Optimization of expression conditions of dengue virus NS2B-NS3 protease in E.coli and determination of enzyme activity / 中国生物制品学杂志

@#Objective To express dengue virus(DENV)NS2B-NS3 protease in E.coli,optimize the expression conditions and determine the enzyme activity,so as to lay a foundation of screening and discovering of lead compounds targeting DENV.Methods Codon-optimized NS2B-NS3 gene was inserted into pET-28a vector to construct recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3,which was transformed E.coli Rosetta(DE3)competent cells and induced by IPTG to express NS2B-NS3 protease. The optimal expression conditions of NS2B-NS3 protease in E.coli were determined by optimizing induction length,induction temperature and IPTG concentration. NS2B-NS3 protease was isolated and purified by HisTrap~(TM) affinity chromatography column and measured for the protease activity by fluorescence resonance energy transfer(FRET)assay.Results The recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3 was constructed correctly as identified by restriction analysis(NheⅠ/XhoⅠ)and sequencing. The optimal expression conditions of NS2BNS3 protease in E.coli were as follows:induction temperature of 20 ℃,induction length of 10 h and IPTG concentration of0. 2 mmol/L. The purified NS2B-NS3 protease showed a purity of more than 90% with a exhibited a of 20 mg/L,which bound to mouse monoclonal antibody against His-tag specifically and had good hydrolytic activity with a specific activity of 16. 111 U/mg,a K_m of 16. 46 μmol/L and a k_(cat) of 0. 028/s.Conclusion DENV NS2B-NS3 protease with high purity and activity was successfully prepared,which laid an experimental foundation of the establishment of high-throughput screening model for inhibitors targeting NS2B-NS3 protease.

Palmitoylation in Regulating Fyn Kinase Activity Based on FRET Imaging / 医用生物力学

Objective To investigate the molecular mechanism of palmitoylation modification in regulating the activity of non-receptor tyrosine kinase Fyn. Methods The intracellular Fyn activity was detected by applying fluorescence resonance energy transfer (FRET) technology, and the mechanism was investigated by combining with Fyn palmitoylation deficiency and C-terminal Src kinase ( CSK ) plasmid co-expression. ResultsExperimental data showed that single loss of either of ( C3, C6) palmitoylation sites resulted in higher Fyn activity, and C6 seemed more significant. It is known that CSK membrane translocation occurred after activation. FRET assay confirmed that CSK could down-regulate the activity of Fyn in cells, but could not effectively regulate the activity of Fyn(GSS) with the loss of palmitoylation sites. Conclusions The results in this study support the hypothesis on Fyn regulation by spatial localization, namely, non-palmitoylated Fyn (GSS) is less effective in the inhibitory regulation by CSK on cell membrane, thus promoting constitutive high activity expression

Progress in research on fluorescence resonance energy transfer technology based on upconversion nanomaterials for biological detection / 中国生物制品学杂志

@#Upconversion nanoparticles(UCNPs)doped with rare earth elements have advantages in biose-nsing because of their good fluorescence stability,biocompatibility and avoidance of background fluorescence. Therefore,fluorescence resonance energy transfer(FRET)system based on upconversion particles(UCNPs based FRET)has been widely used in biological detection. This paper reviews the application and prospect of UCNPs based FRET in biological detection of biotoxins,hormones,proteins,nucleic acids,bacteria,and so on.

Influence of selection of domain shape on parameters of microdosimetric kinetic model / 中华放射医学与防护杂志

