胰蛋白酶与DNA相互作用的共振瑞利散射光谱及其分析应用研究

当胰蛋白酶与鲱鱼精DNA(hsDNA)、 鲑鱼精DNA(sDNA)以及小牛胸腺DNA(ctDNA)之间发生相互作用时,共振瑞利散射(RRS)会显著增强,并产生新的RRS光谱. 三者有近似的光谱特征,其最大RRS峰分别位于307 nm(hsDNA和sDNA体系)和290 nm处(ctDNA体系),另一散射峰位于350 nm处,其散射强度(ΔI)与DNA或者胰蛋白酶的浓度成正比,因此可用于蛋白质和DNA的相互测定. 当用胰蛋白酶测定DNA时,hsDNA,sDNA和ctDNA的检测范围分别为1.4×10^-3～2.3, 2.1×10^-3～2.5和3.5×10^-3～1.9 μg/mL(ctDNA),它们的检出限分别为0.4,0.7和1.1 ng/mL. 当用hsDNA测定胰蛋白酶时,其线性范围为0.1～30.0 μg/mL,检出限为39.0 ng/mL. 研究了最佳的反应条件、 影响因素和结合产物的化学性质,考察了胰蛋白酶与DNA的结合比,推测了它们的结合方式,并以胰蛋白酶作RRS探针,发展了一种高灵敏、 简便和快速测定痕量DNA的新方法.     

Resonance Rayleigh scattering study of the interaction of bleomycinA_5 with nucleic acids and its analytical application

The interaction of bleomycinA5 with nucleic acids has been investigated by using resonance Rayleigh scattering (RRS), molecular absorption and fluorescence spectra. The result shows that in near pH 2.2 buffer medium and absence of any metal ions, nucleic acids are capable of binding with bleomycinA5 (BLMA5) to form complexes which can remarkably enhance the RRS intensity and result in batho- chromic and hyperchromic molecular absorption of nucleic acids and fluorescence quenching of bleomycinA5. The RRS spectral characteristics for the binding products of bleomycinA5 with various DNA and RNA are similar, and the maximum RRS peaks are at 301 nm for ctDNA and sDNA, 370 nm for hsDNA, 310 nm for RNAtypeVI and RNAtypeIII, respectively. The increments of RRS intensity are greatly different in which DNA enhances greatly and RNA enhances lightly. In this work, the optimum condi- tions of the interaction and some influencing factors have been investigated. The reaction mechanism and a binding model for the interaction of BLMA5 with the nucleic acids are discussed. In addition, a highly sensitive, simple and rapid new method for the determination of DNA has been developed. The detection limits (3σ) are 5.7 ng/mL for ctDNA, 7.4 ng/mL for sDNA and 9.2 ng/mL for hsDNA, respectively. The method can be applied to determination of trace amounts of DNA.     

Resonance Rayleigh scattering study of the reaction of nucleic acids with thionine and its analytical application

Resonance Rayleigh scattering (RRS) of the thionine (TH)-nucleic acids system and its analytical application have been studied. In pH 2.2 acidic buffer medium, some nucleic acids can react with TH to form TH-nucleic acids complex. This results in a great enhancement of RRS and the appearance of new RRS spectra. The RRS spectral characteristics of TH-ctDNA system, the affecting factors and the optimum conditions of the reaction have been investigated. The enhancement of the RRS signal is directly proportional to the concentration of nucleic acids in the range 0-10.0 microg/ml for calf thymus DNA and 0-15.0 microg/ml for yeast RNA, and its detection limits (3sigma) are 3.5 ng/ml for calf thymus DNA and 4.9 ng/ml for yeast RNA, respectively. The method shows a wide linear range and high sensitivity, and was applied to the determination of trace amounts of nucleic acid in synthetic samples and practical samples with satisfactory results. The bind properties for the interactions of TH with ctDNA were investigated using a Scatchard plot based on the measurement of the enhanced RRS data at 340 nm, and the binding number and intrinsic binding constant are 4.9 and 2.6 x 10(5) mol/dm(3), respectively.     

