Fluorescence Resonance Energy Transfer Method Research Articles

Highly selective detection of deoxyribonucleic acid in living cells using RecA-green fluorescent protein-single-stranded deoxyribonucleic acid filament fluorescence resonance energy transfer probe.

A fluorescence resonance energy transfer (FRET) method was developed for double-stranded deoxyribonucleic acid (dsDNA) detection in living cells using the RecA-GFP (green fluorescent protein) fusion protein filament. In brief, the thiol-modified single-stranded DNA (ssDNA) was attached to gold nanoparticles (AuNPs); on the contrary, the prepared RecA-GFP fusion protein interacted with ssDNA. Due to the FRET between AuNPs and RecA-GFP, fluorescence of RecA-GFP fusion protein was quenched. In the presence of homologous dsDNA, homologous recombination occurred to release RecA-GFP fusion protein. Thus, the fluorescence of RecA-GFP was recovered. The dsDNA concentration was detected using fluorescence intensity of RecA-GFP. Under optimal conditions, this method could detect dsDNA activity as low as 0.015 optical density (OD) Escherichia coli cells, with a wide linear range from 0.05 to 0.9 OD cells, and the regression equation was ΔF = 342.7c + 78.9, with a linear relationship coefficient of 0.9920. Therefore, it provided a promising approach for the selective detection of dsDNA in living cells for early clinical diagnosis of genetic diseases.

Single-Molecule FRET-Based Multiplexed Detection.

Single-molecule multiplexed detection is a high-promise toolkit for the expanding field ofbiosensing and molecular diagnostics. Among many single-molecule techniques available today for biomarker sensing including fluorescence, force, electrochemical, spectroscopic, barcoding, and other techniques, fluorescence-based approaches are arguably the most widely used methods due to their high sensitivity, selectivity, and readily available fluorophore-labeling schemes for a wide variety of biomolecules. However, multiplexed imaging using fluorescence techniques has proven to be challenging due to the sophisticated labeling schemes often requiring multiple FRET (fluorescence resonance energy transfer) pairs and/or excitation sources, which lead to overlapping signals and complicate data analysis. Here, we describe a single-molecule FRET method that enables multiplexed analysis while still using only one FRET pair, and thus the described approach is a significant step forward from conventional FRET methods.

Amentoflavone, a potent natural matrix metalloproteinase 2 inhibitor

Gelatinase A (MMP-2) has been studied and proven to play a vital role in the intrusion and metastasis of cancer. Flavonoids influence on molecular and cellular functions of MMP-2 and thus a systematic investigation of flavonoids against the metalloproteolytic activity of MMP-2 has been performed in this study. A fluorescence resonance energy transfer method was used to investigate the inhibitory activities of various flavonoids. Flavone, flavonol and isobavachalcone derivatives showed their inhibitory activity against MMP-2. Surprisingly, the most effective inhibitor was Amentoflavone and its blocking function was superior to other flavonoids. Its IC50 value was 0.689 μM. An induced-fit docking study was carried out to survey its extraordinary activity. The binding mode of Amentoflavone is quite similar to that of (2 ∼ {S})-2-[2-[4-(4-methoxyphenyl) phenyl] sulfanylphenyl] pentanedioic acid complexed with MMP-9. Amentoflavone interacts with the functional zinc and catalytic residue, Glu202. Therefore, the docking study reasonably confirmed the strong inhibitory activity of Amentoflavone.

Cationic Conjugated Polymer Fluorescence Resonance Energy Transfer for DNA Methylation Assessment to Discriminate the Geographical Origins of Lonicerae japonicae flos.

The flavor and taste of Lonicerae japonicae flos (LJF) products are heavily influenced by geographical origin. Tracing the geographical origin is an important aspect of LJF quality assessment. Here, DNA methylation analysis coupled with chemometrics revealed that, in 10 CpG islands upstream of genes in the chlorogenic acid and iridoid biosynthetic pathways, DNA methylation differences appear close association with LJF geographical origin. DNA methylation status in these CpG islands was determined using the cationic conjugated polymer fluorescence resonance energy transfer method. As a result, LJFs from 39 geographical origins were classified into four groups corresponding to Northern China, Central Plain of China, Southeast China, and Western China, according to cluster analysis and principal component analysis. Our findings contribute to an understanding of the modulation of LJF taste and can assist in understanding how DNA methylation in LJF varies with geographical origin.

