偶氮氯膦Ⅲ与人血清白蛋白相互作用的电化学研究

偶氮氯膦Ⅲ是一种具有电化学活性的染料,在pH3.5的Britton-Robinson缓冲溶液中,它可以与人血清白蛋白发生相互作用形成一种生物超分子复合物,使溶液中游离的染料浓度降低.以线性扫描二阶导数极谱法对偶氮氯膦Ⅲ-人血清白蛋白的相互作用体系进行了详细的研究,复合物的形成使偶氮氯膦Ⅲ在-0.124V(vs.SCE)的还原峰电流降低,考察了结合反应的最佳条件和测定条件,求解了结合常数和结合比.     

Voltammetric Studies on Chromotrope 2B‐Protein Interaction and Its Analytical Application

In this paper the interaction of chromotrope 2B (Ch2B) with proteins was studied by the electrochemical method. Ch2B is an azo dye and shows irreversible electrochemical responses on the mercury electrode in a pH 3.0 Britton‐Robinson (B‐R) buffer solution. After the addition of human serum albumin (HSA) into the Ch2B solution, an interaction took place, and a supramolecular complex was formed in the mixed solution. The electrochemical parameters of the Ch2B‐HSA interaction system were calculated and compared. The results showed that in the absence and presence of HSA in Ch2B solution, the electrochemical parameters such as the formal potential E⁰, the electrode reaction standard rate constant k_s, etc. showed no significant changes, which indicated that an electro‐inactive supramolecular biocomplex was formed. The free concentration of Ch2B in reaction solution was decreased, and this resulted in the decrease of the peak current. The binding constant and the binding ratio were calculated as 7.85 × 10⁹ and 1:2, respectively, and the interaction mechanism was discussed. Based on the decrease of the peak current, this new electrochemical method was proposed for the determination of HSA in the concentration range of 2.0?25.0 mg/L with the linear regression equation as ΔIp′ (nA) = 50.56C (mg/L) — 6.72 (γ = 0.995). This method was further used to determine other different kinds of proteins, such as bovine serum albumin (BSA), oval albumin, etc‥ The new method was successfully applied to detect the content of albumin in healthy human serum samples with the results in good agreement with the traditional Coomassie Brilliant Blue G‐250 spectrophotometric method.     

Microdetermination of double-stranded DNA by linear sweep voltammetry with phenosafranine.

A novel voltammetric method for the determination of microamounts of fish sperm double-stranded (ds) DNA based on its interaction with phenosafranine (PSF) is proposed in this paper. In a pH 3.5 Britton-Robinson (B-R) buffer solution, PSF had a well-defined second-order derivative linear-sweep voltammetric reductive peak at -0.32 V (vs. SCE) on a mercury electrode. After the addition of dsDNA into the PSF solution, the reductive peak current decreased significantly without a shift of the peak potential, and no new peak appeared. The experiment results showed that a new supramolecular complex was formed after the interaction of dsDNA with PSF, which resulted in a decrease of the diffusion coefficient, and then a decrease of the reductive peak current. The interaction conditions and the electrochemical detection conditions were carefully investigated. Under the optimal conditions, the decrease of the peak current was proportional to the dsDNA concentration in the range 1.0 - 40.0 microg/mL with the linear regression equation DeltaI(p)'(nA) = 32.59C(microg/mL) - 4.03 (n = 13, gamma = 0.998) and a detection limit of 0.25 microg/mL (3 sigma). The interaction mechanism was considered based on the aggregation of the dsDNA-PSF supramolecular complex; the stoichiometry of this supramolecular complex was calculated based on voltammetric data with a binding number of 3 and a binding constant of 2.76 x 10(12). This method was successfully applied to the determination of synthetic samples and the polymerase chain reaction (PCR) product of the nopaline synthase gene (NOS) DNA from genetically modified organisms (GMOs) with satisfactory results.     