Objective:To calculate the single-event dose-averaged specific energy of particles delivered in spherical domains based on the track structure model and using triple integration, and to investigate the influence of the domain shape on the key model parameters of microdosimetric kinetic model (MKM) and its corresponding physical significance.Methods:The domains are assumed to be cylinders and spheres, respectively. With α 0, domain radius, rd, and nucleus radius, Rn, as undetermined coefficients, the nuclear charge numbers, kinetic energies and their corresponding LETs of three kinds of charged particles ( 3He, 12C, 20Ne) as independent variables, D10 as dependent variable, the mean value of squared residuals, J2, between the D10 calculated values and D10 experimental values as the optimization objective, the final fitting values of the above undetermined coefficients of human salivary gland (HSG) cells and Chinese hamster lung (V79) cells obtained after iteration by the robust least square method are the optimal model parameter values of MKM. Results:For HSG cells, cylindrical domain: α 0=0.073/Gy, rd=0.29 μm, Rn=4.1 μm, J2=0.039 7 Gy 2; spherical domain: α 0=0.023/Gy, rd=0.29 μm, Rn=4.4 μm, J2=0.039 3 Gy 2; For V79 cells, cylindrical domain: α 0=0.114/Gy, rd=0.25 μm, Rn=3.8 μm, J2=0.097 4 Gy 2; spherical domain: α 0=0.095/Gy, rd=0.26 μm, Rn=4.1 μm, J2=0.096 9 Gy 2. Conclusions:For the same type of cells, cylindrical and spherical domains were selected respectively, and there are significant differences in MKM parameters obtained by fitting. The fitting values of the domain radius, rd of the two shapes of domains show no significant difference, while the fitting values of α0 of spherical domains are smaller than those of cylindrical domains, the fitting values of nucleus radius, Rn, of spherical domain are larger than those of cylindrical domains, closer to the nucleus radius observed by fluorescence microscopy. In the low LET (<20 keV/μm) region, D10 calculated according to the parameters of the two different shapes of domains are different, so the selection of the domain shape will cause differences in the relative biological effectiveness(RBE) calculation of proton in the region near Bragg peak.

Determination of methylene blue release in plasma virus inactivation bag by fluorescence energy transfer method / 中国输血杂志

【Objective】 To establish a simple, economical and rapid method for the determination of methylene blue (MB) release in virus inactivation bag. 【Methods】 Based on the fluorescence energy transfer between MB and BSA-stabilized gold nanoclusters (BSA-AuNCs), the standard curve of MB determination was established by measuring the fluorescence quenching degree of MB to BSA-AuNCs in different concentrations to conduct the determination of MB release in virus inactivation bag. 【Results】 There was a good linear relationship between the MB concentration (cMB) and the fluorescence quenching degree of BSA-AuNCs[ (I0-I)/I0=0.018cMB+ 0.021(r=0.996)] when the fluorescence emission wavelength was about 620 nm and the cMB was in the range of (0.9-36) μmoL/L. The recovery of MB was 98.00% -101.95 % when applied to determine MB at high, medium, and low concentrations, the obtained intra-day variation coefficients were 0.73%, 0.81% and 0.77% respectively, and the obtained inter-day variation coefficients were 3.92%, 3.81%, and 4.73% respectively. There was no significant difference between the results measured by this method and those measured by combination of solid-phase extraction and spectrophotometry(P>0.05). 【Conclusion】 The fluorescence energy transfer method could achieve simple and rapid determination of MB release in virus inactivation bag with accurate and reliable results.

XCR1 Forms a Heterodimer with CXCR4 and Regulates Its Internalization / 中国生物化学与分子生物学报