Achievements in resonance Raman spectroscopy review of a technique with a distinct analytical chemistry potential

In an extended introduction, key aspects of resonance Raman spectroscopy (RRS) such as enhanced sensitivity and selectivity are briefly discussed in comparison with normal RS. The analytical potential is outlined. Then achievements in different fields of research are highlighted in four sections, with emphasis on recent breakthroughs: (1) The use of visible RRS for analyzing carotenoids in biological matrices, for pigments and dyes as dealt with in art and forensics, and for characterizing carbon nanotubes. (2) The use of RRS in the deep UV (excitation below 260 nm) in the bioanalytical and life sciences fields, including nucleic acids, proteins and protein-drug interactions. Metalloproteins can be studied by visible RRS in resonance with their chromophoric absorption. (3) Progress in theoretical calculations of RRS excitation profiles and enhancement factors, which ultimately might facilitate analytical RRS. (4) Instrumental and methodological achievements including fiber-optic UV-RRS, coupling of RRS to liquid chromatography and capillary electrophoresis. Sensitivities can approach the single-molecule level with surface-enhanced RRS or tip-enhanced RRS. Last but not least, promising fluorescence background rejection techniques based on time-gated detection will be presented. This review ends with a concluding section on future expectations for RRS, in particular its potential as an analytical technique.     

金纳米微粒作探针共振瑞利散射光谱法测定盐酸异丙嗪

在pH 1.8～3.0的酸性介质中,质子化的盐酸异丙嗪(PMZ)可与带负电荷的金纳米微粒依靠静电和疏水作用相互结合,导致共振瑞利散射(RRS)强度显著增强,其最大散射峰位于368 nm,并在284,440,498 nm处有明显的散射峰,在选定的测量波长下,盐酸异丙嗪在0.04～0.10μg/mL的浓度范围内与RRS强度成正比,该法具有高的灵敏度,其检出限为1.34 ng/mL。考察了体系的RRS光谱特征,研究了适宜的反应条件、影响因素,研究了共存物质的影响,据此建立了金纳米微粒作探针RRS法测定盐酸异丙嗪的新方法。     

Resonance Rayleigh scattering study of the interaction of bleomycinA<Subscript>5</Subscript> with nucleic acids and its analytical application

The interaction of bleomycinA5 with nucleic acids has been investigated by using resonance Rayleigh scattering (RRS), molecular absorption and fluorescence spectra. The result shows that in near pH 2.2 buffer medium and absence of any metal ions, nucleic acids are capable of binding with bleomycinA5 (BLMA5) to form complexes which can remarkably enhance the RRS intensity and result in bathochromic and hyperchromic molecular absorption of nucleic acids and fluorescence quenching of bleomycinA5. The RRS spectral characteristics for the binding products of bleomycinA5 with various DNA and RNA are similar, and the maximum RRS peaks are at 301 nm for ctDNA and sDNA, 370 nm for hsDNA, 310 nm for RNAtypeVI and RNAtypeIII, respectively. The increments of RRS intensity are greatly different in which DNA enhances greatly and RNA enhances lightly. In this work, the optimum conditions of the interaction and some influencing factors have been investigated. The reaction mechanism and a binding model for the interaction of BLMA5 with the nucleic acids are discussed. In addition, a highly sensitive, simple and rapid new method for the determination of DNA has been developed. The detection limits (3σ) are 5.7 ng/mL for ctDNA, 7.4 ng/mL for sDNA and 9.2 ng/mL for hsDNA, respectively. The method can be applied to determination of trace amounts of DNA.     

Spectroscopic study on the interaction between methylene blue and chondroitin 4-sulfate and its analytical application.

The interaction of Methylene Blue (MB) with chondroitin-4-sulfate (CHS) has been investigated using spectroscopic techniques, including UV-Vis absorption, Rayleigh resonance scattering (RRS), and circular dichroism (CD). The addition of CHS caused a decrease in the absorbance of MB at 664 nm with a new absorption band appearing at 570 nm, enhanced RRS at 314 nm and 560 nm, and also resulted in an intense CD signal at 568 nm. The Scatchard model has been applied to calculating the binding constant and the number of binding sites. The calculated parameters are consistent with the experimental results. The factors affecting the interaction were investigated. Quantitative spectroscopic methods were developed for the first time. They are based on the fact that a decrease in the absorption at 664 nm and an enhancement of the RRS intensity at 314 nm are proportional to the concentration of CHS added in a certain range. Satisfactory results were obtained on the determination of synthetic samples.     

