Development of FRET and Stress Granule Dual-Based System to Screen for Viral 3C Protease Inhibitors.

3C proteases (3Cpros) of picornaviruses and 3C-like proteases (3CLpros) of coronaviruses and caliciviruses represent a group of structurally and functionally related viral proteases that play pleiotropic roles in supporting the viral life cycle and subverting host antiviral responses. The design and screening for 3C/3CLpro inhibitors may contribute to the development broad-spectrum antiviral therapeutics against viral diseases related to these three families. However, current screening strategies cannot simultaneously assess a compound's cytotoxicity and its impact on enzymatic activity and protease-mediated physiological processes. The viral induction of stress granules (SGs) in host cells acts as an important antiviral stress response by blocking viral translation and stimulating the host immune response. Most of these viruses have evolved 3C/3CLpro-mediated cleavage of SG core protein G3BP1 to counteract SG formation and disrupt the host defense. Yet, there are no SG-based strategies screening for 3C/3CLpro inhibitors. Here, we developed a fluorescence resonance energy transfer (FRET) and SG dual-based system to screen for 3C/3CLpro inhibitors in living cells. We took advantage of FRET to evaluate the protease activity of poliovirus (PV) 3Cpro and live-monitor cellular SG dynamics to cross-verify its effect on the host antiviral response. Our drug screen uncovered a novel role of Telaprevir and Trifluridine as inhibitors of PV 3Cpro. Moreover, Telaprevir and Trifluridine also modulated 3Cpro-mediated physiological processes, including the cleavage of host proteins, inhibition of the innate immune response, and consequent facilitation of viral replication. Taken together, the FRET and SG dual-based system exhibits a promising potential in the screening for inhibitors of viral proteases that cleave G3BP1.

Carbon Dots Conjugated Antibody as an Effective FRET-Based Biosensor for Progesterone Hormone Screening.

In this work, carbon dots (CDs) were synthesized by a one-step hydrothermal method using citric acid and ethylene diamine, and covalently functionalized with antibodies for the sensing of progesterone hormone. The structural and morphological analysis reveals that the synthesized CDs are of average size (diameter 8-10 nm) and the surface functionalities are confirmed by XPS, XRD and FT-IR. Further graphene oxide (GO) is used as a quencher due to the fluorescence resonance energy transfer (FRET) mechanism, whereas the presence of the analyte progesterone turns on the fluorescence because of displacement of GO from the surface of CDs effectively inhibiting FRET efficiency due to the increased distance between donor and acceptor moieties. The linear curve is obtained with different progesterone concentrations with 13.8 nM detection limits (R2 = 0.974). The proposed optical method demonstrated high selectivity performance in the presence of structurally resembling interfering compounds. The PL intensity increased linearly with the increased progesterone concentration range (10-900 nM) under the optimal experimental parameters. The developed level-free immunosensor has emerged as a potential platform for simplified progesterone analysis due to the high selectivity performance and good recovery in different samples of spiked water.

An Integrated Analysis of Mechanistic Insights into Biomolecular Interactions and Molecular Dynamics of Bio-Inspired Cu(II) and Zn(II) Complexes towards DNA/BSA/SARS-CoV-2 3CLpro by Molecular Docking-Based Virtual Screening and FRET Detection.

Novel constructed bioactive mixed-ligand complexes (1b) [CuII(L)2(phen)] and (2b) [ZnII(L)2(phen)] {where, L = 2-(4-morpholinobenzylideneamino)phenol), phen = 1,10-phenanthroline} have been structurally analysed by various analytical and spectroscopic techniques, including, magnetic moments, thermogravimetric analysis, and X-ray crystallography. Various analytical and spectral measurements assigned showed that all complexes appear to have an octahedral geometry. Agar gel electrophoresis's output demonstrated that the Cu(II) complex (1b) had efficient deoxyribonucleic cleavage and complex (2b) demonstrated the partial cleavage accomplished with an oxidation agent, which generates spreadable OH● through the Fenton type mechanism. The DNA binding constants observed from viscosity, UV-Vis spectral, fluorometric, and electrochemical titrations were in the following sequence: (1b) > (2b) > (HL), which suggests that the complexes (1b-2b) might intercalate DNA, a possibility that is supported by the biothermodynamic measurements. In addition, the observed binding constant results of BSA by electronic absorption and fluorometric titrations indicate that complex (1b) revealed the best binding efficacy as compared to complex (2b) and free ligand. Interestingly, all compounds are found to interact with BSA through a static approach, as further attested by FRET detection. The DFT and molecular docking calculations were also performed to realize the electronic structure, reactivity, and binding capability of all test samples with CT-DNA, BSA, and the SARS-CoV-2 3CLPro, which revealed the binding energies were in a range of -8.1 to -8.9, -7.5 to -10.5 and -6.7--8.8 kcal/mol, respectively. The higher reactivity of the complexes than the free ligand is supported by the FMO theory. Among all the observed data for antioxidant properties against DPPH᛫, ᛫OH, O2-• and NO᛫ free radicals, complex (1a) had the best biological efficacy. The antimicrobial and cytotoxic characteristics of all test compounds have been studied by screening against certain selected microorganisms as well as against A549, HepG2, MCF-7, and NHDF cell lines, respectively. The observed findings revealed that the activity enhances coordination as compared to free ligand via Overtone's and Tweedy's chelation mechanisms. This is especially encouraging given that in every case, the experimental findings and theoretical detections were in perfect accord.