Chemokine signal pathways are important for the regulation of tumour metastasis. Chemokine receptors CXCR4 (C-X-C chemokine receptor type 4) and XCR1 (chemokine XC receptor 1) are involved in the metastasis of breast cancer, while the interaction between them remains unclear. Here we first identified the interaction between CXCR4 and XCR1 based on membrane protein yeast two-hybrid assays. Bioluminescence resonance energy transfer (BRET) showed that XCR1 could competitively bind to CXCR4 to form a heterodimer (P < 0.01). Results of wound healing assays via transient transfection of XCR1 and CXCR4 into HEK293 cells showed that 41.55% of the migration area rate in the co-transformation group was lower than 58.75% in the CXCR4-alone group after adding 30 nmol/L S D F-β. The co-expression of XCR1 inhibited the cellular motility, possibly mediated by the SDF-1β (stromal cell-derived factor 1)/CXCR4 signal pathway (P < 0.05). Furthermore, CXCR4 on the cell surface after co-expression of XCR1 in CXCR4-EGFP transgenic HEK293 cells was detected by flow cytometry. And the result suggested that XCR1 could accelerate the internalization of CXCR4 into the heterodimer induced by 30 nmol/L SDF-1β (P<0.05), which increased the internalization rate from 14.38% to 64.10%. Finally, the phosphorylation of Akt and ERK, which were involved in cell proliferation and migration, respectively, were examined. After 10 minutes of SDF-1β stimulation, ERK phosphorylation in the CXCR4-alone group showed a 3.59-fold increase, whereas the increase of ERK phosphorylation in the co-transfected group was only 2.08-fold. Interestingly, heterodimer formation reduced the phosphorylation level of ERK and shortened the activation time, whereas the phosphorylation level of Akt remained unchanged. Collectively, our findings revealed the hetero-dimerization of CXCR4 and XCR1 and its effects on CXCR4-mediated cellular motility, receptor internalization, and ERK pathway phosphorylation. Therefore, XCR1-targeting drugs could be candidates for cross-desensitization of CXCR4 and might represent a possible option for inhibiting breast cancer metastasis.

Histone Variant H2A.Z Enhances the Stability of Nucleosomes under the Effect of Salt Ions / 中国生物化学与分子生物学报

In eukaryotes, the basic structural unit of chromatin is the nucleosome, and genomic DNA iscompressed in chromatin. The presence of nucleosomes usually inhibits the biological processes that occuron the DNA templates, such as transcription, replication, repair, and recombination,. The histonevariant H2A.Z can regulate the structure of chromatin and affect the transcription process of genes, but itsdetailed regulation mechanism remains unclear. In this paper, the method of Förster resonance energytransfer (FRET) was used to detect the influence of sodium chloride, potassium chloride, manganesechloride, calcium chloride and magnesium chloride on the dissociation of nucleosomes. Then, the stabilitydifferences between the nucleosomes containing the histone variant H2A. Z and the conventionalnucleosomes were compared under the action of salt ions. Widom 601 DNA sequence was labeled withdual fluorescence Cy3 and Cy5, and the dissociation of nucleosomes was reflected by the change offluorescence signal value. FRET results showed that the dissociation speed of nucleosomes containing thehistone variant H2A. Z under the action of sodium chloride, potassium chloride, manganese chloride, calcium chloride and magnesium chloride is slower than that of canonical nucleosomes, and the influenceof calcium chloride, manganese chloride and magnesium chloride on dissociation is more obvious thansodium chloride, potassium chloride. The results of electrophoresis analysis showed that the dissociationrate of nucleosomes containing histone variant H2A. Z was significantly lower than that of canonicalnucleosomes at 75℃. Fluorescence thermal shift (FTS) was used to further analyze the stability ofnucleosomes containing histone variant H2A.Z. It was found that the fluorescence signals of the two typesof nucleosomes showed two distinct growth periods, and the fluorescence signals generated in thedissociation process of the two types of nucleosomes showed two distinct growth periods. The temperaturecorresponding to the first increasing period of fluorescence signal in the dissociation process of H2A. Z-containing nucleosome is significantly higher than that in the dissociation process of the canonicalnucleosome, which indicated that the dissociation and denaturation temperature of the H2A.Z / H2B dimerin the nucleosome is higher than that of the canonical H2A / H2B dimer, and the H2A. Z-containingnucleosomes have high thermal stability. The results indicated that the structure of nucleosomes containingthe histone variant H2A.Z is more stable than that of canonical nucleosomes.

