise as next-generation circulating biomarkers for early cancer detection via liquid biopsies. A novel, solid-state nanoplasmonic sensor capable of assaying circulating microRNAs through a combined surface-enhanced Raman scattering (SERS) and plasmon-enhanced fluorescence (PEF) approach has been developed. Here, the unique localized surface plasmon resonance properties of chemically-synthesized gold triangular nanoprisms (Au TNPs) are utilized to create large SERS and PEF enhancements. With careful modification to the surface of Au TNPs, this sensing approach is capable of quantifying circulating microRNAs at femtogram/microliter concentrations. Uniquely, the multimodal analytical methods mitigate both false positive and false negative responses and demonstrate the high stability of our sensors within bodily fluids. As a proof of concept, microRNA-10b and microRNA-96 were directly assayed from the plasma of six bladder cancer patients. Results show potential for a highly specific liquid biopsy method that could be used in point-of-care clinical diagnostics to increase early cancer detection or any other diseases including SARS-CoV-2 in which RNAs can be used as biomarkers.


Subject(s)
Circulating MicroRNA/blood , Fluorescent Dyes/chemistry , Spectrum Analysis, Raman , Urinary Bladder Neoplasms/diagnosis , Betacoronavirus/isolation & purification , Biomarkers, Tumor/blood , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/pathology , Coronavirus Infections/virology , Gold/chemistry , Humans , Limit of Detection , Microscopy, Confocal , Nanostructures/chemistry , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Point-of-Care Systems , SARS-CoV-2 , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology
2.
Cells ; 11(3)2022 01 27.
Article in English | MEDLINE | ID: covidwho-1662647

ABSTRACT

In this contribution, we report on the possibility that cryptococcal protease(s) could activate the SARS-CoV-2 spike (S) protein. The S protein is documented to have a unique four-amino-acid sequence (underlined, SPRRAR↓S) at the interface between the S1 and S2 sites, that serves as a cleavage site for the human protease, furin. We compared the biochemical efficiency of cryptococcal protease(s) and furin to mediate the proteolytic cleavage of the S1/S2 site in a fluorogenic peptide. We show that cryptococcal protease(s) processes this site in a manner comparable to the efficiency of furin (p > 0.581). We conclude the paper by discussing the impact of these findings in the context of a SARS-CoV-2 disease manifesting while there is an underlying cryptococcal infection.


Subject(s)
Aspartic Acid Proteases/metabolism , Bacterial Proteins/metabolism , Cryptococcus neoformans/enzymology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Aspartic Acid Proteases/genetics , Bacterial Proteins/genetics , Binding Sites , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/virology , Cryptococcus neoformans/genetics , Fluorescent Dyes/chemistry , Furin/genetics , Furin/metabolism , Humans , Pandemics , Peptides/chemistry , Peptides/metabolism , Proteolysis , SARS-CoV-2/physiology
3.
Angew Chem Int Ed Engl ; 61(9): e202113617, 2022 02 21.
Article in English | MEDLINE | ID: covidwho-1565164

ABSTRACT

The main protease (Mpro ) and papain-like protease (PLpro ) play critical roles in SARS-CoV-2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of Mpro and PLpro is extremely valuable for SARS-CoV-2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual-color probe (3MBP5) for the simultaneous detection of Mpro and PLpro by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro . 3MBP5-activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid-transfected HEK 293T cells, and SARS-CoV-2-infected TMPRSS2-Vero cells. Results from the dual-color probe first verified the simultaneous detection and intracellular distribution of SARS-CoV-2 Mpro and PLpro . This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.


Subject(s)
Color , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/metabolism , Fluorescent Dyes/chemistry , Protease Inhibitors/pharmacology , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans
4.
PLoS One ; 16(12): e0246916, 2021.
Article in English | MEDLINE | ID: covidwho-1546847

