测定蛋白质的罗丹明B自聚平衡体系

以罗丹明B单体和二聚体平衡转化为基础，提出了罗丹明B自聚平衡体系测定蛋白质方法；实验表明，罗丹明B在阴离子表面活性剂的作用下自聚成二聚体，体系荧光降低，但是蛋白质加入后体系荧光增强；该法灵敏度高，简便，快速，选择性好，实验结果与传统的溴甲酚绿方法一致。     

荧光素二聚体荧光探针测定卡那霉素的研究

基于卡那霉素对荧光素二聚体具有良好的荧光增强作用,以现场形成的荧光素二聚体作为荧光探针,建立了卡那霉素定量分析的新方法。在碱性条件下（pH=9.0）,当溴化十六烷基三甲基铵（CTAB）浓度为0.4-1.0 mmol/L时,荧光素（FL）在CTAB作用下形成弱荧光现场二聚体,导致荧光猝灭。加入卡那霉素,体系荧光明显增强,且荧光强度与卡那霉素浓度呈良好的线性关系。在优化条件下,卡那霉素的测定线性范围为2.5×10^-3 - 0.1 g/L,检出限（3σ/K）为1.4×10^-4g/L。用于实际样品分析,测定回收率为101%-116%,RSD为1.9%-2.6%。     

荧光猝灭测定痕量钯

报道了Pd(Ⅱ）与碘离子进行氧化还原反应生成游离碘使异硫氰酸荧光素荧光猝灭，借此异硫氰酸荧光素为荧光指示剂间接测定钯的新方法。在pH6.00的条件下，异硫氰酸荧光素的最大激发波长为485nm；最大发射波长为515nm。钯含量在20－200ng/L范围内与体系的荧光强度有良好的线性关系；检出限为6.4ng/L。本法具有灵敏度高、费用低、操作简便、快速等优点，用于样品分析，结果令人满意。     

荧光素-曙红Y能量转移荧光猝灭法测定微量白蛋白研究

在λex/λem=405/547 nm,于缓冲溶液和表面活性剂存在的情况下,荧光素和曙红Y能够发生有效能量转移,而牛血清白蛋白(BSA)的加入使得曙红Y荧光猝灭,该体系可用于微量蛋白质的测定。系统探讨了荧光素-曙红Y能量转移体系发生荧光猝灭的条件,最佳条件为:2.0 mL pH=3.8的B-R缓冲溶液,0.4 mL 0.05%曲拉通X-100,1.5 mL 1.0×10-4mol/L的荧光素水溶液,2.0 mL 1.0×10-4mol/L的曙红Y水溶液,最佳实验时间为溶液配制完成静置15 min后60 min内,最佳加入顺序为pH=3.8缓冲溶液+荧光素+曙红Y+曲拉通+蛋白质标准溶液或样品。在优化的实验条件下,蛋白质含量在0～2.0μg/mL范围内与荧光猝灭强度呈良好的线性关系。检出限为6.6 ng/mL;测定样品的相对标准偏差(RSD)在±5%以内;样品加标回收率为90.4%～95.3%。该法可用于人血清、牛奶中蛋白质含量的测定。     

灿烂甲酚蓝二聚体作为荧光探针测定人血清白蛋白

阳离子染料灿烂甲酚蓝(BCB)在适宜浓度的阴离子表面活性剂十二烷基硫酸钠(SDS)或十二烷基磺酸钠(SLS)存在时形成二聚体。研究了以此形成的现场二聚体作为荧光探针测定蛋白质的可能性。结果表明,二聚体具有弱荧光,其荧光强度的回升与体系中蛋白质的量呈线性关系,线性范围为0～7.8μg·ml-1,检出限为3.89×10-3μg·ml-1。用于人血清白蛋白的测定,结果满意。     

新的吲哚类同型二聚体荧光探针测定蛋白质的分析研究

采用新的水溶性吲哚类同型二聚体探针Ⅰ,建立了一种新的蛋白质荧光分析体系.在酸性条件下,蛋白质分子能与吲哚探针发生结合作用,体系荧光明显增强并向长波方向发生红移,荧光强度与蛋白质质量浓度呈良好的线性关系,在最优条件下,牛血清白蛋白BSA的线性响应范围0.80～25.00 μg/mL,检出限(3σ/K)为0. μg/mL;用于模拟样品的测定,1.00,2.00和5.00 μg/mL的样品测定回收率为99.0%～102.0%,相对标准偏差1.8%～2.8%;与国家标准方法比较,结果测定偏差为1.0%～4.1%.     