Modulating effects of RAMPs on signaling profiles of the glucagon receptor family

Receptor activity-modulating proteins (RAMPs) are accessory molecules that form complexes with specific G protein-coupled receptors (GPCRs) and modulate their functions. It is established that RAMP interacts with the glucagon receptor family of GPCRs but the underlying mechanism is poorly understood. In this study, we used a bioluminescence resonance energy transfer (BRET) approach to comprehensively investigate such interactions. In conjunction with cAMP accumulation, Gα q activation and β-arrestin1/2 recruitment assays, we not only verified the GPCR-RAMP pairs previously reported, but also identified new patterns of GPCR-RAMP interaction. While RAMP1 was able to modify the three signaling events elicited by both glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), and RAMP2 mainly affected β-arrestin1/2 recruitment by GCGR, GLP-1R and glucagon-like peptide-2 receptor, RAMP3 showed a widespread negative impact on all the family members except for growth hormone-releasing hormone receptor covering the three pathways. Our results suggest that RAMP modulates both G protein dependent and independent signal transduction among the glucagon receptor family members in a receptor-specific manner. Mapping such interactions provides new insights into the role of RAMP in ligand recognition and receptor activation.

Assessment of biological dose of proton radiotherapy based on different relative biological effect models / 中华放射医学与防护杂志

Objective:To assess proton biological dose with using two kinds of relative biological effect models and to compare them with traditional clinical proton biological dose ( Dose1.1). Methods:Based on Particle simulation tools(TOPAS), physical dose, LET d and LET t were calculated in water phantom and two anthropomorphic phantoms (brain and prostate tumors) respectively. Then DoseLET d and DoseLET t were calculated according to different relative biological effect models, an RBE was 1.1 in traditional clinical proton biological dose calculation. Three kinds of biological doses were compared in the water phantom. To quantify the differences between three method in anthropomorphic phantoms, three points ( D1, D2, D3) were selected according to the physical dose to compare the biological dose. Results:DoseLET d and DoseLET t in water phantom showed the same trend with water depth and both of them were higher than Dose1.1 at the end of proton beam range. The maximal difference between DoseLET d and DoseLET t in the anthropomorphic phantoms was 10.08 cGy, where the relative difference was less than 5%. When DoseLET d and DoseLET t were compared with Dose1.1, the maximal differences in brain tumor target were 71.97 cGy and 61.91 cGy respectively, where the relative differences were less than 25%. The maximal differences in prostate tumor target were 25.95 and 19.96 cGy respectively, where the relative differences were less than 12%. However, the differences outside the target were very small, where the maximal differences in brain and prostate tumors were 5.99 cGy and 9.92 cGy respectively, and the relative differences were less than 5%. Conclusions:Biological doses calculated by two method are of little difference in both water and anthropomorphic phantoms, however, large differences were observed when they were compared with the traditional clinical proton biological dose especially in the high dose area.

Optimization of expression conditions and determination the proteolytic activity of codon-optimized SARS-CoV-2 main protease in Escherichia coli / 生物工程学报

The main protease (Mpro) of SARS-CoV-2 is a highly conserved and mutation-resistant coronaviral enzyme, which plays a pivotal role in viral replication, making it an ideal target for the development of novel broad-spectrum anti-coronaviral drugs. In this study, a codon-optimized Mpro gene was cloned into pET-21a and pET-28a expression vectors. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells and the expression conditions were optimized. The highly expressed recombinant proteins, Mpro and Mpro-28, were purified by HisTrapTM chelating column and its proteolytic activity was determined by a fluorescence resonance energy transfer (FRET) assay. The FRET assay showed that Mpro exhibits a desirable proteolytic activity (25 000 U/mg), with Km and kcat values of 11.68 μmol/L and 0.037/s, respectively. The specific activity of Mpro is 25 times that of Mpro-28, a fusion protein carrying a polyhistidine tag at the N and C termini, indicating additional residues at the N terminus of Mpro, but not at the C terminus, are detrimental to its proteolytic activity. The preparation of active SARS-CoV-2 Mpro through codon-optimization strategy might facilitate the development of the rapid screening assays for the discovery of broad-spectrum anti-coronaviral drugs targeting Mpro.