ABSTRACT

The COVID-19 pandemic has reintroduced questions regarding the potential risk of SARS-CoV-2 exposure amongst passengers on an aircraft. Quantifying risk with computational fluid dynamics models or contact tracing methods alone is challenging, as experimental results for inflight biological aerosols is lacking. Using fluorescent aerosol tracers and real time optical sensors, coupled with DNA-tagged tracers for aerosol deposition, we executed ground and inflight testing on Boeing 767 and 777 airframes. Analysis here represents tracer particles released from a simulated infected passenger, in multiple rows and seats, to determine the exposure risk via penetration into breathing zones in that row and numerous rows ahead and behind the index case. We present here conclusions from 118 releases of fluorescent tracer particles, with 40+ Instantaneous Biological Analyzer and Collector sensors placed in passenger breathing zones for real-time measurement of simulated virus particle penetration. Results from both airframes showed a minimum reduction of 99.54% of 1 µm aerosols from the index source to the breathing zone of a typical passenger seated directly next to the source. An average 99.97 to 99.98% reduction was measured for the breathing zones tested in the 767 and 777, respectively. Contamination of surfaces from aerosol sources was minimal, and DNA-tagged 3 µm tracer aerosol collection techniques agreed with fluorescent methodologies.


Subject(s)
Aircraft , Computer Simulation , Fluorescent Dyes/chemistry , /chemistry , COVID-19/pathology , COVID-19/prevention & control , COVID-19/virology , DNA/chemistry , DNA/metabolism , Humans , Masks , Microspheres , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
5.
J Am Chem Soc ; 143(48): 20095-20108, 2021 12 08.
Article in English | MEDLINE | ID: covidwho-1531986

ABSTRACT

Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. Using this approach, we were able to label various proteins such as BTK, K-RasG12C, and SARS-CoV-2 PLpro with different tags. For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive "turn-on" fluorescent probe, we were able to monitor ligand binding to the active site of BTK. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which installed a CRBN binder onto BTK. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology.


Subject(s)
Agammaglobulinaemia Tyrosine Kinase/chemistry , Fluorescent Dyes/chemistry , Ligands , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Adenine/analogs & derivatives , Adenine/chemistry , Adenine/metabolism , Agammaglobulinaemia Tyrosine Kinase/metabolism , Catalytic Domain , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Half-Life , Humans , Piperidines/chemistry , Piperidines/metabolism , Proteolysis , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Pyrimidines/chemistry , Pyrimidines/metabolism , SARS-CoV-2/enzymology
6.
Acc Chem Res ; 54(21): 3991-4000, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1483068

ABSTRACT

The modern healthcare system faces an unrelenting threat from microorganisms, as evidenced by global outbreaks of new viral diseases, emerging antimicrobial resistance, and the rising incidence of healthcare-associated infections (HAIs). An effective response to these threats requires rapid and accurate diagnostic tests that can identify causative pathogens at the point of care (POC). Such tests could eliminate diagnostic uncertainties, facilitating patient triaging, minimizing the empiric use of antimicrobial drugs, and enabling targeted treatments. Current standard methods, however, often fail to meet the needs of rapid diagnosis in POC settings. Culture-based assays entail long processing times and require specialized laboratory infrastructure; nucleic acid (NA) tests are often limited to centralized hospitals due to assay complexity and high costs. Here we discuss two new POC tests developed in our groups to enable the rapid diagnosis of infection. The first is nanoPCR that takes advantages of core-shell magnetoplasmonic nanoparticles (MPNs): (i) Au shell significantly accelerates thermocycling via volumetric, plasmonic light-to-heat conversion and (ii) a magnetic core enables sensitive in situ fluorescent detection via magnetic clearing. By adopting a Ferris wheel module, the system expedites multisamples in parallel with a minimal setup. When applied to COVID-19 diagnosis, nanoPCR detected SARS-CoV-2 RNA down to 3.2 copy/µL within 17 min. In particular, nanoPCR diagnostics accurately identified COVID-19 cases in clinical samples (n = 150), validating its clinical applicability. The second is a polarization anisotropy diagnostic (PAD) system that exploits the principle of fluorescence polarization (FP) as a detection modality. Fluorescent probes were designed to alter their molecular weight upon recognizing target NAs. This event modulates the probes' tumbling rate (Brownian motion), which leads to changes in FP. The approach is robust against environmental noise and benefits from the ratiometric nature of the signal readout. We applied PAD to detect clinically relevant HAI bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus). The PAD assay demonstrated detection sensitivity down to the single bacterium level and determined both drug resistance and virulence status. In summary, these new tests have the potential to become powerful tools for rapid diagnosis in the infectious disease space. They do not require highly skilled personnel or labor-intensive analyses, and the assays are quick and cost-effective. These attributes will make nanoPCR and PAD well-aligned with a POC workflow to aid physicians to initiate prompt and informed patient treatment.