Methods to investigate nucleosome structure and dynamics with single-molecule FRET.

The nucleosome is the fundamental building block of chromatin. Changes taking place at the nucleosome level are the molecular basis of chromatin transactions with various enzymes and factors. These changes are directly and indirectly regulated by chromatin modifications such as DNA methylation and histone post-translational modifications including acetylation, methylation, and ubiquitylation. Nucleosomal changes are often stochastic, unsynchronized, and heterogeneous, making it very difficult to monitor with traditional ensemble averaging methods. Diverse single-molecule fluorescence approaches have been employed to investigate the structure and structural changes of the nucleosome in the context of its interactions with various enzymes such as RNA Polymerase II, histone chaperones, transcription factors, and chromatin remodelers. We utilize diverse single-molecule fluorescence methods to study the nucleosomal changes accompanying these processes, elucidate the kinetics of these processes, and eventually learn the implications of various chromatin modifications in directly regulating these processes. The methods include two- and three-color single-molecule fluorescence resonance energy transfer (FRET), single-molecule fluorescence correlation spectroscopy, and fluorescence (co-)localization. Here we report the details of the two- and three-color single-molecule FRET methods we currently use. This report will help researchers design their single-molecule FRET approaches to investigating chromatin regulation at the nucleosome level.

Employment of the Phage Cocktail as a Species-Specific Recognition Agent for Wide-Spectrum Detection of Bacterial Strains.

Phages have already been employed to detect bacteria because of their specific recognition capability and strong infectious activity toward their host. However, the reported single-phage-based techniques are inevitably restricted by false negative results that arose from extremely high strain specificity of phages. In this study, a cocktail composed of three Klebsiella pneumoniae (K. pneumoniae) phages was prepared as a recognition agent to broaden the recognition spectrum for detecting this bacterial species. A total of 155 clinically isolated strains of K. pneumoniae collected from four hospitals were adopted to test its recognition spectrum. A superior recognition rate of 91.6% for the strains was achieved due to the complementarity of the recognition spectra of the three phages composed of the cocktail. However, the recognition rate is as low as 42.3-62.2% if a single phage is employed. Based on the wide-spectrum recognition capability of the phage cocktail, a fluorescence resonance energy transfer method was established for detecting K. pneumoniae strains by employing fluorescein isothiocyanate labeled to the phage cocktail and Au nanoparticles labeled to p-mercaptophenylboronic acid as energy donors and acceptors, respectively. The detection process can be completed within 35 min, with a wide dynamic range of 5.0 × 102-1.0 × 107 CFU/mL. The application potential was verified by applying it to quantitate K. pneumoniae in different sample matrixes. This pioneer work opens an avenue for achieving wide-spectrum detection of different strains belonging to the same bacterial species with the phage cocktail.

The Potential of <i>Clerodendrum paniculatum</i> Leaves Fraction as a 3-Chymotrypsin-Like (3CL) Protease Inhibitor of SARS-CoV-2

We described the biological activity of the Clerodendrum paniculatum leaf fraction against the SARS-CoV-2 3-Chymotrypsin-like 3CL protease at the molecular level. This study applied LC-MS/MS to identify bioactive compounds from fractions, computational studies, and fluorescence resonance energy transfer (FRET) assays to ascertain their inhibitory activity. LC-MS/MS analysis of the three samples revealed that sample 1 contained 18 compound peaks. In samples 2 and 3, there were 23 and 25 compounds with different molecular weights, respectively. Docking's study identified that the alkaloids (komarovicine and roemerine) have lower binding energies than other metabolites and standard compounds, with values of -33.47 and -32.63 kJ/mol, respectively. Roemerine demonstrated excellent stability based on dynamic simulation results and confirmed its affinity for 3CL protease predicted by the MM-PBSA approach of -89.44 kJ/mol. The FRET method for testing 3CL protease activity revealed that sample 2 had an enzyme inhibitory activity of 94.3%, which was close to that of GC376 (98.19%). Meanwhile, samples 1 and 3 yielded satisfactory inhibition activity by 89.64% and 85.24%, respectively. The antiviral activity of C. paniculatum leaves was discovered for the first time by inhibiting the 3CL protease SARS-CoV-2, providing an excellent opportunity for its development as an anti-SARS-CoV-2.

