卤代荧光素与西布曲明的荧光猝灭和褪色光谱及其分析应用

在弱酸性缓冲溶液中,乙基曙红(EE)、赤藓红(ET)和荧光桃红(PX)3种卤代荧光素与盐酸西布曲明(SH)形成离子缔合物,导致吸收光谱发生变化和荧光猝灭。研究了反应产物的吸收和荧光光谱特征,适宜的反应条件,据此建立了以卤代荧光素为光谱探针的灵敏、简便、快速测定SH的新方法。其中ET体系褪色反应灵敏度最高,对SH的线性范围为0.1～4.0μg/mL,检出限为0.06μg/mL;PX-SH体系的荧光猝灭法对SH的线性范围是0.2～4.6μg/mL(λex/λem=540nm/560nm),检出限为0.09μg/mL。讨论了离子缔合反应对荧光及吸收光谱的影响及卤代荧光素荧光猝灭和褪色的原因。     

毛细管电泳-激光诱导荧光检测法分离检测谷胱甘肽及其构成氨基酸

以4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD-Cl)为柱前衍生试剂,建立了一种毛细管电泳-激光诱导荧光直接检测氧化型和还原型谷胱甘肽及其构成氨基酸(谷氨酸、半胱氨酸和甘氨酸)的新方法。经过实验条件的优化,采用25 mmol/L硼砂-20 mmol/L聚氧乙烯月桂醚(Brij-35)-5%乙腈(pH 9.5)的缓冲体系,在柱温为25°C、分离电压为20 kV的条件下,压力进样3447.5 Pa(0.5 psi)×3 s,五种物质在11 min内实现高效基线分离。在该方法下,还原型谷胱甘肽、氧化型谷胱甘肽、谷氨酸、半胱氨酸和甘氨酸的线性范围分别为:1～50μg/mL,1～50μg/mL,0.5～25μg/mL,1～50μg/mL,0.1～20μg/mL;检测限分别为:0.1μg/mL,0.1μg/mL,0.005μg/mL,0.1μg/mL,0.001μg/mL。以还原型谷胱甘肽钠粉针剂为样品,方法的加标回收率为99.5%～110.7%,相对标准偏差为0.26%～3.272%(n=3)。该方法准确、快速、灵敏、检测限低,有望用于样品中氧化型和还原型谷胱甘肽及其构成氨基酸的含量分析。     

DNA荧光探针—荧光素-中性红体系的研究

基于DNA对荧光素(FL)-中性红(NR)分子间荧光能量转移的抑制作用，以荧光素-中性红为荧光探针，考察该探针与DNA的结合反应，建立了准确测定DNA的新方法，在pH＝6.5条件下，hsDNA、ctDNA和smDNA的浓度与荧光素-中性红体系的荧光比值变化量Δ(Fd／Fa)成线性关系，响应线性范围分别为0．25-6．25μg／mL、0．10-5．00μg／mL、0．10-4．00μg／mL，0.025μg/mL和0．023μg／mL；分析测定了DNA合成样品，回收率91．3％-101．4％，相对标准偏差小于4．2％．     

光谱法研究核固红-Cu2+-阿莫西林体系及其分析应用

利用阿莫西林与Cu2+络合导致Cu2+对核固红的荧光猝灭程度减弱的特性,建立了荧光增强分析法检测阿莫西林含量的新方法。讨论了体系的紫外-可见吸收光谱、荧光光谱以及酸度、试剂浓度、反应温度、反应时间和离子强度等因素对体系荧光强度的影响。在最佳实验条件下,荧光强度的增强程度与阿莫西林质量浓度呈线性关系,线性范围为0.40~15.0μg/mL,检出限为0.29μg/mL。考察了一些金属离子和药物赋型物对体系的影响。将方法用于市售阿莫西林胶囊分析,回收率在100.4%~103.4%之间。     

荧光猝灭法测定药物中异烟肼含量

在pH 6.5的Walpole缓冲溶液中,当以300 nm的光激发时,1,2-萘醌-4-磺酸钠在468 nm处产生1个较强的荧光峰。加入异烟肼后,由于其与1,2-萘醌-4-磺酸钠发生反应,从而使体系颜色加深且荧光信号减弱。在最佳实验条件下,在0.10~8.0μg/mL范围内,体系的荧光信号△F与异烟肼浓度之间有较好的线性关系,检出限为40.2 ng/mL。方法可直接用于测定药物中异烟肼含量,回收率为96.5%~106.3%。     