Subject(s)
Bacterial Infections/diagnosis , COVID-19 Testing , COVID-19/diagnosis , Fluorescence Polarization , Nanotechnology , Polymerase Chain Reaction , Fluorescent Dyes/chemistry , Humans , Point-of-Care Systems , RNA, Viral/genetics , SARS-CoV-2/genetics
7.
Adv Sci (Weinh) ; 8(23): e2101166, 2021 12.
Article in English | MEDLINE | ID: covidwho-1473797

ABSTRACT

Lipid-based nanoparticles have been applied extensively in drug delivery and vaccine strategies and are finding diverse applications in the coronavirus disease 2019 (COVID-19) pandemic-from vaccine-component encapsulation to modeling the virus, itself. High-throughput, highly flexible methods for characterization are of great benefit to the development of liposomes featuring surface proteins. DNA-directed patterning is one such method that offers versatility in immobilizing and segregating lipid-based nanoparticles for subsequent analysis. Here, oligonucleotides are selectively conjugated onto a glass substrate and then hybridized to complementary oligonucleotides tagged to liposomes, patterning them with great control and precision. The power of DNA-directed patterning is demonstrated by characterizing a novel recapitulative lipid-based nanoparticle model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-S-liposomes-that presents the SARS-CoV-2 spike (S) protein on its surface. Patterning a mixture of S-liposomes and liposomes that display the tetraspanin CD63 to discrete regions of a substrate shows that angiotensin-converting enzyme 2 (ACE2) specifically binds to S-liposomes. Subsequent introduction of S-liposomes to ACE2-expressing cells tests the biological function of S-liposomes and shows agreement with DNA-directed patterning-based assays. Finally, multiplexed patterning of S-liposomes verifies the performance of commercially available neutralizing antibodies against the two S variants. Overall, DNA-directed patterning enables a wide variety of custom assays for the characterization of any lipid-based nanoparticle.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/diagnosis , Liposomes/chemistry , Nanoparticles/chemistry , Oligonucleotides/chemistry , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , COVID-19/virology , Fluorescent Dyes/chemistry , HEK293 Cells , Humans , Liposomes/metabolism , Microscopy, Confocal , Oligonucleotides/metabolism , Protein Binding , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Tetraspanins/chemistry , Tetraspanins/metabolism
8.
Sci Rep ; 11(1): 20428, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1469984

ABSTRACT

Glycosylation is the most common post-translational modification and has myriad of biological functions. However, glycan analysis has always been a challenge. Here, we would like to present new techniques for glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS2 spike (S) glycoproteins. SARS2, a novel coronavirus and the causative agent of the COVID-19 pandemic, has had significant social and economic impacts since the end of 2019. To obtain the N-glycan fingerprint of an S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, then separated by SDS-PAGE, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders are enzymatically generated and run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. O-glycans can also be fingerprinted. Due to the lack of an enzyme for broad O-glycan release, O-glycans on the S protein can be labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated by gel electrophoresis. Glycan fingerprinting could serve as a quick method for globally assessing the glycosylation of a specific glycoprotein.


Subject(s)
COVID-19/virology , Polysaccharides/analysis , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Carbocyanines/chemistry , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes/chemistry , Fucose/analogs & derivatives , Glycosylation , Humans , N-Acetylneuraminic Acid/analogs & derivatives , Optical Imaging
9.
Chem Commun (Camb) ; 57(79): 10222-10225, 2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1408635

ABSTRACT

We developed a one-minute, one-step SARS-CoV-2 antigen assay based on protein-induced fluorescence enhancement of a DNA aptamer. The system showed significant selectivity and sensitivity towards both nucleocapsid protein and SARS-CoV-2 virus lysate, but with marked improvements in speed and manufacturability. We hence propose this platform as a mix-and-read testing strategy for SARS-CoV-2 that can be applied to POC diagnostics in clinical settings, especially in low- and middle-income countries.


Subject(s)
Antigens, Viral/chemistry , Aptamers, Nucleotide/chemistry , COVID-19 Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , SARS-CoV-2 , Biological Assay , Carbocyanines/chemistry , Fluorescence , Fluorescent Dyes/chemistry , Phosphoproteins/chemistry
10.
J Am Chem Soc ; 143(14): 5413-5424, 2021 04 14.
Article in English | MEDLINE | ID: covidwho-1387160

ABSTRACT

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.