Time-Resolved Persistent Luminescence Encoding for Multiplexed Severe Acute Respiratory Syndrome Coronavirus 2 Detection.

Capable of precise simultaneous multitarget identifications within a minimized sample, optical multiplexing is vital for accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) while remaining spectral crowding and background interfering. In merits of an autofluorescence-free background and high-capability throughput, a persistent luminescence (PersL) lifetime/color binary encoding strategy was herein proposed for SARS-CoV-2 diagnosis. Based on luminescence resonance energy transfer processes, the intense lifetimes and representative emissions of PersL nanoplatforms were rationally manipulated to create a temporal coding dimension within a wide seconds-to-minutes range through three individual channels. Particularly, at least four populations of barcoding in a certain channel were successfully decoded by a purpose-built time-resolved PersL technology. As a proof-of-concept, functionalized PersL nanoplatforms were further well developed for the simultaneous quantification of five-plex SARS-CoV-2 biomarkers with limits of detection in the subnanomolar range. Remarkably, PersL nanoplatforms enabled a highly differentiable discrimination of multitargets at various concentrations of ultralow background and high-fidelity resolutions, thereby advancing a powerful tool for optical multiplexing in biomedical applications.

Ultrasensitive FRET-based aptasensor for interleukin-6 as a biomarker for COVID-19 progression using nitrogen-doped carbon quantum dots and gold nanoparticles.

A label-free and specific FRET-based interleukin-6 (IL-6) aptasensor was developed using a DNA aptamer modified with nitrogen-doped carbon quantum dots (NCDs) and gold nanoparticles (AuNPs) as a donor-quencher pair. The assayed target was capable of disrupting the donor-acceptor assemblies yielding a concentration-related fluorescence recovery of NCDs (λem = 445 nm and λex = 350 nm). By designing two different probes, the interaction of DNA aptamers with IL-6 protein was studied using FRET efficiency. It appeared that the sensing probes showed slightly different sensing profiles. One of the aptasensors showed a linear response of 1.5-5.9 pg/mL for IL-6 with a coefficient of determination of R2 ≥ 0.99 and the a detection limit of 0.82 pg/mL (at S/N = 3). The experimental results indicated that the biosensor can be applied to determine IL-6 in human serum (with recovery of 95.7-102.9%). Due to the high sensitivity, excellent selectivity, and simplicity of the procedure, this strategy represents a promising alternative for IL-6 sensing in clinical applications.

Rapid and Highly Sensitive Detection of Target DNA Related to COVID-19 Virus With a Fluorescent Bio-conjugated Probe via a FRET Mechanism.

A novel cyanine 3 (Cy3)-based bio-conjugated sensor has been developed to detect target DNA or extracted RNA from COVID -19 samples using the fluorescence resonance energy transfer (FRET) experiment. A special sequence of the COVID -19 genome was selected as a complementary DNA (target DNA) part. The opposite chain of this target sequence was designed in 2 parts; one part was attached to the Cy3 organic dye (capture DNA or Cy3- DNA), and the other part was attached to the BHQ2 molecule (quencher DNA or BHQ2- DNA). The Cy3 molecule acts as a donor pair, and BHQ2 acts as an acceptor pair in the FRET experiment. The capture DNA and quencher DNA can form a sandwiched complex in the presence of target DNA. The formation of the entitled sandwiched hybrid causes the decrement of emission intensity of the Cy3 donor in bio-conjugated Cy3-DNA via energy transfer from Cy3 (as a donor) to BHQ2 (as an acceptor). Indeed, in the presence of non-complementary DNA, the pairing of DNA strands does not occur, the FRET phenomenon does not exist, and therefore fluorescence intensity of Cy3 does not decrease. Moreover, this biosensor was successfully applied to analyze real samples containing extracted RNA of COVID -19 prepared for the reverse transcriptase-polymerase chain reaction (RT-PCR) test, and the results were promising.