Electrochemical Investigation of Ascorbic Acid Interacting with Bovine Serum Albumin

Abstract

Electrochemical investigation of the interaction of Ascorbic acid (AA) with bovine serum albumin (BSA) on a glassy carbon electrode is reported for the first time. In a 0.1 mol/l, pH 7.3 NaCl solution, AA had a well‐defined voltammetric oxidative peak at +0.2086 V (versus Ag/AgCl) on a GC electrode. After the addition of BSA into the AA solution, the oxidative peak current decreased significantly without a shift of the peak potential, and no new peak appeared. The experimental results showed that a new nonelectrochemical supramolecular complex was formed after the interaction of BSA with AA, which resulted in a decrease of the diffusion coefficient, and then a decrease of the oxidative peak current. The interaction conditions and the electrochemical detection conditions were carefully investigated. The stoichiometry of this supramolecular complex was calculated based on voltammetric data with a binding number of two and a binding constant of 1.38×10⁷.     

A Linear Sweep Voltammetric Determination of DNA with Methyl Violet

A new quantitative method for the determination of DNA in aqueous solution based on the interaction of methyl violet (MV) with fsDNA by linear sweep voltammetric technique is proposed in this paper. The interaction of MV with fsDNA caused the decrease of the reductive peak current of MV at ?0.650 V (vs. SCE) on a mercury electrode in pH 1.5 Britton‐Robinson (B‐R) buffer solution. Factors including the acidity of the buffer, the reaction time, the optimal concentration of MV, the reaction temperature, the influences of the foreign substances, etc. were all carefully investigated. Under the optimal conditions, the decrease of the reductive peak current of MV was proportional to the concentration of fsDNA in the linear range of 0.90～20.0 mg/L and the detection limit for fsDNA was 0.25 mg/L with the RSD of 4.00%. Synthetic samples were determined with satisfactorily results. The stoichiometry of MV with DNA was calculated by voltammetric data and the results showed that the binding ratio of DNA:MV was 1:2 with the binding constant β as 7.36 × 10⁸.     

Studies on the interaction of protein with acid chrome blue K by electrochemical method and its analytical application

An electrochemical investigation on the interaction of acid chrome blue K (ACBK) with protein on the mercury electrode with different electrochemical methods such as cyclic voltammetry and linear sweep voltammetry was reported in this paper. In pH 3.0 Britton-Robinson (B-R) buffer solution, ACBK has an irreversible voltammetric reductive peak at -0.23 V (vs. SCE). The addition of human serum albumin (HSA) into the ACBK solution resulted in the decrease of reductive peak currents without the change of the peak potential and no new peaks appeared on the cyclic voltammogram. In the absence and presence of HSA, the electrochemical parameters such as the formal potential E0, the electrode reaction standard rate constant k(s) and the charge transfer coefficient alpha of the interaction system were calculated and the results showed that there were no significant changes between each other. Thus, the interaction of ACBK with protein forms an electro-inactive supramolecular bio-complex, which induces the decrease of the free concentration of ACBK in the reaction solution, and the decrease of the reductive peak current of ACBK. The binding constant and the binding ratio are calculated as 1.29 x 10(8) and 1:2, respectively, and the interaction mechanism is discussed. Based on the binding reaction, this new electrochemical method is further applied to the determination of HSA with the linear range from 3.0-20.0 mg/L and the linear regression equation as deltaIp"(nA) = 10.08 + 19.90 C (mg/L). This method was further applied to determinate the content of protein in the healthy human serum samples with the results in good agreement with the traditional Coomassie brilliant blue G-250 spectrophotometric method.     