Estabishment and evaluation of a new method for rapid detection of CA16 hand, foot and mouth disease pathogens based on fluorescence resonance energy transfer technique / 吉林大学学报(医学版)

Objective: To establish a new method for rapid detection of Coxsachie virus A16 (CA16) hand, foot and mouth disease pathogens based on fluorescence resonance energy transfer (FRET) technique, to evaluate the detection effect and to make the method to meet the requirements of large sample size detection during the outbreak of disease. Methods: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and bicinchoninic acid (BCA) protein assay were used to identify the purity of CA16 chicken yolk antibody (CA16-IgY) and the protein level. Indirect enzyme-linked immunosorbent assay (iELISA) was used to detect the titer and specificity of anti-CA16 IgY antibody. The size, morphology and characterization of gold nanoparticles (AuNPs) and their biological probes (IgY-AuNPs) were determined by UV-visible spectroscopy (UV-Vis), infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The CA16 detection system was constructed based on FRET technique. The sensitivity and specificity of the detection method and clinical sample detection were evaluated by optimizing the IgY-AuNPs concentration, sodium chloride (NaCl) dosage, fluorescence recovery time and other indicators. Results: The CA16-IgY had high purity, the titer was 1:128 000, the average protein level was 12. 15 mg · L-1, and CA16-IgY had good specificity. The results of UV-Vis, FTIR and TEM of AuNPs and IgY-AuNPs showed that IgY was successfully labeled onto the surface of AuNPs, which suggested that IgY-AuNPs could specially recognize CA16 was successfully prepared by electrostatic self-assembly. The CA16 detection system was constructed based on FRET technology, after optimization of the detection system, the optimal dosage of IgY-AuNPs was determined to be 0.52 X 10-3 g · L-1, the optimal dosage of NaCl was 40 μL and the optimal fluorescence recovery time was 90 min. The standard curve of the established detection method was I525 ntu= 15. 452 IgC-9. 746, R2 = 0.993 2, the detection limit was as 1 X 104 PFU · ml-1. Compared with qRT-PCR, the agreement rate reached 93. 75%. Conclusion: A new rapid detection method for CA16 hand, foot and mouth disease pathogens is successfully established, which can be applied to laboratory and clinical tests.

Progress in fluorescent visualization techniques for neurotransmitter detection / 生物工程学报

Neurotransmitters play an important role in nervous system. Temporal and spatial changes of neurotransmitter distribution are crucial to information processing in neural networks. Biosensors that can visually monitor neurotransmitters are one of the vital tools to explore a variety of physiological and pathological activities. This article reviews recent advances in monitoring neurotransmitters with high temporal and spatial resolution, and introduces the latest fluorescent imaging methods for typical neurotransmitters, including glutamate, dopamine, γ-aminobutyric acid and acetylcholine. The article also summarizes the basic principles, advantages and disadvantages of various visually detection methods, and provides systematic suggestions for designing neurotransmitter sensors with high temporal and spatial resolution.

Advances in the research and applications of orange fluorescent protein / 生物工程学报

Fluorescent proteins can be used as probes to investigate intercellular molecular interactions and trace the pathway of specific metabolites, thus providing a detailed and accurate description of various metabolic processes and cellular pathways in living cells. Nowadays, the existing fluorescent proteins cover almost all spectral bands from ultraviolet to far-red. These fluorescent proteins have been applied in many fields of bioscience with the help of high-resolution microscopy, making great contributions to the development of biology. It is generally agreed that orange fluorescent proteins refer to the fluorescent proteins at the spectral range of 540-570 nm. In recent years, researches on orange fluorescent proteins have made great progress, and they have been widely applied in the field of biology and medicine as reporter protein and fluorescence resonance energy transfer as fluorescent receptor. This paper reviews the studies in the field of orange fluorescent proteins over the last 15 years, with the special focus on the development and application of orange fluorescent proteins to provide the basis for the future studies.