[Discovery of SARS-CoV-2 main protease inhibitors using an optimized FRET-based high-throughput screening assay].

For rapid discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors, an optimized fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay was developed. The recombinant Mpro was expressed in Escherichia coli Rosetta (DE3) cells and the specific activity of purified Mpro was assessed by a FERT assay using a fluorescently labeled substrate. Subsequently, the reaction buffer, working concentration of Mpro, incubation temperature and length, and DMSO tolerance were systematically optimized. The Mpro was solubly expressed in E. coli cells and exhibited an expected enzymatic activity (40 000 U/mg) in a FRET assay. Through these systematic optimizations, we selected 0.4 µmol/L Mpro and 5 µmol/L FRET substrate as the optimal working concentrations in this FRET screening assay, and a high Z' factor of 0.79 was achieved. More importantly, the addition of reducing reagent 1, 4-dithiothreitol in reaction buffer is necessary to faithfully assess the reliability of the screening assay. Using this assay, plumbagin (PLB) and ginkgolic acid (GA) were identified as potential Mpro inhibitors in vitro from a natural product library. In summary, we developed an optimized FRET-based HTS assay for the discovery of Mpro inhibitors, and PLB and GA could serve as the promissing lead compounds to generate more potent antiviral agents targeting SARS-CoV-2 Mpro.

Portable FRET-Based Biosensor Device for On-Site Lead Detection.

Most methods for measuring environmental lead (Pb) content are time consuming, expensive, hazardous, and restricted to specific analytical systems. To provide a facile, safe tool to detect Pb, we created pMet-lead, a portable fluorescence resonance energy transfer (FRET)-based Pb-biosensor. The pMet-lead device comprises a 3D-printed frame housing a 405-nm laser diode-an excitation source for fluorescence emission images (YFP and CFP)-accompanied by optical filters, a customized sample holder with a Met-lead 1.44 M1 (the most recent version)-embedded biochip, and an optical lens aligned for smartphone compatibility. Measuring the emission ratios (Y/C) of the FRET components enabled Pb detection with a dynamic range of nearly 2 (1.96), a pMet-lead/Pb dissociation constant (Kd) 45.62 nM, and a limit of detection 24 nM (0.474 µg/dL, 4.74 ppb). To mitigate earlier problems with a lack of selectivity for Pb vs. zinc, we preincubated samples with tricine, a low-affinity zinc chelator. We validated the pMet-lead measurements of the characterized laboratory samples and unknown samples from six regions in Taiwan by inductively coupled plasma mass spectrometry (ICP-MS). Notably, two unknown samples had Y/C ratios significantly higher than that of the control (3.48 ± 0.08 and 3.74 ± 0.12 vs. 2.79 ± 0.02), along with Pb concentrations (10.6 ppb and 15.24 ppb) above the WHO-permitted level of 10 ppb in tap water, while the remaining four unknowns showed no detectable Pb upon ICP-MS. These results demonstrate that pMet-lead provides a rapid, sensitive means for on-site Pb detection in water from the environment and in living/drinking supply systems to prevent potential Pb poisoning.

Specific length and structure rather than high thermodynamic stability enable regulatory mRNA stem-loops to pause translation.

Translating ribosomes unwind mRNA secondary structures by three basepairs each elongation cycle. Despite the ribosome helicase, certain mRNA stem-loops stimulate programmed ribosomal frameshift by inhibiting translation elongation. Here, using mutagenesis, biochemical and single-molecule experiments, we examine whether high stability of three basepairs, which are unwound by the translating ribosome, is critical for inducing ribosome pauses. We find that encountering frameshift-inducing mRNA stem-loops from the E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) hinders A-site tRNA binding and slows down ribosome translocation by 15-20 folds. By contrast, unwinding of first three basepairs adjacent to the mRNA entry channel slows down the translating ribosome by only 2-3 folds. Rather than high thermodynamic stability, specific length and structure enable regulatory mRNA stem-loops to stall translation by forming inhibitory interactions with the ribosome. Our data provide the basis for rationalizing transcriptome-wide studies of translation and searching for novel regulatory mRNA stem-loops.