Application of Cupferron‐lead(II) Complex for the Electrochemical Determination of dsDNA

Based on the interaction of cupferron and lead(II) complex [Cup‐Pb(II)] with double‐stranded DNA (dsDNA) a new voltammetric method for the detection of DNA was described in this paper. In pH 4.0 HAc‐hexamine buffer solution, [Cup‐Pb(II)] complex showed a sensitive second order derivative polarographic reductive peak at ‐0.554 V (vs. SCE). After the addition of dsDNA into [Cup‐Pb(II)] mixture solution the reductive peak current decreased with the positive shift of reductive peak potential, which was the typical characteristic of intercalation mode. Under the optimum conditions, the decrease of reductive peak current was directly proportional to the dsDNA concentration in the range from 1.0 to 25.0 mg/L with the linear regression equation as ΔIp″ (nA) = 129.30 + 62.51 C (mg/L) (n = 13, γ = 0.991). The detection limit of 0.90 mg/L (3σ) and the relative standard derivation (RSD) of 2.43% for 10 parallel determinations of 10.0 mg/L dsDNA were found. The method was successfully applied to synthetic samples with good results, and the stoichiometry of dsDNA with [Cup‐Pb(II)] complex was calculated by the voltammetic data with the binding number as 2 and the binding constant as 2.82 × 10⁹.     

Electrochemical studies on the interaction of heparin with crystal violet and its analytical application

In this paper, crystal violet (CV) was used to determine heparin concentration by linear sweep voltammetry on a dropping mercury electrode (DME). In Britton-Robinson (B-R) buffer solution, pH 3.0, CV had a well-defined second-order derivative linear sweep voltammetric reductive wave at −0.74 V (vs. SCE). After the addition of heparin to the CV solution, the reductive peak current decreased greatly with the positive movement of the peak potential and without appearance of new peaks in the scanning potential range. Based on the decrease in the reductive peak current, a new voltammetric method for the determination of heparin was established. The conditions for the interaction and the electrochemical detection were optimized, and interfering substances were investigated. Under the optimal conditions, the decrease in reductive peak currents of CV was proportional to heparin concentration in the range 0.1–8.0 mg/L with the linear regression equation Δip″(nA) = 400.42 + 1563.11c (mg/L), (n = 14, γ = 0.993). The detection limit was 0.092 mg/L. This new method was further successfully applied to the determination of heparin content in heparin sodium injection samples with satisfactory results. The binding ratio and binding mechanism were also studied by the electrochemical method. The text was submitted by the authors in English.     

Electrochemical Studies of the Interaction of 2‐Nitroacridone with DNA and Determination of DNA

A new acridone derivate 2‐nitroacridone (NAD) was synthesized and a new method of electrochemical probe has been proposed for the determination of salmon sperm DNA based on its interaction with NAD. The electrochemical behavior of interaction of NAD with DNA was investigated on glassy carbon electrode (GCE). In the presence of DNA, the peak current of NAD decreases and the peak potential shifts to a more positive potential without appearance of a new peak. The binding ratio between NAD and salmon sperm DNA was calculated to be 2 : 1 and the binding constant was 3.19×10⁵ L/mol. The decrease of the peak current (ΔI_p) of NAD was proportional to the concentration of DNA in the range from 1.55×10^?7 M to 2.02×10^?6 M with the detection limit of 3.10×10^?8 M, and DNA of synthetic sample was determined satisfactorily. Additionally, the binding mechanism was preliminarily discussed. The mode of interaction between NAD and DNA was found to be intercalation binding.     

甲基紫与肝素钠结合反应的电化学研究及分析应用

应用电化学分析法研究了在pH 1.5的酸性反应条件下肝素钠与甲基紫的结合反应. 甲基紫在滴汞电极上有一个不可逆的还原峰, 峰电位为－0.58 V (vs. SCE), 加入肝素钠后峰电位发生正移且峰电流下降, 利用电化学方法对电极反应过程进行了研究, 结果发现两者结合后生成了一种电化学活性的复合物, 导致溶液的电化学参数发生了变化, 求解出结合比为1∶3, 结合常数为2.47×10¹⁴, 对结合反应条件和电化学检测条件进行了优化, 在最佳条件下峰电流的降低同肝素钠的浓度在0.2～4.0 mg/L范围内呈线性关系, 线性回归方程为∆I_p″ (nA)＝－724.9＋1741.4c (mg/L) (n＝11, γ＝0.994), 检测限为0.072 mg/L. 将本方法应用于肝素钠样品的测定, 结果令人满意. 对常见干扰物质的影响进行了考察, 表明本方法具有较好的选择性.     

