甲基蓝与血红蛋白相互作用的共振瑞利散射光谱研究及小分子的影响

在弱酸性条件下,甲基蓝(MB)可以通过静电和疏水作用聚集到血红蛋白(Hb)上并形成离子缔合物,不仅使得吸收光谱发生变化,而且使得共振瑞利散射增强。据此发展了一种测定Hb的RRS便捷方法,其线性范围和检出限分别为0.2~20μg/mL和0.012μg/mL。与RRS法相比,分光光度法的线性范围和检出限分别为8~60μg/mL和0.688μg/mL(344 nm),5~60μg/mL和0.398μg/mL(408 nm)。此RRS法可用于尿样中Hb的痕量检测。实验考察了体系的吸收、散射光谱性质和最佳聚合条件,并在最佳条件下研究了分析参数及作用机理。并初步讨论了有机小分子对体系的影响,结果表明分子间氢键是主要作用力不容忽视。     

Using supramolecular hydrogels to discover the interactions between proteins and molecular nanofibers of small molecules

Here we report the first example of the use of supramolecular hydrogels to discover the protein targets of aggregates of small molecules.     

Study on molecular interactions between proteins on live cell membranes using quantum dot-based fluorescence resonance energy transfer

Mouse anti-human CD71 monoclonal antibody (anti-CD71) was conjugated with red quantum dots (QDs; 5.3 nm, emission wavelength λ _em = 614 nm) and used to label HeLa cells successfully. Then green QD-labeled goat anti-mouse immunoglobulin G (IgG; the size of the green QDs was 2.2 nm; λ _em = 544 nm) was added to bind the red-QD-conjugated anti-CD71 on the cell surface by immunoreactions. Such interaction between anti-CD71 and IgG lasted 4 min and was observed from the fluorescence spectra: the fluorescence intensity of the “red” peak at 614 nm increased by 32%; meanwhile that of the “green” one at 544 nm decreased by 55%. The ratio of the fluorescence intensities (I _544 nm/I _614 nm) decreased from 0.5 to 0.2. The fluorescence spectra as well as cell imaging showed that fluorescence resonance energy transfer took place between these two kinds of QDs on the HeLa cells through interactions between the primary antibody and the secondary antibody.     

Determination of proteins with Fast Red VR by a corrected resonance light-scattering technique.

A simple corrected resonance light-scattering (CRLS) technique was established to correct for any distortion of the resonance light scattering (RLS) spectra resulting from molecular absorption. By using an absorption cell holder to change the propagation direction of the incident light beam of a common spectrofluorometer, the molecular absorption was directly measured through a spectrofluorometer. With measurements of the CRLS signals of the interaction of Fast Red VR (FRV) and proteins, we proved that the present correction for the RLS spectra in terms of the molecular absorption of excitation and scattering radiation can improve the detection sensitivity by about two fold.     

Study on the interaction between diphenhydramine and erythrosin by absorption,fluorescence and resonance Rayleigh scattering spectra

In pH 4.5 Britton-Robinson(BR)buffer solution,erythrosin(ET)can react with diphenhydramine(DP)to form a 1:1 ion-association complex,which not only results in the change of the absorption spectra,but also results in the great enhancement of resonance Rayleigh scattering(RRS)and the quenching of fluorescence.Furthermore,a new RRS spectrum will appear,and the maximum RRS wavelength was located at about 580 nm.In this work,the spectral characteristics of the absorption,fluorescence and RRS,the optimum conditions of the reaction and the properties of an analytical chemistry were inves- tigated.A sensitive,simple and new method for the determination of DP by using erythrosin as a probe has been developed.The detection limits for DP were 0.0020μg/mL for RRS method,0.088μg/mL for absorption method and 0.094μg/mL for fluorophotometry.There was a linear relationship between the absorbance,RRS and fluorescence intensities and the drug concentration in the range of 0.0067-2.0, 0.29-6.4 and 0.31-3.2μg/mL,respectively.The effects of the interaction of diphenhydramine and erythrosin on the absorption,fluorescence and resonance Rayleigh scattering spectra were discussed. In light polarization experiment,the polarization of RRS at maximum wavelength was measured to be P =0.9779,and it revealed that the RRS spectrum of DP-ET complex consists mostly of resonance scat- tering and few resonance fluorescence.In this study,enthalpy of formation and mean polarizability were calculated by AM1 quantum chemistry method.In addition,the reaction mechanism and the rea- sons for the enhancement of scattering spectra and the energy transfer between absorption,fluores- cence and RRS were discussed.     