Exploration of SARS-CoV-2 3CLpro Inhibitors by Virtual Screening Methods, FRET Detection, and CPE Assay.

COVID-19 caused by a novel coronavirus (SARS-CoV-2) has been spreading all over the world since the end of 2019, and no specific drug has been developed yet. 3C-like protease (3CLpro) acts as an important part of the replication of novel coronavirus and is a promising target for the development of anticoronavirus drugs. In this paper, eight machine learning models were constructed using naïve Bayesian (NB) and recursive partitioning (RP) algorithms for 3CLpro on the basis of optimized two-dimensional (2D) molecular descriptors (MDs) combined with ECFP_4, ECFP_6, and MACCS molecular fingerprints. The optimal models were selected according to the results of 5-fold cross verification, test set verification, and external test set verification. A total of 5766 natural compounds from the internal natural product database were predicted, among which 369 chemical components were predicted to be active compounds by the optimal models and the EstPGood values were more than 0.6, as predicted by the NB (MD + ECFP_6) model. Through ADMET analysis, 31 compounds were selected for further biological activity determination by the fluorescence resonance energy transfer (FRET) method and cytopathic effect (CPE) detection. The results indicated that (+)-shikonin, shikonin, scutellarein, and 5,3',4'-trihydroxyflavone showed certain activity in inhibiting SARS-CoV-2 3CLpro with the half-maximal inhibitory concentration (IC50) values ranging from 4.38 to 87.76 µM. In the CPE assay, 5,3',4'-trihydroxyflavone showed a certain antiviral effect with an IC50 value of 8.22 µM. The binding mechanism of 5,3',4'-trihydroxyflavone with SARS-CoV-2 3CLpro was further revealed through CDOCKER analysis. In this study, 3CLpro prediction models were constructed based on machine learning algorithms for the prediction of active compounds, and the activity of potential inhibitors was determined by the FRET method and CPE assay, which provide important information for further discovery and development of antinovel coronavirus drugs.

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.

Discovery of Zafirlukast as a novel SARS-CoV-2 helicase inhibitor using in silico modelling and a FRET-based assay.

The coronavirus helicase is an essential enzyme required for viral replication/transcription pathways. Structural studies revealed a sulphate moiety that interacts with key residues within the nucleotide-binding site of the helicase. Compounds with a sulphoxide or a sulphone moiety could interfere with these interactions and consequently inhibit the enzyme. The molecular operating environment (MOE) was used to dock 189 sulphoxide and sulphone-containing FDA-approved compounds to the nucleotide-binding site. Zafirlukast, a leukotriene receptor antagonist used to treat chronic asthma, achieved the lowest docking score at -8.75 kcals/mol. The inhibitory effect of the compounds on the SARS-CoV-2 helicase dsDNA unwinding activity was tested by a FRET-based assay. Zafirlukast was the only compound to inhibit the enzyme (IC50 = 16.3 µM). The treatment of Vero E6 cells with 25 µM zafirlukast prior to SARS-CoV-2 infection decreased the cytopathic effects of SARS-CoV-2 significantly. These results suggest that zafirlukast alleviates SARS-CoV-2 pathogenicity by inhibiting the viral helicase and impairing the viral replication/transcription pathway. Zafirlukast could be clinically developed as a new antiviral treatment for SARS-CoV-2 and other coronavirus diseases. This discovery is based on molecular modelling, in vitro inhibition of the SARS-CoV helicase activity and cell-based SARS-CoV-2 viral replication.

Detection of SARS-CoV-2 spike protein binding to ACE2 in living cells by TR-FRET.

The SARS-CoV-2 coronavirus infects human cells through the interaction of the viral envelope spike protein (IPR044366) with the human angiotensin-converting enzyme 2 (ACE2), expressed at the surface of target cells. Here, we describe a detailed protocol to measure the binding of the receptor binding domain (RBD) of spike to ACE2 by time-resolved fluorescence resonance energy transfer (TR-FRET). The assay detects the spike/ACE2 interaction in physiologically relevant cellular contexts and is suitable for high-throughput investigation of interfering small-molecule compounds and antibodies. For complete details on the use and execution of this protocol, please refer to Cecon et al. (2021).