基于荧光增强效应的可逆型牛血清白蛋白光化学生物传感器研究

研究了一种具有可逆响应的蛋白质生物传感器。敏感层由丙烯酰胺荧光素固定于硅烷化的平面波导上形成，然后装配在自制的流通池中并用光导纤维连入荧光分光光度计，结果发现牛血清白蛋白（BSA）对此敏感膜有明显的荧光增强反应，在0.4-20μmol/L范围内敏感膜的荧光增强度与BSA的浓度呈线性关系，检测下限为0.1μmol/L。并且此传感器无需特殊的试剂及其进行再生，等电点不同的蛋白质引起的荧光增强程度也不同，常见的金属离子对测定无影响，但重金属离子对测量有较大的影响。对传感器的响应机理也进行了多方面的分析，推测传感器的响应可能是由蛋白质上的氨基与丙烯酰胺荧光素上的羧基发生反应引起的。     

番红花红T与表面活性剂的作用及其在标记DNA中的应用

对阳离子染料番红花红Ｔ（ＳＴ）在阴离子表面活性剂存在时的溶液状态的吸收光谱和荧光光谱进行了研究。结果表明，低浓度阴离子表面活性剂与ＳＴ形成缔合物，导致ＳＴ的吸收与荧光强度降低；增大表面活性剂的浓度，其分子胶束前预聚集促使染料形成非荧光二聚体，导致荧光急剧猝灭，吸收光谱出现新的特征吸收峰；当表面活性剂浓度大于临界胶束浓度（ＣＭＣ）时，染料二聚体离解，ＳＴ单体增溶于胶团中形成新的高量子产率荧光体。本文     

高效液相色谱法测定水体中的痕量一氧化氮

基于4,5-二氨基荧光素与一氧化氮（NO）反应生成高荧光三唑荧光素的原理，建立了一种测定水体中溶解NO的高效液相色谱方法。通过不同流动相（乙腈∶磷酸盐缓冲溶液）配比实验和色谱柱温度梯度实验，确定了该方法测定水体中NO的最佳实验条件：流动相配比为乙腈∶磷酸盐缓冲溶液=8∶92(V/V)；柱温为25℃。在优化条件下，测得NO浓度与荧光强度在0.33～3.32 nmol/L和13.3～266 nmol/L范围内具有良好的线性关系，检出限为90 pmol/L，相对标准偏差为5.1%，平均加标回收率为122.4%。方法可用于测定可口可乐、植物饮料、海水和日本星杆藻藻液中的NO浓度。     

四种荧光素酯的化学物理性质

在紫外线的照射下,荧光素酯能与生物体细胞作用发出鲜艳的黄绿色荧光,由于生物体细胞的生活力不同,细胞内水解酶的含量也不同,因而显出的荧光强度不同。借此性质可以测定细胞内荧光素水解酶的活性以及细胞的生活状态。由于它们是一类选择性好,灵敏度较高的生物荧光染色剂,所以深入研究四种荧光素酯的化学物理性质是有意义的。四种荧光素酯的分子结构可用下面通式表示。     

荧光猝灭法测定壳聚糖含量

在pH 6.3的NaH2PO4-Na2HPO4缓冲溶液中,壳聚糖对荧光素的荧光强度具有明显的猝灭作用,且在一定浓度范围内,其猝灭程度与加入的壳聚糖浓度成线性关系,据此建立了一种新的测定壳聚糖含量的荧光猝灭分光光度法。该方法的回归方程为ΔF=64.02+42.28ρ(mg/L),R2=0.9942,线性范围为0.50~10.0 mg/L,检出限为0.27 mg/L。样品测定的RSD为4.5%(n=6),平均回收率为99.3%。采用该方法可测定复杂样品中的壳聚糖含量。     

一种近红外花菁染料的合成及其应用于生物大分子的测定

光谱性质;蛋白质;核酸测定;一种近红外花菁染料的合成及其应用于生物大分子的测定     

罗丹明6G的溶液状态和荧光特性的研究

罗丹明6G的荧光性质和它的分子聚集态研究是一个重要的科研领域,所得之k_d值表明水溶液中形成罗丹明6G的双聚体。本文提出用荧光法测定k_d聚合常数,该法用于测定罗丹明6G的溶液状态.实验结果表明罗丹明6G双聚体解聚的某些规律,当单体与双聚体平衡趋于单体时,荧光灵敏度就会增加。     

Fiber-optic biosensors based on fluorescence energy transfer

A new optical homogeneous biochemical method for the assay of glucose has been developed, based on fluorescence energy transfer between a glucose analog, dextran labeled with fluorescein isothiocyanate (FITC-dextran), and a glucose-receptor protein, Rhodamine-labeled Concanavalin A (Rh-ConA). When FITC-dextran binds to Rh-ConA in solution, and is light-activated, the FITC label transfers its absorbed energy to the Rhodamine label, which then emits light according to its own characteristic fluorescence spectrum. When glucose is added to this solution, the FITC fluorescence intensity increases as FITC-dextran is released from the Rh-ConA and is replaced by glucose. Thus it is possible to determine glucose concentrations directly from the level of FITC fluorescence.     

Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescence

Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining.     

Studies on the Toluidine Blue Dimer as a Fluorescence Probe for Protein Assays

Abstract

A spectrofluorometric method was developed for the determination of total serum protein by exploring toluidine blue (TB) as the fluorescence probe. The fluorescence intensity of TB at 648 nm was significantly quenched in the presence of sodium dodecylbenzene sulfonate (SDBS) by forming a dimer of the dye, which can afterwards reconvert to monomer when proteins were added accompanied by the recovery of the fluorescence. This might be attributed to the modulated transferring of the dimer‐monomer equilibrium of TB in the anionic surfactant caused by the addition of protein. A linear calibration graph was obtained in the range of 0.5–50 mg/l BSA, with a detection limit of 0.15 mg/l and a RSD of 1.3% (n=11, 5.0 mg/l BSA). Total proteins in human serums were analyzed by using the present procedure and the results agreed well with those obtained by the Biuret method.     

ISOLATION OF UNIQUE DIMER SPECTRA OF DYES FROM THE COMPOSITE SPECTRA OF AGGREGATED SOLUTIONS

Abstract— Aggregation of fluorescein, eosin and rose bengal in aqueous solutions has been studied. Mathematical expressions are derived to calculate the monomer mole fractions in moderately and highly concentrated solutions. The average values of the dimer dissociation constant, K_d , for fluorescein, eosin and rose bengal in aqueous solutions at pH 12 are 0.20, 9.0×10^-3 and 40 × 10^-3 moles/l respectively. A method is developed here to isolate the dimer spectrum from the composite spectrum. The uniqueness of this method is established by comparison with other known methods. The dimer spectra of all the three dyes are reported.     

荧光黄的示波极谱特性研究及其在催化反应-示波极谱分析中的应用

在pH4.9HOAc-NaOAc缓冲溶液中, 荧光黄于-0.50V(vs.SCE)产生一灵敏的单扫描示波极谱波。在100℃弱碱性介质中, 铱(IV)对KIO~4氧化荧光黄这一反应具有强烈催化作用。本文研究了荧光黄的示波极谱行为及利用该催化反应测定痕量铱的各种影响因素,拟定了铱的催化反应-示波极谱分析新方法, 它们的检出限和测定范围分别为0.04ng/mL和0.08-8.0ng/mL。     

Capillary electrophoretic analysis of hydroxyl radicals produced by respiring mitochondria

Here, we report the use of a capillary electrophoretic method with laser-induced fluorescence detection to evaluate hydroxyl radicals produced by respiring mitochondria. The probe, hydroxyphenylfluorescein (HPF), is separated from the product, fluorescein, in under 5 min with zeptomole and attomole limits of detection for fluorescein and HPF, respectively. Purification of the probe with a C-18 SPE column is necessary to reduce the fluorescein impurity in the probe stock solution from 0.4 % to less than 0.001 %. HPF was responsive to hydroxyl radicals produced by isolated mitochondria from L6 cells, and this signal was blunted when DMSO was added to scavenge hydroxyl radicals and when carbonyl cyanide m-chlorophenylhydrazone was added to depolarize the mitochondria. The method was used to compare hydroxyl radical levels in mitochondria isolated from brown adipose tissue of lean and obese mice. Mitochondria from obese mice produced significantly more hydroxyl radicals than those from lean mice.

Study on the formation and depolymerization of acridine orange dimer in acridine orange-sodium dodecyl benzene sulfonate-protein system

Experiment indicates that the fluorescence of acridine orange (AO) can be greatly quenched by anionic surfactant sodium dodecyl benzene sulfonate (SDBS), but when protein is added into the AO-SDBS system, the fluorescence intensity of the latter is enhanced. It is considered that SDBS can promote the formation of AO dimer, resulting in the quenching of the fluorescence of AO. When bovine serum albumin (BSA) is added into AO-SDBS system, BSA and SDBS can interact and form negative micelle-like cluster complex with "aromatic ring stacking," which destroys the formation conditions of AO dimer and makes some AO dimers turn into monomer, resulting in the fluorescence enhancement of AO-SDBS system. Whereas the positive AO and residual AO dimer are dissolved in the negative BSA-SDBS cluster through electrostatic and hydrophobic forces and form a large association. Here, the fluorescence enhancement of AO-SDBS is considered to originate from the hydrophobic microenvironment provided by BSA and SDBS, the depolymerization of AO dimer and intermolecular energy transfer between BSA and AO.