A conformational switch in clathrin light chain regulates lattice structure and endocytosis at the plasma membrane of mammalian cells

Conformational changes in endocytic proteins are regulators of clathrin-mediated endocytosis. Three clathrin heavy chains associated with clathrin light chains (CLC) assemble into triskelia that link into a geometric lattice that curves to drive endocytosis. Structural changes in CLC have been shown to regulate triskelia assembly in solution, yet the nature of these changes, and their effects on lattice growth, curvature, and endocytosis in cells are unknown. Here, we develop a new correlative fluorescence resonance energy transfer (FRET) and platinum replica electron microscopy method, named FRET-CLEM. With FRET-CLEM, we measure conformational changes in clathrin at thousands of individual morphologically distinct clathrin-coated structures. We discover that the N-terminus of CLC repositions away from the plasma membrane and triskelia vertex as coats curve. Preventing this conformational switch with chemical tools increases lattice sizes and inhibits endocytosis. Thus, a specific conformational switch in the light chain regulates lattice curvature and endocytosis in mammalian cells.

A conformational switch in clathrin light chain regulates lattice structure and endocytosis at the plasma membrane of mammalian cells.

The nanoscale localizations, interactions, and conformations of endocytic proteins are key regulators of clathrin-mediated endocytosis. Although super-resolution microscopy has revealed the detailed organization of endocytic proteins, the resolution of super-resolution imaging cannot assess molecular interaction and conformational changes. Fluorescence resonance energy transfer (FRET) occurs when dyes are separated by less than 10 nm for most fluorescent proteins pairs. Thus, it can be used to map close-range molecular complexes and dynamics. Yet, to understand how endocytosis works, measurements from FRET must be correlated to the distinct stages of endocytosis. To accomplish this, we developed a new correlative lifetime-based FRET (FLIM-FRET) and platinum replica transmission electron microscopy (PREM) method, named FRET-CLEM. Here, FRET-based atomic distances can be mapped directly to individual cellular structures visualized in EM at the plasma membrane. We used this method to measure the conformational changes in clathrin light chain (CLC), a component of the clathrin triskelion and assembled clathrin lattice. CLC conformational changes have been proposed to regulate the assembly of clathrin in solution. However, CLC conformational changes and their effects on clathrin lattice growth, curvature, and endocytosis at the membrane of living cells are unknown. Using FRET-CLEM, we discovered that CLC undergoes a conformational switch as clathrin lattices curve. Preventing this conformational switch with acute chemical tools increased clathrin lattice sizes and inhibited endocytosis. Therefore, a specific conformational switch in CLC regulates lattice curvature and endocytosis in mammalian cells. These new correlative light and EM data will help develop a complete mechanistic model of endocytosis. More generally, FRET-CLEM can map molecular interactions and conformational changes at targeted membrane-associated proteins at identified cellular compartments including exocytic sites and neuronal synapses.

Aptamer-based DNA-catalyzed amplification strategy for sensitive fluorescence resonance energy transfer detection of Acinetobacter baumannii

Acinetobacter baumannii (A. baumannii) is a common pathogen that causes hospital-acquired infections and is resistant to a wide variety of antibiotics. Consequently, the rapid and highly sensitive detection of A. baumannii is required during the early stages of infection. Therefore, we developed a DNA-catalyzed amplification mechanism based on aptamers, combined with a novel fluorescence resonance energy transfer (FRET) method based on graphene oxide (GO), for the detection of A. baumannii. In the presence of A. baumannii, an aptamer bound to A. baumannii, releasing the template strand, which triggered an entropy-driven catalysis (EDC) reaction. One EDC product was then used as the catalyst for catalytic hairpin assembly (CHA) on a GO nanosheet. Finally, the GO released a huge amount of FAM-labeled DNA duplices, which could be detected with FRET. This strategy circumvented the extraction of nucleic acids and was easy to execute, with a detection time of ≤1.5 h. The detection of A. baumannii with this method ranges from 5 cfu/mL to 1 × 105 cfu/mL, with a detection limit of 1.1 cfu/mL. The method was sufficiently sensitive and specific to detect A. baumannii rapidly in cerebrospinal fluid. In summary, our strategy provides a new option for the early detection and point-of-care testing (POCT) of A. baumannii infections, allowing their earlier and more precise treatment.