Identification of the SARS-CoV-2 Delta variant C22995A using a high-resolution melting curve RT-FRET-PCR.

Knowledge of SARS-CoV-2 variants is essential for formulating effective control policies. Currently, variants are only identified in relatively small percentages of cases as the required genome sequencing is expensive, time-consuming, and not always available. In countries with facilities to sequence the SARS-CoV-2, the Delta variant currently predominates. Elsewhere, the prevalence of the Delta variant is unclear. To avoid the need for sequencing, we investigated a RT-FRET-PCR that could detect all SARS-CoV-2 strains and simultaneously identify the Delta variant. The established Delta RT-FRET-PCR was performed on reference SARS-CoV-2 strains, and human nasal swab samples positive for the Delta and non-Delta strains. The Delta RT-FRET-PCR established in this study detected as few as ten copies of the DNA target and 100 copies of RNA target per reaction. Melting points of products obtained with SARS-CoV-2 Delta variants (around 56.1°C) were consistently higher than products obtained with non-Delta strains (around 52.5°C). The Delta RT-FRET-PCR can be used to diagnose COVID-19 patients and simultaneously identify if they are infected with the Delta variant. The Delta RT-FRET-PCR can be performed with all major thermocycler brands meaning data on Delta variant can now be readily generated in diagnostic laboratories worldwide.

The Mpro structure-based modifications of ebselen derivatives for improved antiviral activity against SARS-CoV-2 virus.

The main protease (Mpro or 3CLpro) of SARS-CoV-2 virus is a cysteine enzyme critical for viral replication and transcription, thus indicating a potential target for antiviral therapy. A recent repurposing effort has identified ebselen, a multifunctional drug candidate as an inhibitor of Mpro. Our docking of ebselen to the binding pocket of Mpro crystal structure suggests a noncovalent interaction for improvement of potency, antiviral activity and selectivity. To test this hypothesis, we designed and synthesized ebselen derivatives aimed at enhancing their non-covalent bonds within Mpro. The inhibition of Mpro by ebselen derivatives (0.3 µM) was screened in both HPLC and FRET assays. Nine ebselen derivatives (EBs) exhibited stronger inhibitory effect on Mpro with IC50 of 0.07-0.38 µM. Further evaluation of three derivatives showed that EB2-7 exhibited the most potent inhibition of SARS-CoV-2 viral replication with an IC50 value of 4.08 µM in HPAepiC cells, as compared to the prototype ebselen at 24.61 µM. Mechanistically, EB2-7 functions as a noncovalent Mpro inhibitor in LC-MS/MS assay. Taken together, our identification of ebselen derivatives with improved antiviral activity may lead to developmental potential for treatment of COVID-19 and SARS-CoV-2 infection.

Versatile and rapid microfluidics-assisted antibody discovery.

Recent years have seen unparalleled development of microfluidic applications for antibody discovery in both academic and pharmaceutical research. Microfluidics can support native chain-paired library generation as well as direct screening of antibody secreting cells obtained by rodent immunization or from the human peripheral blood. While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, microfluidics can expedite therapeutic antibody discovery for cancer or immunological disease indications. In this study, a commercially available microfluidic device, Cyto-Mine, was used for the rapid identification of natively paired antibodies from rodents or human donors screened for specific binding to recombinant antigens, for direct screening with cells expressing the target of interest, and, to our knowledge for the first time, for direct broad functional IgG antibody screening in droplets. The process time from cell preparation to confirmed recombinant antibodies was four weeks. Application of this or similar microfluidic devices and methodologies can accelerate and enhance pharmaceutical antibody hit discovery.

Discovery and Mechanism of SARS-CoV-2 Main Protease Inhibitors.

The emergence of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an urgent public health crisis. Without available targeted therapies, treatment options remain limited for COVID-19 patients. Using medicinal chemistry and rational drug design strategies, we identify a 2-phenyl-1,2-benzoselenazol-3-one class of compounds targeting the SARS-CoV-2 main protease (Mpro). FRET-based screening against recombinant SARS-CoV-2 Mpro identified six compounds that inhibit proteolysis with nanomolar IC50 values. Preincubation dilution experiments and molecular docking determined that the inhibition of SARS-CoV-2 Mpro can occur by either covalent or noncovalent mechanisms, and lead E04 was determined to inhibit Mpro competitively. Lead E24 inhibited viral replication with a nanomolar EC50 value (844 nM) in SARS-CoV-2-infected Vero E6 cells and was further confirmed to impair SARS-CoV-2 replication in human lung epithelial cells and human-induced pluripotent stem cell-derived 3D lung organoids. Altogether, these studies provide a structural framework and mechanism of Mpro inhibition that should facilitate the design of future COVID-19 treatments.