A New Electrochemical Method for the Determination of Proteins with Cupferron‐Cadmium(II) Complex

A new electrochemical method was proposed for the determination of trace amounts of proteins based on the cupferron (Cup) and cadmium(II) complex [Cup‐Cd(II)] as the voltammetric probe. In the selected pH 6.5 Britton–Robinson (B–R) buffer solution, Cup can interact with Cd(II) to form a stable complex of [Cup‐Cd(II)], which had a sensitive linear sweep voltammetric reductive peak at ?0.654 V (vs. SCE). The addition of human serum albumin (HSA) into [Cup‐Cd(II)] complex solution could greatly decrease the reductive peak current without the change of the reductive peak potential, which indicated that HSA could interact with [Cup‐Cd(II)] complex to form a supramolecular biocomplex. The interaction mechanism was discussed and the decrease of reductive peak current was proportional to the concentration of HSA, which could be further used for the proteins determination. The optimal conditions of the binding reaction and the electrochemical detection were carefully investigated. Under the optimal conditions a new quantitative determination method for different kinds of proteins such as HSA, bovine serum albumin (BSA) and bovine hemoglobin (BHb) etc. was developed. The proposed method was simple, practical and relatively free from the interferences of coexisting substances, and it was further applied to the samples determination with satisfactory results. The binding constant (β_s) and the binding number (m) of HSA with [Cup‐Cd(II)] complex were calculated by the voltammetric data with the results as β_s=1.12×10⁶ and m=1.     

Electrochemical Study of the Interaction Mechanism of Proflavine (PF) with DNA Using Carbon Paste (CPE) and Hanging Mercury Drop (HMDE) Electrode

Abstract

Proflavine binds with DNA in a complicated manner. This work involves the electrochemical study of this interaction using differential pulse voltammetry at a carbon paste electrode (CPE) and alternating current voltammetry at a hanging mercury drop electrode (HMDE). At the CPE the peak current intensity at 1.0 V (corresponding to the oxidation of the guanine residues) decreased by increasing the concentration of proflavine. At the HMDE, a decrease in the current intensity of the DNA peak at ? 1.2 V (corresponding to segmental desorption) was also observed by increasing the concentration of proflavine. These results confirmed, electrochemically, that proflavine intercalates within the DNA double helix and changes its conformation.     

Comparison of the voltammetric studies at mercury and glassy carbon electrodes for the interaction of lumichrome with DNA and analytical applications

The determination of the interaction between lumichrome (LC), one of the products of decomposition of the biologically important flavins, and calf thymus double-stranded DNA was performed by using cyclic voltammetry (CV) and differential pulse stripping voltammetry (DPSV) in connection with a hanging mercury drop electrode (HMDE) or glassy carbon electrode (GCE). The nature of the process taking place at both electrode surfaces was clarified. It was found that the addition of DNA to a buffered LC solution results in the decrease of redox peak currents with changes in the peak potentials at both electrodes. We assume that LC interacting with DNA produces an electrochemically inactive supramolecular complex via intercalation. There was a difference between the electrochemical parameters determined at the HMDE and those at the GCE. The binding constants ( K) of the LC-DNA complex at HMDE and GCE were determined through the changes of peak currents and their values at the 10(5) level and 10(4) level with each nucleotide residue of DNA binding one LC molecule, respectively. Furthermore, the calibration graph for the determination of DNA was obtained by the decrease in the DPSV peak current of LC in the presence of DNA. Different variables, such as the concentration of LC, the accumulation time and solution conditions, were studied and optimised to maximize the sensitivity; in addition, the detection limit and the reproducibility were determined.     