荧光光度法测定饮料中的铅

对荧光光度法测定饮料中的铅进行实验研究,在3.0mol/L盐酸介质中,Pb2 与Cl-形成PbCl2-4配合物,此配合物在紫外光照射时发出蓝色荧光,应用荧光光度计检测出铅的含量.采用HNO3-HClO4混合酸体系(体积比为8:1)消化饮料样品.试验了盐酸用量、显色时间、干扰离子对铅测定的影响.该方法的检出限为9.0×10-3μg/mL,线性范围为0.3～9.0μg/mL,回收率为97.3%～100.2%.     

流式细胞术散射光谱法测定痕量银的研究

以TritonX_100为乳化剂,用AgNO3与NaCl反应制备了AgCl微粒体系。应用流式细胞仪和荧光光度计分别研究了液相流式AgCl微粒体系的流式细胞术(FCM)光散射及动态散射光谱(DLS)特性,建立了流式细胞术散射光检测痕量Ag+的新方法。实验发现,Ag+质量浓度分别在1.50×10-4~5.00μg/mL、4.80×10-3~20.99μg/mL范围内,流式、动态AgCl微粒体系散射光强度与Ag+质量浓度都呈现线性关系(r=0.995 0和0.999 8),方法检出限分别为0.045、1.50 ng/mL。     

碘化物-CTMAB体系共振光散射法测定电镀废水中的钯

用简易荧光计研究了在微酸性介质中钯-碘化钾-溴化十六烷基三甲基铵体系的共振光散射光谱,考查了光谱特征、影响因素和适宜的反应条件,确定了散射光强度与溶液中钯浓度的关系,提出了共振光散射法测定钯的方法。钯的线性范围为0.0~0.5μg/mL,检出限为0.005μg/mL。该法可用于电镀废水中钯浓度的测定。     

某些芳香族氨基酸作探针荧光猝灭法测定安乃近及其代谢产物

在pH3.2的缓冲介质中,安乃近(ANG)及其代谢产物4-甲氨基安替比林(MAA)、4-乙酰氨基安替比林(AAA)与色氨酸(Trp)、酪氨酸(Tyr)和苯丙氨酸(Phe)等芳香族氨基酸反应并形成结合产物,引起上述氨基酸的荧光发生猝灭,最大猝灭波长分别位于352nm(ANG-Trp体系)、304nm(ANG-Tyr,MAA-Tyr和AAA-Tyr体系)和284nm(ANG-Phe体系).其荧光猝灭值(ΔF)在一定范围内与ANG,MAA和AAA成正比.荧光猝灭反应具有较高灵敏度,对于ANG,MAA和AAA的检出限为13.3ng/mL(ANG-Trp体系)、15.8ng/mL(ANG-Tyr体系)、64.5ng/mL(ANG-Phe体系)、150.0ng/mL(MAA-Tyr体系)和230.8ng/mL(AAA-Tyr体系).实验研究了荧光猝灭反应的适宜条件和影响因素,考察了共存物质的影响,表明方法具有良好的选择性,可用于ANG片剂及其代谢物尿药浓度的快速测定.从吸收光谱的变化、温度的影响以及Stern-Volmer作图,判断该反应为静态猝灭反应,氨基酸和安乃近通过静电引力和芳基堆积作用而形成1:1的复合物.     

浊点萃取-同步荧光法测定痕量荧光增白剂VBL

根据表面活性剂和荧光增白剂之间的相互作用,提出了浊点萃取-同步荧光法测定纸制样品中痕量荧光增白剂VBL的新方法。研究发现,相比于传统荧光发射光谱,当最佳波长差为30 nm时,VBL在402 nm处有一强度高、峰型窄的同步荧光峰。体系的相对荧光峰强度与VBL的浓度在0.001~0.053μg/mL范围内呈良好的线性关系,相关系数0.9993,线性方程为IF=10729.48ρ(μg/mL)+42.36,检测限(3S/K)为8.415×10-4μg/mL。方法用于样品测定,回收率在90.6%~102%之间,相对标准偏差在1.3%~5.2%之间。     

Fluorescence quenching method for determination of trace tungsten in environmental samples with dibromohydroxyphenylfluorone