Fine-tuning of aptamer complementary DNA for fluorescence resonance energy transfer assay

When developing a fluorescence resonance energy transfer (FRET) method to detect target analytes, nucleic acid aptamers with high affinity and specificity are often used as capture probes, and complementary DNA (cDNA) competing with the target analyte for binding with the aptamers is used to detect the analyte. During the development of such methods, the position, length, and base sequence of cDNA play a key role in improving detection sensitivity. Based on relevant research and understanding of the conformation and binding site of nucleic acid aptamers, we established a ratio-based FRET analysis method for an ochratoxin A (OTA)-oriented aptamer and its cDNAs by combining circular dichroism spectroscopy with molecular dynamic simulation. The influence of the position, length, and base sequence of cDNA on the fluorescence response ratio was explored, and a cDNA sequence was fine-tuned, designed, and optimized. Compared with the unoptimized cDNA, the fluorescence response ratio increased 13-fold, and the detection limit was as low as 3.9 nM. This study provides a general reference for the use of aptamers and cDNA to establish a method to determine analytes.

Fluorescence Resonance Energy Transfer (FRET) as a Spectroscopic Ruler for the Investigation of Protein Induced Lipid Membrane Curvature: Bacteriorhodopsin and Bacteriorhodopsin Analogs in Model Lipid Membranes.

Bacteriorhodopsin (bR) is a light-driven proton pump existing in the purple membranes (PM) of Halobacterium salinarum. The effects associated with changes in proton distribution (proton gradient, membrane electric potential) play a key role in ATPase stimulation. However, how the bioenergetic modulus (bR-PM-ATPase) functions remains unclear. One can find indications that hydrophobic matching and the curvature of the lipid membrane may form a functional link between bR and ATPase. To verify whether an interaction between bR and lipids can lead to curvature of the lipid membrane, a spectroscopic ruler, that is, a fluorescence resonance energy transfer (FRET) tool, was used. The distances from fluorescent lipid probes [octadecyl rhodamine B chloride (RhB), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 16-(9-anthroyloxy) palmitic acid (16AP), and hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH), to the retinal chromophore of bR incorporated into phospholipid vesicles, were measured. The incorporation of retinal analogues with changed shape and/or altered electronic properties into the binding site of a bR or bR mutant were used to strengthen the feedback between the protein surrounding and chromophore. The experiments were performed with wild-type and D96N-mutated bR carrying retinal or 14-(12-,10-, 13,14-bi-) fluororetinal. As far as it is known, this is the first time that results obtained by the FRET method show that bR can induce a change in lipid structure interpreted as hydrophobically induced curving of the lipid membrane. Evidence was provided that the chromophore contributed to this effect. The extent of contribution was dependent on the chromophore structure in close vicinity to the place of its link with opsin. The implications of these findings for bR-PM-ATPase module functioning are also discussed.

Oligomerization of the HBV/HDV functional receptor NTCP expressed in Sf9 insect cell

BackgroundSodium taurocholate co-transportering polypeptide (NTCP, SLC10A1) is a vital bile acid transporter and the functional receptor of hepatitis B and D virus. The oligomerization of NTCP is important for the structural study of its interaction with HBV preS1 peptide. MethodsRecombinant NTCPs were expressed in Sf9 host cell using baculoviruses. Function of recombinant NTCP was verified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. A quantitative fluorescence resonance energy transfer (FRET) method was established to analyze the interaction between NTCP wild type (WT) and mutants. Co-immunoprecipitation (Co-IP) was used to test the interaction between NTCP variants. ResultsSub-cellular location of recombinant NTCPs varies with the modification of NTCP. Bands of monomer, dimer and oligomers were shown in gel analysis of NTCP. Significant FRET was observed between cyan florescence protein (CFP) tagged NTCP and yellow florescence protein (YFP) tagged NTCP. FRET efficiency between CFP- and YFP-NTCP S267F mutants was lower than WT. Co-IP results showed that S267F interacts with WT NTCP when co-expressed in cell. ConclusionDimer is the predominant form of NTCP expressed in Sf9 when solubilized with detergent. FRET and Co-IP analysis support that NTCP forms oligomers in Sf9 cell. General significanceOur results showed that NTCP formed oligomers in Sf9 cell. Meanwhile the FRET analysis of NTCP variants further elucidated the molecular mechanism of NTCP oligomerization.