A carbon nanoparticle-peptide fluorescent sensor custom-made for simple and sensitive detection of trypsin / 药物分析学报

Herein, we report a novel sensor to detect trypsin using a purpose-designed fluorescein-labelled peptide with negatively charged carbon nanoparticles (CNPs) modified by acid oxidation. The fluorescence of the fluorescein-labelled peptide was quenched by CNPs. The sensor reacted with trypsin to cleave the peptide, resulting in the release of the dye moiety and a substantial increase in fluorescence intensity, which was dose-and time-dependent, and trypsin could be quantified accordingly. Correspondingly, the biosensor has led to the development of a convenient and efficient fluorescent method to measure trypsin activity, with a detection limit of 0.7μg/mL. The method allows rapid determination of trypsin activity in the normal and acute pancreatitis range, suitable for point-of-care testing. Furthermore, the applicability of the method has been demonstrated by detecting trypsin in spiked urine samples.

In vivo fate study of drug nanocarriers: the applications of environment-responsive fluorescent dyes / 药学学报

The in vivo fate is a crucial factor that governs the successful translation of nanoformulations. However, one of the current biggest challenges is with the real-time monitoring of the body of the nanoparticles themselves. Conventional radioactive or fluorescent probes give signals even after they are disassociated from the particle matrix, generating interference to bioimaging and leading to misjudgment of results. Environment-responsive fluorescent dyes are regarded as promising tools due to signal switching in response to the changes in the environment. Currently, there are three categories of dyes in bioimaging of nanoparticles based on Förster resonance energy transfer (FRET), aggregation-induced emission (AIE) and aggregation-caused quenching (ACQ). They have similar characteristics that strong fluorescence is emitted when they are embedded in the matrix of nanocarriers, whereas the fluorescence quenches upon release from the matrix due to dissociation of nanocarriers. The fluorescence switching reflects the existing status of the nanocarriers and therefore helps to interpret the in vivo behaviors. FRET and AIE probes have been widely used in elucidating the interactions between nanoparticles and cell models. However, they show intrinsic defects in studying in vivo fate of nanoparticles. ACQ-based dyes are sensitive to water, a universal factor in the biological environment. Therefore, with the help of bioimaging equipment, the in vivo trafficking process of nanoparticles can be unraveled. This review article tends to provide an overview on the rationale, pros and cons and applications of the three categories of environment-responsive fluorescent dyes in the investigation of the in vivo fate of nanocarriers.

A one-step specific assay for continuous detection of sirtuin 2 activity

Sirtuins (SIRTs) are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases with diverse physiological functions. A variety of small molecules have been developed to interrogate the physiological function of SIRTs. Therefore, it is desirable to establish efficient and convenient assays to screen SIRTs modulators. In this study, we designed a series of fluorescent nonapeptide probes derived from substrates of SIRT1-SIRT3. Fluorescence increment of these probes is based on SIRT-mediated removal of the acyl side chain with fluorophore, which makes this system free of lysine-recognizing protease. Comparing the reaction of these fluorescent nonapeptide substrates with SIRT1-SIRT3 and SIRT6, it was confirmed that this assessment system was the most suitable for SIRT2 activity detection. Thus, SIRT2 was used to modify substrates by truncating the amino acids or lysine side chain of nonapeptide. Finally, two specific and efficient fluorescent probes for SIRT2, ne-D9 and ne-K4a, were developed. Evaluation of the results revealed that ne-K4a based assay was more suitable for modulators screening , while the other specific substrate ne-D9 was stable in cell lysate and could detect the activity of SIRT2 in the same. In summary, this study presents a novel strategy for detecting SIRT2 activity and in cell lysate.