Binding equilibrium of I- to serum albumin with resonance Rayleigh scattering

The binding equilibrium between l- and human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by means of the resonance Rayleigh scattering (RRS) and equilibrium dialysis. It has been found for the first time that RRS and multiple frequency scattering (MFS) are enhanced as the l- binding to the HSA and BSA, but fluorescence quenches. The equilibrium dialysis results suggest that the binding of l- to HSA and BSA fits a phase-distribution model other than Scsitchard model, and that the order of magnitude of its phase-distribution constant was found to be 104. It is most probable that Cl~ or other anion ions influence the binding of P by changing the ionic strength in the solution. The dialysis at different pH indicates that the binding mechanism is due to the electrostatic forces between the T-and protonated basic amino-acid residues.     

Resonance Rayleigh scattering method for the determination of polyvinylpyrrolidone using eosin Y as the probe

The intensities of resonance Rayleigh scattering (RRS) of poly (vinyl pyrrolidone) (PVP) and of eosin Y (EY) are weak in solutions of pH 2.9 to 3.4. If reacted with each other, the intensities of RRS are largely enhanced and new RRS bands appear at 276 nm and 320 nm. The intensity at 276 nm is linearly related to the concentration of PVP in the range from 0.10 to 1.6 μg mL^-1. The correlation coefficient is 0.9987, and the detection limit is 28.7 ng mL^-1. The binding mode between PVP and EY was studied by RRS, and by absorption and fluorescence spectroscopy. The optimum reaction conditions and some potential interferences were investigated. The method displays good selectivity and was applied to the determination of PVP in beer.     

Study on the interactions of chlorpromazine hydrochloride and promethazine hydrochloride with nucleic acids by resonance Rayleigh scattering spectrum

The interaction between nucleic acids and medicine molecule is one of the important research fields of nucleic acids, which is very valuable for investigating the interactive mechanisms of anti-cancer and anti-virus medicine, screening medicine in vitro, …     

Absorption,Fluorescence and Resonance Rayleigh Scattering Spectra of Interaction of Papain with Calf Thymus DNA

In weak acidic medium, interaction between papain and calf thymus DNA (ctDNA) resulted in absorption spectral change, fluorescence quenching of papain and remarkable enhancement of resonance Rayleigh scattering (RRS). The interaction types and binding modes were discussed by characteristics of RRS, absorption, fluorescence and circular dichroism spectra combining thermodynamic data. Four interaction types include electrostatic attraction, hydrophobic force, hydrogen bonding and aromatic stacking interaction. Papain interacted with the major groove of ctDNA. Aromatic stacking interaction is the main reason of change of absorption spectrum and fluorescence quenching of papain. Surface enhanced scattering effect, resonance energy transfer effect, increase of molecular volume and conformational change make contribution to RRS enhancement. The enhanced RRS intensity (ΔI) is directly proportional to the concentration of ctDNA or papain. The detection limit (3σ) is 5.2 ng·mL^?1 for ctDNA and 5.6 ng·mL^?1 for papain. This creates conditions for determination of papain and ctDNA.     

A novel and sensitive method for recognition and indirect determination of Al(III) in biological fluid based on the quenching of resonance Rayleigh scattering intensities of "Al(III)-EV-DNA" complexing system

A novel method for recognition and indirect determination of Al(III) by using biological molecules has been established based on the quenching of RRS intensity. In the weak acidic medium, the reaction of ethyl violet (EV) and DNA would result in great enhancement of RRS intensity. However, the presence of Al(III) would lead to the decrease of the RRS intensity owing to the competition coordination of Al with DNA. The decreased intensity of RRS is directly proportional to the concentration of Al(III) in the range of (0.1-2.5)x10(-6) and (0.30-4.5)x10(-5)M, respectively. The method has high sensitivity and its detection limit (3sigma) is 3.6x10(-8)M. The characteristics of RRS spectra of the system, the optimum conditions of the reaction, and the reaction mechanism have been investigated. The method can recognize Al(III) selectively owing to its strong binding to the phosphate backbone of DNA, and has been applied to the determination of Al(III) concentration in synthetic biological samples with satisfactory results. Therefore, the proposed method is promising as an effective means for selective recognition and sensitive determination in situ of Al(III). Furthermore, this study would contribute to further understanding of the biological significance of Al neurotoxicity.     