Precise Quantification of Molybdate In Vitro by the FRET-Based Nanosensor 'MolyProbe'.

Molybdenum (Mo) is an essential trace element in all kingdoms of life. Mo is bioavailable as the oxyanion molybdate and gains biological activity in eukaryotes when bound to molybdopterin, forming the molybdenum cofactor. The imbalance of molybdate homeostasis results in growth deficiencies or toxic symptoms within plants, fungi and animals. Recently, fluorescence resonance energy transfer (FRET) methods have emerged, monitoring cellular and subcellular molybdate distribution dynamics using a genetically encoded molybdate-specific FRET nanosensor, named MolyProbe. Here, we show that the MolyProbe system is a fast and reliable in vitro assay for quantitative molybdate determination. We added a Strep-TagII affinity tag to the MolyProbe protein for quick and easy purification. This MolyProbe is highly stable, resistant to freezing and can be stored for several weeks at 4 °C. Furthermore, the molybdate sensitivity of the assay peaked at low nM levels. Additionally, The MolyProbe was applied in vitro for quantitative molybdate determination in cell extracts of the plant Arabidopsis thaliana, the fungus Neurospora crassa and the yeast Saccharomyces cerevisiae. Our results show the functionality of the Arabidopsis thaliana molybdate transporter MOT1.1 and indicate that FRET-based molybdate detection is an excellent tool for measuring bioavailable Mo.

Synthesis, crystal structure, BSA binding and antibacterial studies of Ni(II) complexes derived from dithiocarbazate based ligands

A series of nickel(II) bis-complexes of the type [NiL2] C1-C4, where L is a tridentate (NNS) ligand have been isolated and characterized by conductivity studies, elemental analyses, IR and UV–Vis spectral measurements. The single crystal X-ray diffraction studies on [Ni(L1)2] C1 and [Ni(L3)2] C3 reveal that the deprotonated tridentate (NNS) ligands are meridionally coordinated to ascertain a distorted octahedral coordination geometry around nickel(II). The [Ni(L1)2] complex crystallizes in the monoclinic space group P21/c with a = 9.5009(4) Å, b = 13.3354(4) Å, c = 16.7988(7) Å, α = 90 °, β = 98.946(4)°, γ = 90° and Z = 4 while [Ni(L3)2] crystallizes in the monoclinic space group P21/c with a = 16.1362(19), b = 20.2502(11), c = 8.5462(5), α = 90 °, β = 102.778(8)°, γ = 90 ° and Z = 4. The antibacterial potencies of the compounds C1-C4 were estimated on the growth of P. aeruginosa, a Gram-negative bacterium, and S. mutans, a Gram-positive bacterium by broth culture and biofilm formation assay. The interaction between bovine serum albumin (BSA) was investigated using fluorescence quenching techniques. The results revealed that the fluorescence quenching of BSA is through a static quenching process. The binding constants and the number of binding sites were determined using fluorescence quenching data. The binding distance between BSA and Ni-complexes has been determined by using the fluorescence resonance energy transfer method.

P133 Point-of-care measurement of infliximab and adalimumab trough levels by the fluorescence resonance energy transfer (FRET) assay

Abstract Background Therapeutic drug monitoring (TDM) is the clinical practice of measuring serum drug concentrations to guide clinical decision-making. In IBD patients, it is applied mainly in infliximab (IFX) and adalimumab (ADL) therapy. TDM is either performed reactively in response to disease activity, or proactively in the absence of symptoms. The aim of the present study was to compare two analytical methods for the detection of trough infliximab and adalimumab levels (TL): point-of-care (POCT) fluorescence resonance energy transfer (FRET) method, and the enzyme- linked immunosorbent immunoassay (ELISA). Methods TL IFX were measured in n=24 and TL ADL in n= 21 blood samples. For ELISA, standard peripheral venous samples were used, and for POCT FRET measurements, capillary finger puncture was performed. Two assay results were compared evaluating the regression relationship using Spearman’s correlation coefficient and the Bland-Altman plots. Cohen’s kappa analysis was used to compare the degree of qualitative agreement between the two data sets. Repeatability was assessed by the difference of the repeated measurements, their means and standard deviations. Results The high degree of correlation was found between the measured values, both qualitative and quantitative (p < 0.0001 for both IFX and ADL), either expressed by the Spearman’s correlations, or by Bland-Altman analysis. Cohen’s weighted kappa 0.903 for IFX and 0.834 for ADL showed substantial/almost perfect agreement of qualitative results. Closeness of the agreement of successive measurements was very acceptable for both TLs of both drugs. Conclusion Point-of-care detection of IFX and ADL trough levels by FRET is accurate, comparable to the reference method, analysis is sufficiently consistent. For clinical sites with a proactive approach to TDM, FRET may be a useful tool for decision-making regarding individualization of biologic therapy. Acknowledgements: We thank ProciseDX company for the ProciseDX analyzer rental, and for providing the laboratory diagnostics.