Evaluation method for compatibility of compounds in fluorescence resonance energy transfer model to screen β-secretase inhibitors / 中国药理学与毒理学杂志

OBJECTIVE To develop a method to evaluate the compatibility of compounds in the fluorescence resonance energy transfer (FRET) model for β-secretase (BACE1) inhibitor screening.METHODS Two commercially available BACE1 inhibitor screening systems based on FRET were selected to evaluate the BACE1 inhibitory activities of (-)-epigallocatechin-3-gallate (EGCG) and Compound 1 according to the supplier's protocol.The inhibitory rates and slopes of the catalytic curves of the inhibitors were calculated.The effect of inhibitors on the fluorescence intensity of the systems were quantitatively calculated and the comparatively evaluated.RESULTS EGCG,a reported non-competitive inhibitor of BACE1,directly induced the reduction of fluorescence intensity of one of the systems.The slope of the line with the addition of EGCG (10.8±2.6) conformed to that of the line of EGCG inhibition (10.2±3.4),which indicated that EGCG was a pseudo-positive inhibitor of BACE1.Compound 1 had little effect on the fluorescence intensity of the systems,so the inhibitory activity of Compound 1 was confirmed.The compounds which showed inhibitory activity in preliminary screening should be checked in the blank control without BACE1 to calibrate the effect of compound on the system fluorescence intensity.The applicability of the tested compounds in the screening system could thus be evaluated to prevent pseudo-positive results.CONCLUSION This fluorescence calibration method with compound control can be universally used for assays based on FRET theory to evaluate the applicability of tested BACE1 inhibitors.

Insight into the interaction of inhaled corticosteroids with human serum albumin:A spectroscopic-based study / 药物分析学报

It is well known that the safety and efficacy profile of an inhaled cortocosteroid (ICS) is influenced by the pharmacokinetic properties and associated pharmacodynamic effects of the drug. Freely circulating, protein unbound, and active ICS can cause systemic adverse effects. Therefore, a detailed investigation of drug-protein interaction could be of great interest to understand the pharmacokinetic behaviour of corticosteroids and for the design of new analogues with effective pharmacological properties. In the present work, the interaction between some corticosteroids and human serum albumin (HSA) has been studied by spectroscopic approaches. UV–Vis spectroscopy confirmed that all the investigated corticos-teroids can bind to HSA forming a protein-drug complex. The intrinsic fluorescence of HSA was quenched by all the investigated drugs, which was rationalized in terms of a static quenching mechanism. The thermodynamic parameters determined by the Van't Hoff analysis of the binding constants (negativeΔH andΔS values) clearly indicate thathydrogen bonds and van der Waals forces play a major role in the binding process between albumin and betamethasone, flunisolide and prednisolone, while hydrophobic forces may play a major role in stabilizing albumin-triamcinolone complexes.

Detection of Arginine by AuNPs / CQDs Nanoprobes Based on Fluorescence Resonance Energy Transfer Effect / 分析化学

A novel method for rapid detection of arginine based on fluorescence resonance energy transfer effect (FRET) between carbon quantum dots ( CQDs) and gold nanoparticles ( AuNPs) was developed. Firstly, the CQDs with excellent fluorescence properties were synthesized by one-step microwave assisted method. The AuNPs/ CQDs composites were characterized and their quenching mechanism was analyzed. Then the amount of AuNPs/ CQDs, the pH value and the reaction time were optimal. Under the optimum conditions, the fluorescence system was used to detect the content of arginine, showing a good linear relationship ( R2 = 0. 993 ) between fluorescence intensity and concentration of arginine in the range of 0. 1-10. 0 μmol/ L, and the detection limit was 5. 8 nmol/ L. Finally, the content of arginine in grape juice was determined by this method with recoveries of 105. 4％ -110. 8％ , which indicated that the proposed FRET system had the potential for practical detection of arginine in fruit juice.

Progresses of Single Molecular Fluorescence Resonance Energy Transfer in Studying Biomacromolecule Dynamic Process / 分析化学

Biomacromolecules participate in various kinds of vital processes. Observing and analyzing their structural dynamic and the dynamic processes of intermolecular interaction at molecular level is important for understanding the action mechanism. Since its advent, single molecular fluorescence resonance energy transfer (SM-FRET) has demonstrated its great potential in studying the conformational change and interaction process of biomacromolecules, and a series of new mechanisms have been revealed. This review summarized recent progresses of SM-FRET in studying protein structural dynamic, nucleic acid structural dynamic, protein-protein and protein-nucleic acid interactions.