Evaluation of interactions between RAW264.7 macrophages and small molecules by capillary electrophoresis

In this study, the affinity interactions between RAW 264.7 macrophages and three small molecules including naringin, oleuropein and paeoniflorin were evaluated by affinity capillary electrophoresis (ACE), partial filling affinity capillary electrophoresis (PFACE) and frontal analysis capillary electrophoresis (FACE), respectively. The result indicated that ACE (varying concentrations of cell suspension were filled in the capillary as receptor) may not be suitable for the evaluation of interactions between cell and small molecules due to the high viscosity of cell suspension; PFACE can qualitatively evaluate the interaction, but the difference in viscosity between RAW264.7 suspension and buffer effects on the liner relationship between filling length and injection time, which makes the calculation of binding constant difficult. Furthermore, based on the PFACE results, naringin showed stronger interaction with macrophages than the other two molecules; taking advantage of the aggregation phenomenon of cell induced by electric field, FACE was successfully used to determine the stoichiometry (n = 5×10⁹) and binding constant (K_b = 1×10⁴ L/mol) of the interaction between RAW264.7 and naringin.     

Solvent effect on fluorescence spectra of a spirooxazine

Fluorescence and excitation spectra of spironaphthoxazine (SNO) in various solvents have been investigated systematically. The fluorescence in low-polar solvents normally originated from SNO in the excited state. In protic high-polar solvents, fluorescence from an excited intermediate species (X^* _h) relaxed from a part of the excited SNO by breaking a C-O bond in the oxazine ring was also observed. It has been found that a ground-state intermediate species (X_s), transformed from a part of ground-state SNO, is directly excited to its excited state (X_s ^*), which displays fluorescence in aprotic high-polar solvents.     

The effect of fluctuations on emission spectra: practical distinctions between raman and fluorescence spectra

By the use of stochastic theory it is clearly shown that two types of emission should to fluctuations such as would occur during interactions with their environment are resonantly driven by light. In our approach the electromagnetic field is treated in a classical manner and decay is introduced by a Wigner-Weisskopfansatz. The two types of emission should occur for a three-level system and relaxation to other levels is not involved in the mechanism. Also included is a discussion of operational definitions of fluorescence (and other spontaneous emissions) and of the relationship between absorption and scattering in terms of dephasing or T₂ processes, especially as this relates to conventional emission phenomena.     

Determination of proteins at nanogram levels by a resonance light-scattering technique with tetra-substituted sulphonated aluminum phthalocyanine

This is the first report on the determination of proteins with tetra-substituted sulphonated aluminum phthalocyanine (AlS₄Pc) by resonance light-scattering (RLS). At pH 3.0, the weak RLS of AlS₄Pc can be enhanced by the addition of proteins. Based on this, a novel quantitative method has been developed for the determination of proteins in aqueous solutions. Under optimal conditions, the linear ranges of the calibration curves were 0.050–2.0 μg ml⁻¹ for both human serum albumin (HSA) and human r-IgG. The detection limits were 12.7 ng ml⁻¹ for HSA and 16.1 ng ml⁻¹ for human r-IgG. The method has been applied to the analysis of total protein in human serum samples collected from the hospital and the results were in good agreement with those reported by the hospital, which indicates that the method presented here is not only sensitive, simple, but also reliable and suitable for practical applications.     

Matrix IR spectra and quantum-chemical calculations of the products of small nickel cluster interactions with water molecules

Reactions of Ni _n clusters with water molecules were studied by IR spectroscopy in inert matrices and quantum chemistry methods. The geometric configurations, total energies, and vibrational frequencies of all the possible Ni₂(H₂O) and Ni₃(H₂O) isomers were calculated. For both systems, the main minima and transition states were found. Water was shown to dissociate to hydrogen and hydroxyl in the reactions, and, in all the complexes formed, hydrogen is situated in the bridge position on the Ni-Ni bond.     

Study on the interaction between fluoroquinolones and erythrosine by absorption, fluorescence and resonance Rayleigh scattering spectra and their application

In pH 4.4-4.5 Britton-Robinson (BR) buffer solution, fluoroquinolone antibiotics (FLQs) including ciprofloxacin (CIP), norfloxacin (NOR), levofloxacin (LEV) and lomefloxacin (LOM) could react with erythrosine (Ery) to form 1:1 ion-association complexes, which not only resulted in the changes of the absorption spectra and the quenching of fluorescence, but also resulted in the great enhancement of resonance Rayleigh scattering (RRS). These offered some indications of the determination of fluoroquinolone antibiotics by spectrophotometric, fluorescence and resonance Rayleigh scattering methods. The detection limits for fluoroquinolone antibiotics were in the range of 0.097-0.265 microg/mL for absorption methods, 0.022-0.100 microg/mL for fluorophotometry and 0.014-0.027 microg/mL for RRS method, respectively. Among them, the RRS method had the highest sensitivity. In this work, the spectral characteristics of the absorption, fluorescence and RRS, the optimum conditions of the reactions and the properties of the analytical chemistry were investigated. The methods have been successfully applied to determination of some fluoroquinolone antibiotics in human urine samples and tablets. Taking CIP-Ery system as an example, the charge distribution, the enthalpy of formation and the mean polarizability were calculated by density function theory (DFT) method. In addition, the reasons for the enhancement of scattering spectra were discussed.     