Development and optimization of heavy metal lead biosensors in biomedical and environmental applications.

The detrimental impact of the heavy metal lead (Pb) on human health has been studied for years. The fact that Pb impairs human body has been established from countless painful and sad historical events. Nowadays, World Health Organization and many developmental countries have established regulations concerning the use of Pb. Measuring the blood lead level (BLL) is so far the only way to officially evaluate the degree of Pb exposure, but the so-called safety value (10 µg/dL in adults and 5 µg/dL in children) seems unreliable to represent the security checkpoint for children through daily intake of drinking water or physical contact with a lower contaminated level of Pb contents. In general, unsolved mysteries about the Pb toxicological mechanisms still remain. In this review article, we report on the methods to prevent Pb poison for further Pb toxicological research. We establish high-sensitivity Pb monitoring, and also report on the use of fluorescent biosensors such as genetically-encoded fluorescence resonance energy transfer-based biosensors built for various large demands such as the detection of severe acute respiratory syndrome coronavirus 2. We also contribute to the development and optimization of the FRET-based Pb biosensors. Our well-performed version of Met-lead 1.44 M1 has achieved a limit of detection of 10 nM (2 ppb; 0.2 µg/dL) and almost 5-fold in dynamic range (DR) supported for the real practical applications-that is, the in-cell Pb sensing device for blood and blood-related samples, and the Pb environmental detections in vitro. The perspective of our powerful Pb biosensor incorporated with a highly sensitive bio-chip of the portable device for quick Pb measurements will be addressed for further manipulation.

Spectral and time-resolved photoluminescence of human platelets doped with platinum nanoparticles.

This paper describes a detailed study of spectral and time-resolved photoprocesses in human platelets and their complexes with platinum (Pt) nanoparticles (NPs). Fluorescence, quantum yield, and platelet amino acid lifetime changes in the presence and without femtosecond ablated platinum NPs have been studied. Fluorescence spectroscopy analysis of main fluorescent amino acids and their residues (tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe)) belonging to the platelet membrane have been performed. The possibility of energy transfer between Pt NPs and the platelet membrane has been revealed. Förster Resonance Energy Transfer (FRET) model was used to perform the quantitative evaluation of energy transfer parameters. The prospects of Pt NPs usage deals with quenching-based sensing for pathology's based on platelet conformations as cardiovascular diseases have been demonstrated.

Droplet Microarray Based on Nanosensing Probe Patterns for Simultaneous Detection of Multiple HIV Retroviral Nucleic Acids.

Multiplexed detection of viral nucleic acids is important for rapid screening of viral infection. In this study, we present a molybdenum disulfide (MoS2) nanosheet-modified dendrimer droplet microarray (DMA) for rapid and sensitive detection of retroviral nucleic acids of human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2) simultaneously. The DMA platform was fabricated by omniphobic-omniphilic patterning on a surface-grafted dendrimer substrate. Functionalized MoS2 nanosheets modified with fluorescent dye-labeled oligomer probes were prepatterned on positively charged amino-modified omniphilic spots to form a fluorescence resonance energy transfer (FRET) sensing microarray. With the formation of separated microdroplets of sample on the hydrophobic-hydrophilic micropattern, prepatterned oligomer probes specifically hybridized with the target HIV genes and detached from the MoS2 nanosheet surface due to weakening of the adsorption force, leading to fluorescence signal recovery. As a proof of concept, we used this microarray with a small sample size (<150 nL) for simultaneous detection of HIV-1 and HIV-2 nucleic acids with a limit of detection (LOD) of 50 pM. The multiplex detection capability was further demonstrated for simultaneous detection of five viral genes (HIV-1, HIV-2, ORFlab, and N genes of SARS-COV-2 and M gene of Influenza A). This work demonstrated the potential of this novel MoS2-DMA FRET sensing platform for high-throughput multiplexed viral nucleic acid screening.