以溴甲酚紫为电化学探针测定血清白蛋白

常见的蛋白质定量分析方法、电化学行为及其分析应用已有报道．本文基于酸性条件下蛋白质可与溴甲酚紫(BP)结合生成一种超分子复合物,使溴甲酚紫还原峰电流降低,建立了一种测定蛋白质的新方法,并用于测定人血清样品中的白蛋白含量．     

Linear sweep voltammetric studies on the interaction of brilliant cresyl blue with nucleic acids and its analytical application

In this paper, the interaction of brilliant cresyl blue (BCB) with nucleic acids was studied and further applied for the microdetermination of nucleic acids. In aqueous Britton-Robinson (B-R) buffer solution, BCB can be easily reduced on the hanging mercury drop electrode (HMDE) and had a sensitive voltammetric reduction peak at -0.09 V (vs. SCE). The reduction peak current of BCB could be greatly decreased by the addition of DNA. The results of voltammetric measurements had indicated that a binding reaction was occurred between BCB and DNA and a new supramolecular complex was formed, which resulted in the decrease of the diffusion coefficient of the reaction solution and the decrease of the reduction peak current correspondingly. The conditions of interaction and the electrochemical detection were carefully investigated. Under the selected conditions, the calibration curves for the detection of fish sperm (fs)DNA, calf thymus (ct)DNA and yeast (y)RNA were established. The linear range of this assay was 1.0-30.0 microg/mL for fsDNA, 1.0-45.0 microg/mL for ctDNA and 1.0-25.0 microg/mL for yRNA, respectively. The detection limits were 0.38 microg/mL fsDNA, 0.43 microg/mL ctDNA, 0.64 microg/mL yRNA. The interaction parameters such as the equilibrium constant and the binding number were calculated by electrochemical method. The results showed that the 2:3 type of complex was formed in the fsDNA-BCB complex with the binding constant as 2.51 x 10(7). The proposed method was further applied to the synthetic samples determination with satisfactory results.     

A simple linear sweep voltammetric method for the determination of double-stranded DNA with malachite green

In pH 3.5 Britton—Robinson buffer solution double-stranded (ds) DNA can react with malachite green (MG) to form an interaction complex, which resulted in the decrease of the electrochemical response of MG, MG had a well-defined second-order derivative linear sweep voltammetric peak at −0.73 V (vs. SCE). After the addition of dsDNA into MG solution, the reductive peak current decreased with the positive shift of peak potential, which was the typical characteristic of intercalation. Based on the interaction, an indirect electrochemical determination method for dsDNA was established. The optimum conditions for the reaction were investigated and there were little or no interferences from the commonly coexisting substances. The decrease of peak current was linear with the concentration of dsDNA over the range of 0.8–12.0 μg cm⁻³ with the linear regression equation as ΔI _p″/nA = 91.70 C/(μg cm⁻³) + 74.55 (n = 10, γ = 0.990). The detection limit was calculated as 0.46 μg cm⁻³ (3σ). The method had high sensitivity and was further applied to the dsDNA synthetic samples with satisfactory result. The interaction mechanism was discussed with the intercalation of DNA-MG to form a supramolecular complex and the stoichiometry of the supramolecular complex was calculated by electrochemical method with the binding number 3 and the binding constant 2.35 × 10¹⁵ (mol dm⁻³)⁻³.     

Linear sweep voltammetric determination of protein based on its interaction with Alizarin Red S

In this paper, the electrochemical behavior of the interaction of Alizarin Red S (ARS) with bovine serum albumin (BSA) was investigated on the hanging mercury drop electrode (HMDE). In the acidic solution (pH 4.2), ARS can be easily reduced on the HMDE and it has a well-defined polarographic wave at −0.29 V (SCE). On addition of BSA or human serum albumin (HAS) into the ARS solution, the reduction peak current of ARS decreases without the movement of the peak potential and the appearance of new peaks. The study shows that a new electrochemically non-active complex is formed via intercalation of ARS with BSA or HSA, which can not be reduced on the Hg electrode. The decrease of reductive peak current of ARS is proportional to BSA and HSA concentration in the range of 2.0–60 and 2.0–40 mg l⁻¹, respectively. The detection limit of BSA and HSA is 1.0-mg l⁻¹. The analytical results of human serum and urine samples by this method were in good agreement with the Coomassie brilliant blue G-250 assay. The binding number and the binding interaction mechanism are also discussed.     