Investigation of microcontact transfer of proteins from a selectively plasma treated elastomer stamp by fluorescence microscopy and force microscopy

Selective plasma treatment of the recessed regions of the elastomer stamps is shown to alter the resultant protein patterns. Fluorescence microscopy is demonstrated to be an excellent tool to discriminate between regions of microcontact printed fluorescent dye-labelled albumin in polystyrene. Atomic force microscopy and shear force microscopy are used to provide high-resolution images of the patterned protein layers. The formation and characteristics of the patterns formed by these alternative strategies is discussed.     

Herzberg-teller effect and intensity distribution in the absorption and raman scattering resonance spectra of polyatomic molecules

This paper studies the influence of the Herzberg-Teller effect on the intensity distribution in one- and two-photon absorption and Raman scattering resonance spectra of substituted benzenes. A quantum mechanical analysis of the intensity distribution in the spectra of phenylacetylene and pyrazine is accomplished using the general approach suggested in [1, 2]. Due to the inclusion of vibronic coupling in the calculation, one can explain the presence of lines (as well as their combinations and overtones) arising from one-quantum excitation of nontotally symmetric vibrations. The calculated data are in good agreement with the experimental data. Significantly, the two-photon absorption spectrum of pyrazine in the region of the¹A_1g→¹B_3u long-wave singlet-singlet transition is explained only by the Herzberg-Teller effect. Saratov State Pedagogical Institute. Translated fromZhurnal Strukturnoi Khimii, Vol. 36, No. 2, pp. 304–309, March–April, 1995. Translated by L. Smolina     

QM/MM study of the interaction between zigzag SnC nanotube and small toxic gas molecules

Adsorptions of small toxic molecules such as CO, N₂, HCN, SO₂, H₂CO, and NH₃ on a single‐walled (6,0) SnC nanotube (SnCNT) are investigated using Quantum Mechanics/Molecular Mechanics (QM/MM) methodology. The calculations are carried out at the B3LYP/6‐311++G(d,p)//LANL2DZ:UFF level of theory. The high layer of the model consists of a pyrene‐type ring on the nanotube surface as the adsorption site, where one gas molecule is allowed to interact. Conversely, for the adsorption of the two molecules, a larger site like a coronene ring is used for the high layer. Adsorption energy, Gibbs free energy change, Mulliken charge transfer, and total electron‐density maps are computed in each case. The adsorption strength of the gas molecule on the SnCNT surface is also analyzed from the density of states projected to different atoms (PDOS) of the nanotube–adsorbate complexes. The adsorptions of CO and N₂ on the (6,0) SnCNT surface require to cross potential barriers, and the corresponding transition structures are identified by ONIOM‐IRC calculations. For the remaining four molecules, the processes of adsorption are predicted to be barrier‐less. The calculations for the adsorption of H₂CO on (5,0) and (7,0) SnCNT surfaces are extended to study the effect of the size of the nanotube. Results for the adsorption of a single molecule on (6,0) SnCNT using B3LYP functional are compared with those obtained from a dispersion corrected functional such as M06‐2X. © 2015 Wiley Periodicals, Inc.     

Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method

The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants (K_a) obtained by the double-logarithm curve for esculin–BSA and esculin–HSA were 1.02 × 10⁷ and 2.07 × 10⁴ L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S₀ → S₁ transition of esculin () appears, which is similar to the of BSA and HSA. The critical distance (R₀) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.     

Accurate steady-state and zero-time fluorescence spectra of large molecules in solution by a first-principle computational method

We report the first-principle calculation and analysis of the vibrationally resolved steady-state absorption and fluorescence spectra, and of the zero-time fluorescence spectrum of a sizable molecule, coumarin C153, in two different solvents. Our approach, bringing together the most recent developments in the fields of time-dependent density functional theory and of polarizable continuum solvent models, with an efficient method for the computation of vibrational contributions to transition intensities, allows a remarkable agreement with experiments, both concerning the line shapes and the solvatochromic and Stokes shifts. The method is also able to nicely describe the solvent relaxation effect on the fluorescence spectra, perfectly reproducing the energy shift between zero-time and steady-state fluorescence.     