A highly sensitive and selective fluorescence quenching method has been developed for the determination of trace tungsten in environmental samples using dibromohydroxyphenylfluorone (DBHPF) as an emission reagent. In the presence of 0.04?mol/L of sulphuric acid and acetyltrimethylammonium bromide, tungsten(VI) reacts with DBHPF to form a 1?:?3 red complex within 5.0?min. In order for the DBHPF–tungsten(VI) complex to form, the fluorescence intensity of the reagent solution was quenched linearly by adding 0.1 to 1.0?µg of tungsten(VI) in 25?mL of solution. This was measured at 528?nm with excitation at 495?nm. In this work, a standard addition method was investigated and used for sample analysis. The decrease in fluorescence intensity of the reagent solution (ΔF) was linear for 0?～?0.9?µg of tungsten(VI) in 25?mL of solution, and the detection limit (3?s) of the standard addition method was found to be 0.012?ng/mL of tungsten(VI). The effects of various metal and nonmetal ions were studied in detail. The experiments clearly showed that most foreign ions can be tolerated in considerable amounts; in particular, 50-fold Mo(VI), V(V), Zr(VI) and Ti(IV) do not interfere, and the selectivity of the proposed method is better than other previously described methods. Moreover, the method proposed here is very stable and simple, the fluorescence intensity of the solution can remain almost unchanged for 2.0?h at room temperature, and the method has been used successfully to determine tungsten in environmental samples.     

氨基酸的2,3-二甲醛基喹喔啉荧光衍生及高效液相色谱分离分析

将荧光试剂2,3-二甲醛基喹喔啉首次应用到氨基酸的高效液相色谱分离分析中。考察了该荧光试剂与伯胺氨基酸衍生反应的pH值、比例、反应时间等影响因素,确定衍生反应的最佳条件为:100 mmol/L硼砂缓冲液(pH 9.5),二巯基乙醇、衍生试剂与氨基酸的比例为9∶3∶1,室温下反应5 min。采用C18色谱柱,经二元梯度洗脱,荧光检测,17种伯胺蛋白氨基酸得到较好分离。氨基酸的浓度在0.01～1.00 mmol/L范围内与其衍生物峰面积呈较好的线性关系,氨基酸衍生物的检出限为0.02～0.07μg/L。应用该方法对麦冬中游离氨基酸的含量进行测定,结果满意。此方法快速,高效,灵敏度高,精密度好。     

The Application of Gibberellic Acid to the Determination of Trace Amounts of Lead by Spectrofluorimetry

The ionization constant of fluorescent reagent gibberellic acid (GA) was established spectrophotometrically. The fluorescent reaction of this reagent with lead was studied. Based on this chelation reaction, a sensitive, direct spectrofluorimetric method for the determination of trace lead with use of GA has been developed. The reaction conditions for the fluorescence system of lead with GA were studied. The lead ion can form a stable binary chelate with GA, having a ratio of 1:2 in the pH range 7.0‐8.0. The maximum excitation and emission wavelengths are 205.0 nm and 308.8 nm for the lead chelate, respectively. The reaction is instantaneous and the fluorescence intensity of the lead chelate remains stable from 20 to 150 min. Under the optimal experimental conditions the fluorescence intensity is a linear function of concentration in the range 1.0‐10.0 ng/mL of lead and the detection limit is 0.52 ng/mL of lead. Interferences of other ions were studied. The method has been successfully applied to the determination of lead in common paint.     

A Study of Bioactivity of Corn Peptides with Low Molecular Weight Ⅱ： Effect on Plasma Free Amino Acid Concentrations in Rats

The effects of the ingestion of corn peptides with a low molecular weight(LMCP) prepared from zein on some plasma free amino acid concentrations in rats that had taken ethanol were investigated. LMCP(1.0g/kg body weight) in 15~ ethanol(10 mL/kg body weight) was given to Wister rats by intragastrical administration. The amino acid analysis showed that the concentrations of aianine, leucine, and proline in the plasma reached their maximum levels at 30 min for the LMCP-intake group. They are 582.39, 99.60 and 272.51 μg/L, respectively. But in the control group, the plasma free amino acid levels were not changed obviously.Therefore, LMCP could cause an increase in concentration of some free amino acids such as alanine, leucine and proline etc. in plasma of the rats that have taken ethanol.     