Subject(s)
Fluorescence , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Molecular Imaging , RNA, Messenger/analysis , RNA, Messenger/metabolism , COVID-19/drug therapy , Cell Line, Tumor , Cytosine/analogs & derivatives , Cytosine/analysis , Cytosine/chemical synthesis , Cytosine/chemistry , Fluorescent Dyes/chemical synthesis , Green Fluorescent Proteins/metabolism , Histones/metabolism , Humans , Molecular Structure , RNA, Messenger/chemistry , RNA, Messenger/therapeutic use , Spectrometry, Fluorescence
11.
Biosensors (Basel) ; 11(9)2021 Aug 28.
Article in English | MEDLINE | ID: covidwho-1374295

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , CRISPR-Associated Proteins/metabolism , Endodeoxyribonucleases/metabolism , Middle East Respiratory Syndrome Coronavirus/isolation & purification , SARS Virus/isolation & purification , SARS-CoV-2/isolation & purification , Virus Diseases/diagnosis , CRISPR-Cas Systems , Fluorescent Dyes/chemistry , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Nose/virology , Point-of-Care Testing , RNA, Guide/chemistry , RNA, Guide/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS Virus/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Sputum/virology
12.
Nanotheranostics ; 5(4): 461-471, 2021.
Article in English | MEDLINE | ID: covidwho-1369901

ABSTRACT

The gold nanoclusters (Au NCs) are a special kind of gold nanomaterial containing several gold atoms. Because of their small size and large surface area, Au NCs possess macroscopic quantum tunneling and dielectric domain effects. Furthermore, Au NCs fluorescent materials have longer luminous time and better photobleaching resistance compared with other fluorescent materials. The synthetic process of traditional Au NCs is complicated. Traditional Au NCs are prepared mainly by using polyamide amine type dendrites, and sixteen alkyl trimethylamine bromide or sulfhydryl small molecule as stabilizers. They are consequently synthesized by the reduction of strong reducing agents such as sodium borohydride. Notably, these materials are toxic and environmental-unfriendly. Therefore, there is an urgent need to develop more effective methods for synthesizing Au NCs via a green approach. On the other hand, the self-assembly of protein gold cluster-based materials, and their biomedical applications have become research hotspots in this field. We have been working on the synthesis, assembly and application of protein conjugated gold clusters for a long time. In this review, the synthesis and assembly of protein-gold nanoclusters and their usage in cell imaging and other medical research are discussed.


Subject(s)
Fluorescent Dyes , Gold , Green Fluorescent Proteins , Metal Nanoparticles , Optical Imaging , Theranostic Nanomedicine , Fluorescent Dyes/chemistry , Fluorescent Dyes/therapeutic use , Gold/chemistry , Gold/therapeutic use , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/therapeutic use , Humans , Metal Nanoparticles/chemistry , Metal Nanoparticles/therapeutic use
13.
ACS Appl Mater Interfaces ; 13(34): 40342-40353, 2021 Sep 01.
Article in English | MEDLINE | ID: covidwho-1366784

ABSTRACT

Sensitive point-of-care methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in clinical specimens are urgently needed to achieve rapid screening of viral infection. We developed a magnetic quantum dot-based dual-mode lateral flow immunoassay (LFIA) biosensor for the high-sensitivity simultaneous detection of SARS-CoV-2 spike (S) and nucleocapsid protein (NP) antigens, which is beneficial for improving the detection accuracy and efficiency of SARS-CoV-2 infection in the point-of-care testing area. A high-performance magnetic quantum dot with a triple-QD shell (MagTQD) nanotag was first fabricated and integrated into the LFIA system to provide superior fluorescence signals, enrichment ability, and detectability for S/NP antigen testing. Two detection modes were provided by the proposed MagTQD-LFIA. The direct mode was used for rapid screening or urgent detection of suspected samples within 10 min, and the enrichment mode was used for the highly sensitive and quantitative analysis of SARS-CoV-2 antigens in biological samples without the interference of the "hook effect." The simultaneous detection of SARS-CoV-2 S/NP antigens was conducted in one LFIA strip, and the detection limits for two antigens under direct and enrichment modes were 1 and 0.5 pg/mL, respectively. The MagTQD-LFIA showed high accuracy, specificity, and stability in saliva and nasal swab samples and is an efficient tool with flexibility to meet the testing requirements for SARS-CoV-2 antigens in various situations.