Label-free quantitative analysis for studying the interactions between nanoparticles and plasma proteins

A shotgun proteomics approach was used to compare human plasma protein binding capability with cationic liposomes, DNA–cationic lipid complexes (lipoplexes), and lipid–polycation–DNA (LPD) complexes. Nano-high-performance liquid chromatography coupled with a high-resolution LTQ Orbitrap XL mass spectrometer was used to characterize and compare their protein corona. Spectral counting and area under curve methods were used to perform label-free quantification. Substantial qualitative and quantitative differences were found among proteins bound to the three different systems investigated. Protein variety found on lipoplexes and LPD complexes was richer than that found on cationic liposomes. There were also significant differences between the amounts of protein. Such results could help in the design of gene-delivery systems, because some proteins could be more selectively bound rather than others, and their bio-distribution could be driven in vivo for more efficient and effective gene therapy.     

Studying the interaction between CdTe quantum dots and Nile blue by absorption,fluorescence and resonance Rayleigh scattering spectra

Thioglycolic acid (TGA) capped CdTe quantum dots (QDs) with the diameter of 2–3 nm were synthesized. The interaction between CdTe QDs and Nile blue (NB) was investigated by ultraviolet–visible (UV–vis) absorption, resonance Rayleigh scattering (RRS) and fluorescence spectroscopy. UV–vis absorption spectrum of CdTe QDs and NB obviously changed, showing that CdTe QDs could associate with NB to form a new complex. At pH 6.8, NB effectively quenched the fluorescence of CdTe QDs. It was proved that the fluorescence quenching of CdTe QDs by NB was mainly result of the formation of CdTe QDs–NB complex, electrostatic attraction and hydrophobic forces played a major role in stabilizing the complex. The binding molar ratio of CdTe QDs and NB was 5:1 by a mole-ratio method. The interaction between CdTe QDs and NB lead to the remarkable enhancement of RRS and the enchantments were in proportional to the concentration of NB in a certain range. The mechanism of the interaction between CdTe QDs and NB, reasons for the enhancement of RRS intensity were also discussed. The obtained results suggested the more satisfactory mechanism for the interaction between CdTe QDs and NB.     

Quantitative determination of proteins at nanogram levels by the resonance light-scattering technique with macromolecules nanoparticles of PS-AA

The polystyrene-acrylic acid (PS-AA) nanoparticles have been prepared by ultrasonic polymerization, characterized by FT-IR and TEM. It is the first report on the determination of proteins with macromolecules nanoparticles of PS-AA by resonance light-scattering (RLS). At pH 6.9, the RLS of macromolecules nanoparticles of PS-AA can be enhanced by proteins. Based on this, a novel quantitative assay of proteins at the nanogram levels has been proposed. At pH 6.9, the RLS signals of PS-AA were greatly enhanced by proteins in the region of 250-700 nm characterized by the peak at 342 nm. Under optimal conditions, the linear ranges of the calibration curves were 0.02-11.0 microgml-1, 0.04-10.0 microgml-1 and 0.03-10.0 microgml-1 for gamma-globulin (gamma-IgG), bovine serum albumin (BSA) and human serum albumin (HSA), respectively. The detection limits were 16.0 ngml-1, 19.0 ngml-1, and 15.0 ngml-1 for gamma-IgG, BSA and HSA, respectively. The method has been applied to the analysis of total proteins in human serum samples collected from the hospital and the results were in good agreement with those reported by the hospital, which indicates that the method presented here is not only sensitive, simple, but also reliable and suitable for practical application.     

Investigation of the effect of pressure on retention of small molecules using reversed-phase ultra-high-pressure liquid chromatography

The effect of inlet pressure on the retention of a series of low molecular weight acids, bases and neutrals, was investigated at constant temperature in reversed-phase liquid chromatography using a commercial ultra-high-pressure system (Waters UPLC instrument). For neutral compounds, relatively small increases in retention factor of up to approximately 12% for a pressure increase of 500bar were noted; the largest values were obtained for polar solutes, or solutes of higher molecular weight. Ionisable acids and bases gave much larger increases in retention with pressure, in some cases as high as 50% for a pressure increase of 500bar. Thus, such compounds could show increases in retention factor approaching 100% over the pressure range available in the commercial UPLC instrument. Due to these differential increases, significant selectivity effects can be obtained for mixtures of different types of solute merely by changing the pressure.