Interaction of polymyxin B with ds-DNA, and determination of DNA or polymyxin B via resonance Rayleigh scattering and resonance non-linear scattering spectra

Assays were developed for DNA or polymyxin B (PMB) based on enhanced resonance Rayleigh scattering (RRS) and resonance nonlinear scattering (including second order scattering and frequency doubling scattering) that result from the interaction of PMB with DNA. A minor-groove binding mechanism is suggested from the results obtained with RRS and from absorption and circular dichroism spectroscopy. The types of interaction and reasons of RRS enhancement are discussed. Linear relationships do exist over a wide range between the intensity of enhanced scattering and the concentrations of either DNA or PMB. When ctDNA is used as a probe to determine PMB, the detection limit (3σ) is 9.8 ng mL^?1. When PMB is used as a probe to determine DNA, the detection limit (3σ) is in the range from 3.8 to 9.0 ng mL^?1.

A Highly Sensitive Resonance Rayleigh Scattering Method for the Determination of Vitamin B1 with Gold Nanoparticles Probe

In weakly acidic buffer medium, vitamin B₁ (VB₁) interacts with gold nanoparticles to form a binding product, which resulted in a significant enhancement of resonance Rayleigh scattering (RRS) intensity and the appearance of a new RRS spectrum. The maximum RRS peak was at 368 nm, and there are three smaller scattering peaks that were at 284 nm, 440 nm and 495 nm, respectively. The enhanced RRS intensity (ΔI) was directly proportional to the concentration of VB₁ in the range of 0–2.8 × 10⁻⁷ mol L⁻¹. The method had high sensitivity and its detection limit (3σ) was 0.9 ng mL⁻¹. The optimum conditions and the influencing factors have been investigated. The method had good selectivity, which could be observed from the influence of coexisting substances. A sensitive, simple and fast RRS method for the determination of VB₁ with gold nanoparticle probe has been developed. In addition, the reasons for RRS enhancement were discussed.     

The resonance Rayleigh scattering of the interaction of copper(II)-bleomycinA2 with DNA and its analytical application

The forming of bleomycinA2-Cu(II) cationic chelate and the interaction of the chelate with DNA have been investigated by using resonance Rayleigh scattering (RRS), molecular absorption and fluorescence spectra. The result shows that in aqueous solution, bleomycinA2 (BLMA2) can react with Cu(II) to form 1:1 cationic chelate which contributes to the changes of the absorption spectra and the quenched fluorescence of BLMA2. When the cationic chelate further bound with DNA to form ternary ion-association complexes, the remarkable enhancement of the RRS intensity was observed. In this work, the optimum conditions for the coordination reaction of BLMA2 with Cu(II) and some influencing factors have been investigated. The reaction mechanism of BLMA2-Cu(II) binding with DNA was suggested and a binding model was proposed. In addition, the fluorescence quenching type of BLMA2 was investigated. A highly sensitive, simple and rapid new method for the determination of DNA by using BLMA2-Cu(II) as RRS probe has been developed. The detection limits (3σ) are 7.2 ng/mL for ctDNA, 7.1 ng/mL for sDNA and 18 ng/mL for hsDNA. The method can be applied to the determination of trace amounts of DNA.     

Binding equilibrium of I− to serum albumin with resonance Rayleigh scattering

The binding equilibrium between l^? and human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by means of the resonance Rayleigh scattering (RRS) and equilibrium dialysis. It has been found for the first time that RRS and multiple frequency scattering (MFS) are enhanced as the l^? binding to the HSA and BSA, but fluorescence quenches. The equilibrium dialysis results suggest that the binding of l^? to HSA and BSA fits a phase-distribution model other than Scatchard model, and that the order of magnitude of its phase-distribution constant was found to be 10⁴. It is most probable that Cl^? or other anion ions influence the binding of l^? by changing the ionic strength in the solution. The dialysis at different pH indicates that the binding mechanism is due to the electrostatic forces between the l^? and protonated basic amino-acid residues.     