Electrochemical studies of the interaction of Basic Brown G with DNA and determination of DNA

A new method of electrochemical probe has been proposed for the determination of Herring Sperm DNA (DNA) based on its interaction with Basic Brown G (BBG). The electrochemical behavior of interaction of BBG with DNA was investigated on Hg electrode. In 0.1 mol L⁻¹ NH₃-NH₄Cl buffer solution (pH 8.0), BBG can be reduced on Hg electrode with a well-defined voltammetric peak at −0.67 V (versus SCE). In the presence of DNA, the reduction peak current of BBG decreases and the peak potential shifts to a more positive potential without the appearance of new peak. The study shows that a new BBG-DNA complex is formed by linear sweep voltammetry (LSV) and spectrophotometry. The decrease of the second order derivative of reductive peak current (Δi^″p) of BBG is proportional to the concentration of DNA in the range of 0.10-36 μg mL⁻¹. Limit of detection of DNA is 0.04 μg mL⁻¹. DNA of Hepatitis B Virus in serum samples was determined satisfactorily. Additionally, the binding mechanism was preliminarily discussed. The mode of interaction between BBG and DNA was found to be intercalation binding.     

Application of linear-sweep voltammetry to the determination of nucleic acids using crystal violet as an electrochemical probe

A new linear-sweep voltammetric assay of nucleic acids (NAs) based on their interaction with crystal violet (CV) is proposed. In a pH 3.5 Britton—Robinson (B-R) buffer solution, CV had an irreversible voltammetric reductive peak at −0.77 V and the peak current greatly decreased by the addition of NAs. Under the experimental conditions, the decrease in the peak current was used for the NAs assay 0.5–18.0 μg/mL of fish sperm DNA, 0.6–15.0 μg/mL of calf thymus DNA, and 0.8–12.0 μg/mL of yeast RNA. The detection limits (3σ) were 0.32, 0.47, and 0.61 μg/mL for fsDNA, ctDNA, and yRNA, respectively. The binding reaction can be completed after mixing DNA with CV within 10 min and the electrochemical response is stable for 2 h. There are seldom interferences in this method and three synthetic samples were analyzed with satisfactory results. The stoichiometry of the supramolecular complex with the binding number 3 and the binding constant 2.78 × 10¹⁴ is calculated using electrochemical data. The text was submitted by the authors in English.     

Voltammetric determination of hyaluronic acid based on its interaction with phenosafranine

In this paper, a cationic dye of phenosafranine (PSF) was selected as a bioprobe to determine hyaluronic acid (HA) by linear sweep voltammetry on the dropping mercury working electrode (DME). In pH 4.5 Britton-Robinson (B-R) buffer solution, PSF has a well-defined second order derivative linear sweep voltammetric reductive peak at −0.42 V (vs. SCE). After the addition of HA into the PSF solution, the reductive peak current decreased apparently without the movement of reductive peak potential. Based on the decrease of the reductive peak current, a new voltammetric method for HA determination was established. The conditions for the interaction and the electrochemical detection were optimized and the interference substances for the detection were investigated. Under the optimal experimental conditions the difference of peak current was directly proportional to the concentration of HA in the range from 0.8 to 50.0 mg/l with the linear regression equation as Δip″ (nA) = 93.85 + 22.92c (mg/l) (n = 14, γ = 0.995) and the detection limit was calculated as 0.901 mg/l (3). This new method was further applied to determine the HA content in the synthetic samples with satisfactory results and good recovery. The stoichiometry of PSF-HA complexes was calculated and the binding mechanism was also discussed.