Subject(s)
Antigens, Viral/analysis , Biosensing Techniques/methods , Coronavirus Nucleocapsid Proteins/analysis , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Antibodies, Immobilized/immunology , Antibodies, Monoclonal/immunology , Antigens, Viral/immunology , Coronavirus Nucleocapsid Proteins/immunology , Fluorescence , Fluorescent Dyes/chemistry , Humans , Immunoassay/methods , Limit of Detection , Magnetite Nanoparticles/chemistry , Nasopharynx/virology , Phosphoproteins/analysis , Phosphoproteins/immunology , Quantum Dots/chemistry , Saliva/virology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology
14.
ACS Appl Mater Interfaces ; 13(33): 38990-39002, 2021 Aug 25.
Article in English | MEDLINE | ID: covidwho-1351922

ABSTRACT

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10 min, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 and 80 pM limits of detection in 1× phosphate-buffered saline (mock swab) and saliva matrices spiked with cell-culture-generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way toward the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases.


Subject(s)
Antigens, Viral/analysis , COVID-19 Serological Testing/methods , Nucleocapsid Proteins/analysis , SARS-CoV-2/chemistry , Biomarkers/analysis , Biosensing Techniques , COVID-19/prevention & control , Cellulose/chemistry , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Dyes/chemistry , Humans , Microfluidic Analytical Techniques/methods , Peptide Library , Protein Binding
15.
ACS Appl Mater Interfaces ; 13(26): 30295-30305, 2021 Jul 07.
Article in English | MEDLINE | ID: covidwho-1337092

ABSTRACT

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.


Subject(s)
Hydrogels/chemistry , Multiplex Polymerase Chain Reaction/methods , Nanotubes, Carbon/chemistry , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Boron Compounds/chemistry , Coronavirus/chemistry , DNA Primers/chemistry , DNA, Single-Stranded/chemistry , Fluorescent Dyes/chemistry , Graphite/chemistry , Influenza A virus/chemistry , Newcastle disease virus/chemistry , Proof of Concept Study , RNA, Viral/chemistry , Virus Diseases/diagnosis
16.
Carbohydr Polym ; 269: 118345, 2021 Oct 01.
Article in English | MEDLINE | ID: covidwho-1271581

ABSTRACT

This work reports novel chitosan functionalized graphene oxide (GO) nanocomposites combined fluorescence imaging and therapeutic functions in one agent, which can serve as a promising alternative to alleviate related diseases caused hyperinflammation. Briefly, GO was designed to be conjugated with chitosan, fluorescein-labeled peptide, toll-like receptor 4 antibody and hydroxycamptothecin/aloe emodin. We have demonstrated that such nanocomposites could effectively achieve active targeted delivery of pro-apoptotic and anti-inflammatory drugs into inflammatory cells and cause cells apoptosis by acid-responsive drug release. Moreover, confocal fluorescence imaging confirms that the drug-induced inflammatory cells apoptosis could be visualized the light-up fluorescence of fluorescein activated by caspase-3. Meanwhile, inflammatory-related biomarkers have down-regulated after the nanocomposites' treatment in both vitro and vivo experiments consistent with the results in histological sections. In summary, the bifunctional nanocomposites that possess anti-inflammation and fluorescence imaging could serve as a promising therapeutic agent for reducing hyperinflammation caused by numerous diseases.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Apoptosis/physiology , Drug Carriers/chemistry , Inflammation/drug therapy , Nanocomposites/chemistry , Animals , Anti-Inflammatory Agents/chemistry , Antibodies/immunology , Camptothecin/analogs & derivatives , Camptothecin/chemistry , Camptothecin/therapeutic use , Cattle , Cell Line , Chitosan/chemistry , Drug Liberation , Emodin/chemistry , Emodin/therapeutic use , Fluorescent Dyes/chemistry , Graphite/chemistry , Humans , Lipopolysaccharides , Mammary Glands, Human/drug effects , Mammary Glands, Human/pathology , Mastitis/chemically induced , Mastitis/drug therapy , Mastitis/pathology , Mice , Toll-Like Receptor 4/immunology
17.
Molecules ; 26(13)2021 Jun 22.
Article in English | MEDLINE | ID: covidwho-1288958

ABSTRACT

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.