用蛋白质作探针共振瑞利散射和共振非线性散射法测定硫酸软骨素A

在pH值为2.5~4.0的BR缓冲溶液介质中,牛血清白蛋白(BSA)、糜蛋白酶(Chy)和α-淀粉酶(α-Amy)等蛋白质与酸性多糖硫酸软骨素A(CS)形成结合物。 此时将会使共振瑞利散射(RRS)和二级散射(SOS)、倍频散射(FDS)等共振非线性散射的强度显著增大。 在蛋白质过量时,3种散射增强(ΔIRRS、ΔISOS和ΔIFDS)均在一定范围内与CS的浓度成正比,方法具有高灵敏度。 当用Chy、BSA和α-Amy作探针时,3种散射法对于CS的检出限分别在1.4~5.8 μg/L、2.0~13.2 μg/L和1.8~9.6 μg/L。 其中以Chy-CS体系的RRS法最灵敏(检出限1.4 μg/L),可用于痕量CS的测定。 研究了反应体系的RRS、SOS和FDS的光谱特征、适宜的反应条件和影响因素,并以Chy-CS体系为例考察了共存物质的影响,方法有良好的选择性,将其用于滴眼液中CS的测定,取得了较好的结果。     

Resonance Rayleigh-scattering method for the determination of sildenafil citrate in a pharmaceutical formulation using Evans blue.

A highly sensitive resonance Rayleigh-scattering (RRS) method for the determination of sildenafil citrate has been developed, based on the fact that sildenafil (Sild) reacted with Evans Blue (EB) to form an ion-association complex in pH 1.1 - 4.6 aqueous solution. This resulted in a significant enhancement of the RRS intensity, and a new spectrum appeared. The wavelength of the maximum RRS was at 365 nm, and other scattering peaks were at 400, 442, 470 and 534 nm, respectively. The intensity of RRS was directly proportional to the concentration of Sild in the range 0 - 11.5 microg ml(-1), and the detection limit for Sild (3 sigma) was 30.3 ng ml(-1). The composition of the ion-association complex was Sild:EB = 1:1, as established by Job's method. The method had good selectivity and could be applied to the determination of Sild in the aqueous phase without using organic solvent extraction. The method was simple and rapid. In addition, the reaction mechanism and the reason for RRS enhancement were considered.     

Stereochemistry of a spherand-type calixarene

The stereochemistry of the spherand-type calixarene 2a is analyzed in terms of the configuration of the three 2,2'-dihydroxybiphenyl subunits (R or S) and the disposition of the methylene groups (crown or twist). X-ray crystallography indicates that the neutral 2a and its mono- or dianion (in form of the salts 2a(-) x C(5)H(5)NH(+) and 2a(2)(-) x 2Et(3)NH(+)) exist essentially in the same conformation (RRS/SSR-twist). This asymmetric RRS/SSR-twist form is the lowest energy conformer according to molecular mechanics calculations. Low-temperature (1)H NMR data indicate the presence of a major conformer of C(1) symmetry, in agreement with a RRS/SSR-twist form. The lowest energy topomerization pathway mutually exchanges two pairs of methylene protons and is ascribed to an enantiomerization process involving rotation around an Ar-Ar bond.     

Binding equilibrium of I? to serum albumin with resonance Rayleigh scattering

The binding equilibrium between l⁻ and human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by means of the resonance Rayleigh scattering (RRS) and equilibrium dialysis. It has been found for the first time that RRS and multiple frequency scattering (MFS) are enhanced as the l⁻ binding to the HSA and BSA, but fluorescence quenches. The equilibrium dialysis results suggest that the binding of l⁻ to HSA and BSA fits a phase-distribution model other than Scatchard model, and that the order of magnitude of its phase-distribution constant was found to be 10⁴. It is most probable that Cl⁻ or other anion ions influence the binding of l⁻ by changing the ionic strength in the solution. The dialysis at different pH indicates that the binding mechanism is due to the electrostatic forces between the l⁻ and protonated basic amino-acid residues.