Diagnosis of Thrombotic Thrombocytopenic Purpura by ADAMTS13 Activity Quantification.

Rapid quantification of ADAMTS13 activity in plasma is essential for establishing a diagnosis of thrombotic thrombocytopenic purpura (TTP); a rare, but potentially lethal disorder. The current methods for quantitating ADAMTS13 activity are manual and only available at specialized laboratories, which often results in initiation of specific treatments long before a diagnosis of TTP is established. We compared the performance of the HemosIL, a novel and rapid automated method, and the current standard FRET (fluorescence resonance energy transfer) method in quantitating ADAMTS13 activity using 706 consecutive plasma samples collected over a period of 14 years. The clinical accuracy of both methods was further examined using 212 diagnostic samples. The correlation between the FRET and HemosIL methods in all 706 samples and in the 212 diagnostic samples was excellent (Pearson's r of 0.919 and 0.912, respectively). Both methods displayed a high degree of clinical accuracy using the current cutoff of ADAMTS13 activity <0.10 kIU/L (<10%) as diagnostic for TTP: the area under the curve (AUC) was 97.7% for the FRET method and 99.5% for the HemosIL method. When applying a lower cutoff (ADAMTS13 activity <0.05 kIU/L or <5%), the diagnostic accuracy of the HemosIL method increased further (AUC = 99.7%). A novel, rapid method for ADAMTS13 quantification is comparable to the more laborious FRET method in patients with possible TTP. A rapid analysis available in the acute setting assessing patients with possible TTP allows for improved care and optimized treatment of a life-threatening condition.

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.

Driving co-precipitation of hydrophobic drugs in water by conjugating alkyl chains

Co-precipitated nanoaggregates (NAs) of small-molecule hydrophobic drugs have been widely used as carrier-free drug co-delivery systems, but how to construct stable NAs for potential co-delivery of combination drugs has been remaining unclear. Herein, we performed such investigation using our previously reported fluorescence resonance energy transfer (FRET) method, which was based on the fluorescent hydrophobic drugs of camptothecin (CPT), curcumin (CUR), and their lipophilic derivatives containing different alkyl chains. It is shown that unmodified CPT and CUR failed to form stable co-assembling NAs. Increasing hydrophobicity of one drug by conjugating an alkyl chain could promote the co-assembling ability of CPT and CUR, but the resulting NAs were very unstable and undergone a burst disassembly in the plasma. Only simultaneously enhancing the hydrophobicity of CPT and CUR could obtain stable co-assembling NAs. These results highlighted the co-assembly and disassembly of NAs formed by various hydrophobic drugs, which can effectively guide the design of co-assembling small-molecule drug co-delivery systems with high stability to resist the dilution of blood after intravenous injection.

Fluorescence Detection of Glutathione Using N-Doped Graphene Quantum Dots–MnO2 Nanoarchitecture

We build a novel fluorescence resonance energy transfer (FRET) method based on N-doped graphene quantum dots (NGQDs)–MnO2 nanocomposite for rapid, sensitive detection of glutathione (G SH) levels in human serum. In this strategy, MnO2 nanosheets on the NGQDs surface serve as a quencher. NGQDs fluorescence can make a recovery by the addition of GSH, which can reduce MnO2 to Mn2+, and thus the GSH can be monitored. The MnO2 platform affords minimal background and high sensitivity for detecting GSH in this proposed scheme. Meanwhile, relevant fluorescence on–off–on processes were monitored, and the sensing mechanism was explored.