Subject(s)
Antiviral Agents/pharmacology , Fluorescence Resonance Energy Transfer/methods , High-Throughput Screening Assays/methods , Protease Inhibitors/pharmacology , RNA Viruses/enzymology , COVID-19/drug therapy , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/genetics , Coronavirus Papain-Like Proteases/metabolism , Drug Evaluation, Preclinical , Encephalitis Viruses, Tick-Borne/enzymology , Fluorescent Dyes/chemistry , Humans , RNA Helicases/antagonists & inhibitors , RNA Helicases/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , SARS-CoV-2/enzymology , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
18.
Int J Mol Sci ; 22(12)2021 Jun 18.
Article in English | MEDLINE | ID: covidwho-1273462

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus-cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy-which in our opinion can provide important information about the molecular biology of SARS-CoV-2-are discussed.


Subject(s)
Microscopy, Fluorescence , SARS-CoV-2/physiology , COVID-19/pathology , COVID-19/virology , Endocytosis , Fluorescent Dyes/chemistry , Genes, Reporter , Humans , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Virus Internalization
19.
Chem Commun (Camb) ; 57(51): 6229-6232, 2021 Jun 24.
Article in English | MEDLINE | ID: covidwho-1246405

ABSTRACT

Tracking the viral progression of SARS-CoV-2 in COVID-19 infected body tissues is an emerging need of the current pandemic. Imaging at near infrared second biological window (NIR-II) offers striking benefits over the other technologies to explore deep-tissue information. Here we design, synthesise and characterise a molecular probe that selectively targets the N-gene of SARS-CoV-2. Highly specific antisense oligonucleotides (ASOs) were conjugated to lead sulfide quantum dots using a UV-triggered thiol-ene click chemistry for the recognition of viral RNA. Our ex vivo imaging studies demonstrated that the probe exhibits aggregation induced NIR-II emission only in presence of SARS-CoV-2 RNA which can be attributed to the efficient hybridisation of the ASOs with their target RNA strands.


Subject(s)
COVID-19/diagnosis , COVID-19/virology , Fluorescent Dyes/chemistry , Oligonucleotides, Antisense/chemistry , Quantum Dots/chemistry , SARS-CoV-2/isolation & purification , Spectroscopy, Near-Infrared/methods , Animals , COVID-19/diagnostic imaging , COVID-19/metabolism , Click Chemistry/methods , Fluorescent Dyes/chemical synthesis , Humans , Lung/diagnostic imaging , Lung/metabolism , Lung/virology , Metal Nanoparticles/chemistry , Mice , Mice, Inbred BALB C , Models, Animal , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
20.
ACS Appl Mater Interfaces ; 13(21): 24477-24486, 2021 Jun 02.
Article in English | MEDLINE | ID: covidwho-1219585

ABSTRACT

The pseudovirus strategy makes studies of highly pathogenic viruses feasible without the restriction of high-level biosafety facility, thus greatly contributing to virology and is used in the research studies of SARS-CoV-2. Here, we generated a dual-color pseudo-SARS-CoV-2 virus using a human immunodeficiency virus-1 pseudovirus production system and the SARS-CoV-2 spike (S) glycoprotein, of which the membrane was labeled with a lipophilic dye (DiO) and the genomic RNA-related viral protein R (Vpr) of the viral core was fused with mCherry. With this dual-color labeling strategy, not only the movement of the whole virus but also the fate of the labeled components can be traced. The pseudovirions were applied to track the viral entry at a single-particle level in four types of the human respiratory cells: nasal epithelial cells (HNEpC), pulmonary alveolar epithelial cells (HPAEpiC), bronchial epithelial cells (BEP-2D), and oral epithelial cells (HOEC). Pseudo-SARS-CoV-2 entered into the host cell and released the viral core into the cytoplasm, which clearly indicates that the host entry mainly occurred through endocytosis. The infection efficiency was found to be correlated with the expression of the known receptor of SARS-CoV-2, angiotensin-converting 2 (ACE2) on the host cell surface. We believe that the dual-color fluorescently labeled pseudovirus system created in this study can be applied as a useful tool for many purposes in SARS-CoV-2/COVID-19.


Subject(s)
Fluorescent Dyes/chemistry , Pulmonary Alveoli/virology , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/metabolism , Endocytosis , Epithelial Cells/virology , Fluorescence , HEK293 Cells , HIV-1/genetics , Humans , Nasal Mucosa/virology , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL