Rapid One-Pot Microwave Assisted Green Synthesis Nitrogen Doped Carbon Quantum Dots as Fluorescent Precursor for Estimation of Modafinil as Post-Covid Neurological Drug in Human Plasma with Greenness Assessments.

The neuro-stimulant anti-narcoleptic drug as modafinil (MOD) is used to treatment neurological conditions caused by COVID-19. MOD was used to treatment narcolepsy, shift-work sleep disorder, and obstructive sleep apnea-related sleepiness. So, an innovative, quick, economical, selective, and ecologically friendly procedure was carried out. A highly sensitive N@CQDs technique was created from green Eruca sativa leaves in about 4 min using microwave synthesis at 700 w. The quantum yield of the synthesized N@CQDs was found to be 41.39%. By increasing the concentration of MOD, the quantum dots' fluorescence intensity was gradually quenched. After being excited at 445 nm, the fluorescence reading was recorded at 515 nm. The linear range was found to be in the range 50 - 700 ng mL-1 with lower limit of quantitation (LOQ) equal to 45.00 ng mL-1. The current method was fully validated and bio analytically according to (US-FDA and ICH) guidelines. Full characterization of the N@CQDs has been conducted by high resolution transmission electron microscope (HRTEM), Zeta potential measurement, fluorescence, UV-VIS, and FTIR spectroscopy. Various experimental variables including pH, QDs concentration and the reaction time were optimized. The proposed study is simply implemented for the therapeutic drug monitoring system (TDMS) and various clinical laboratories for further pharmacokinetic research.

Spatially Addressable Multiplex Biodetection by Calibrated Micro/Nanostructured Surfaces.

A challenge of any biosensing technology is the detection of very low concentrations of analytes. The fluorescence interference contrast (FLIC) technique improves the fluorescence-based sensitivity by selectively amplifying, or suppressing, the emission of a fluorophore-labeled biomolecule immobilized on a transparent layer placed on top of a mirror basal surface. The standing wave of the reflected emission light means that the height of the transparent layer operates as a surface-embedded optical filter for the fluorescence signal. FLIC extreme sensitivity to wavelength is also its main problem: small, e.g., 10 nm range, variations of the vertical position of the fluorophore can translate in unwanted suppression of the detection signal. Herein, we introduce the concept of quasi-circular lenticular microstructured domes operating as continuous-mode optical filters, generating fluorescent concentric rings, with diameters determined by the wavelengths of the fluorescence light, in turn modulated by FLIC. The critical component of the lenticular structures was the shallow sloping side wall, which allowed the simultaneous separation of fluorescent patterns for virtually any fluorophore wavelength. Purposefully designed microstructures with either stepwise or continuous-slope dome geometries were fabricated to modulate the intensity and the lateral position of a fluorescence signal. The simulation of FLIC effects induced by the lenticular microstructures was confirmed by the measurement of the fluorescence profile for three fluorescent dyes, as well as high-resolution fluorescence scanning using stimulated emission depletion (STED) microscopy. The high sensitivity of the spatially addressable FLIC technology was further validated on a diagnostically important target, i.e., the receptor-binding domain (RBD) of the SARS-Cov2 via the detection of RBD:anti-S1-antibody.

Role of the Probe Sequence/Structure in Developing an Ultra-Efficient Label-Free COVID-19 Detection Method Based on Competitive Dual-Emission Ratiometric DNA-Templated Silver Nanoclusters as Single Fluorescent Probes.

We report the development of a label-, antibody-, enzyme-, and amplification-free ratiometric fluorescent biosensor for low-cost and rapid (less than 12 min) diagnosis of COVID-19 from isolated RNA samples. The biosensor is designed on the basis of cytosine-modified antisense oligonucleotides specific for either N gene or RdRP gene that can form silver nanoclusters (AgNCs) with both green and red emission on an oligonucleotide via a one-step synthesis process. The presence of the target RNA sequence of SARS-CoV-2 causes a dual-emission ratiometric signal transduction, resulting in a limit of detection of 0.30 to 10.0 nM and appropriate linear ranges with no need for any further amplification, fluorophore, or design with a special DNA fragment. With this strategy, five different ratiometric fluorescent probes are designed, and how the T/C ratio, the length of the stem region, and the number of cytosines in the loop structure and at the 3' end of the cluster-stabilizing template can affect the biosensor sensitivity is investigated. Furthermore, the effect of graphene oxide (GO) on the ratiometric behavior of nanoclusters is demonstrated and the concentration-/time-dependent new competitive mechanism between aggregation-caused quenching (ACQ) and aggregation-induced emission enhancement (AIE) for the developed ssDNA-AgNCs/GO nanohybrids is proposed. Finally, the performance of the designed ratiometric biosensor has been validated using the RNA extract obtained from more than 150 clinical samples, and the results have been confirmed by the FDA-approved reverse transcription-polymerase chain reaction (RT-PCR) diagnostic method. The diagnostic sensitivity and specificity of the best probe is more than >90%, with an area under the receiver operating characteristic (ROC) curve of 0.978.

Bead-Based Multiplexed Droplet Digital Polymerase Chain Reaction in a Single Tube Using Universal Sequences: An Ultrasensitive, Cross-Reaction-Free, and High-Throughput Strategy.

The multiplexed digital polymerase chain reaction (PCR) is widely used in molecular diagnosis owing to its high sensitivity and throughput for multiple target detection compared with the single-plexed digital PCR; however, current multiplexed digital PCR technologies lack efficient coding strategies that do not compromise the sensitivity and signal-to-noise (S/N) ratio. Hence, we propose a fluorescent-encoded bead-based multiplexed droplet digital PCR method for ultra-high coding capacity, along with the creative design of universal sequences (primer and fluorescent TaqMan probe) for ultra-sensitivity and high S/N ratios. First, pre-amplification is used to introduce universal primers and universal fluorescent TaqMan probes to reduce primer interference and background noise, as well as to enrich regions of interest in targeted analytes. Second, fluorescent-encoded beads (FEBs), coupled with the corresponding target sequence-specific capture probes through streptavidin-biotin conjugation, are used to partition amplicons via hybridization according to the Poisson distribution. Finally, FEBs mixed with digital PCR mixes are isolated into droplets generated via Sapphire chips (Naica Crystal Digital PCR system) to complete the digital PCR and result analysis. For proof of concept, we demonstrate that this method achieves high S/N ratios in a 5-plexed assay for influenza viruses and SARS-CoV-2 at concentrations below 10 copies and even close to a single molecule per reaction without cross-reaction, further verifying the possibility of clinical actual sample detection with 100% accuracy, which paves the way for the realization of digital PCR with ultrahigh coding capacity and ultra-sensitivity.

Application of meso-CF3-Fluorophore BODIPY with Phenyl and Pyrazolyl Substituents for Lifetime Visualization of Lysosomes.

A bright far-red emitting unsymmetrical meso-CF3-BODIPY fluorescent dye with phenyl and pyrazolyl substituents was synthesized by condensation of trifluoropyrrolylethanol with pyrazolyl-pyrrole, with subsequent oxidation and complexation of the formed dipyrromethane. This BODIPY dye exhibits optical absorption at λab ≈ 610-620 nm and emission at λem ≈ 640-650 nm. The BODIPY was studied on Ehrlich carcinoma cells as a lysosome-specific fluorescent dye that allows intravital staining of cell structures with subsequent real-time monitoring of changes occurring in the cells. It was also shown that the rate of uptake by cells, the rate of intracellular transport into lysosomes, and the rate of saturation of cells with the dye depend on its concentration in the culture medium. A concentration of 5 µM was chosen as the most suitable BODIPY concentration for fluorescent staining of living cell lysosomes, while a concentration of 100 µM was found to be toxic to Ehrlich carcinoma cells.

Fluorescent Platforms for RNA Chemical Biology Research.

Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating.

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.

Detection of SARS-CoV-2 RNA Using a DNA Aptamer Mimic of Green Fluorescent Protein.

RNA detection is important in diverse diagnostic and analytical applications. RNAs can be rapidly detected using molecular beacons, which fluoresce upon hybridizing to a target RNA but require oligonucleotides with complex fluorescent dye and quencher conjugations. Here, we describe a simplified method for rapid fluorescence detection of a target RNA using simple unmodified DNA oligonucleotides. To detect RNA, we developed Lettuce, a fluorogenic DNA aptamer that binds and activates the fluorescence of DFHBI-1T, an otherwise nonfluorescent molecule that resembles the chromophore found in green fluorescent protein. Lettuce was selected from a randomized DNA library based on binding to DFHBI-agarose. We further show that Lettuce can be split into two separate oligonucleotide components, which are nonfluorescent on their own but become fluorescent when their proximity is induced by a target RNA. We designed several pairs of split Lettuce fragments that contain an additional 15-20 nucleotides that are complementary to adjacent regions of the SARS-CoV-2 RNA, resulting in Lettuce fluorescence only in the presence of the viral RNA. Overall, these studies describe a simplified RNA detection approach using fully unmodified DNA oligonucleotides that reconstitute the Lettuce aptamer templated by RNA.

A novel liquid biopsy-based approach for highly specific cancer diagnostics: mitigating false responses in assaying patient plasma-derived circulating microRNAs through combined SERS and plasmon-enhanced fluorescence analyses.

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.

Cryptococcal Protease(s) and the Activation of SARS-CoV-2 Spike (S) Protein.

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.

A Dual-Color Fluorescent Probe Allows Simultaneous Imaging of Main and Papain-like Proteases of SARS-CoV-2-Infected Cells for Accurate Detection and Rapid Inhibitor Screening.

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.

Aerosol tracer testing in Boeing 767 and 777 aircraft to simulate exposure potential of infectious aerosol such as SARS-CoV-2.

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.

Site-Specific Labeling of Endogenous Proteins Using CoLDR Chemistry.

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.

Development of Integrated Systems for On-Site Infection Detection.

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.

DNA-Directed Patterning for Versatile Validation and Characterization of a Lipid-Based Nanoparticle Model of SARS-CoV-2.

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.

Fluorescent glycan fingerprinting of SARS2 spike proteins.

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.

Mix-and-read, one-minute SARS-CoV-2 diagnostic assay: development of PIFE-based aptasensor.

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.

Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

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.

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.

Nanotheranostic Application of Fluorescent Protein-Gold Nanocluster Hybrid Materials: A Mini-review.

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.