Simultaneous analysis of aspartame and its hydrolysis products of Coca-Cola Zero by on-line postcolumn derivation fluorescence detection and ultraviolet detection coupled two-dimensional high-performance liquid chromatography

An innovative two-dimensional high-performance liquid chromatography system was developed for the simultaneous analysis of aspartame and its hydrolysis products of Coca-Cola Zero. A C8 reversed-phase chromatographic column with ultraviolet detection was used as the first dimension for the determination of aspartame, and a ligand-exchange chromatographic column with on-line postcolumn derivation fluorescence detection was employed as the second dimension for the analysis of amino acid enantiomers. The fluorimetric derivative reagent of amino acid enantiomers was o-phthaldialdehyde. The hydrolysis of aspartame in Coca-Cola Zero was induced by electric-heating or microwave heating. Aspartame was quantified by the matrix matched external standard calibration curve with a linear concentration range of 0-50 μg mL(-1) (r(2)=0.9984). The limit of detection (LOD) and the limit of quantification (LOQ) were 1.3 μg mL(-1) and 4.3 μg mL(-1), respectively. The amino acid enantiomers was analyzed by the matrix matched internal standard calibration method (D-leucine as the internal standard) with a linear concentration range of 0-10 μg mL(-1) (r(2)=0.9988-0.9997). The LODs and LOQs for L- and D-aspartic acid and L- and D-phenylalanine were 0.16-0.17 μg mL(-1) and 0.52-0.55 μg mL(-1), respectively, that was 12-13 times more sensitive than ultraviolet detection. The overall analysis accuracy for aspartame and amino acid enantiomers was 90.2-99.2% and 90.4-96.2%, respectively. The overall analysis precision for aspartame and amino acid enantiomers was 0.1-1.7% and 0.5-6.7%, respectively. Generally, the extent of aspartame hydrolysis increases with the increase of electro-thermal temperature, microwave power, and the duration of hydrolysis time. D-aspartic acid and D-phenylalanine can be observed with the electro-thermal racemization at the hydrolysis temperature 120°C for 1 day and only D-aspartic acid can be observed at the hydrolysis temperature 90°C for 2 and 3 days. For the microwave induced hydrolysis, only L-aspartic acid was detected at the power 560 W for 1 min and 320 W for 3 min.     

Fluorimetric determination of secondary amino acids by 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole

The fluorigenic reaction of proline with 7-fluoro-4-nitrobenzo-2-oxa-l,3-diazole (NBD—F) is superior, in terms of reactivity and fluorescence yield, to the reactions with the analogous 7-chloro and 7-bromo derivatives. With NBD—F, the reagent blank fluorescence can be suppressed by adjusting the medium to around pH 1 with hydrochloric acid. Many secondary amino acids can be determined by reaction with NBD—F at pH 7.5 at 70°C for 5 min and subsequent acidification to pH 1. The detection limits for proline, hydroxyproline and sarcosine are 0.08, 0.04 and 0.17 nmol ml^-1, respectively. Under the same conditions, the primary amino acids, alanine, arginine and aspartic acid, are detected at 1.7, 1.7 and 3.4 nmol ml^-1, respectively.     

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。     

铝试剂的荧光光谱与荧光量子产率

首次研究了铝试剂的荧光光谱和荧光量子产率,发现pH3至pH12条件下,用紫外光照射铝试剂溶液可以产生荧光,最大激发波长和最大发射波长分别为297nm和409nm,荧光强度与铝试剂浓度之间存在良好的线性关系,线性范围为0.01～3μg/mL,检测下限为0.01μg/mL,以硫酸奎宁为参比,测得铝试剂的荧光量子产率为0.16。     

功能性CdS纳米荧光探针荧光增敏法测定人血清白蛋白

目前 ,以荧光分析法对蛋白质进行研究主要采用有机荧光探针[1～ 3 ] .与传统的有机染料 (如罗丹明 )探针相比 ,半导体纳米晶体探针的光强度要高 2 0倍 ,光稳定性要高 1 0 0倍 ,谱线宽度只是有机染料谱线宽度的 1 /3 [4 ] .将半导体纳米晶体作为探针用于测定生物分子 ,将大大提高分析的灵敏度和选择性 ,而目前其应用于生物染色、医疗诊断、DNA序列测定和免疫分析等方面的研究很少 [4 ,5] .本文合成了胶态纳米粒子 Cd S,并在其外表面修饰一层巯基乙酸 ,使其具有水溶性 ,并能与生物分子作用 ,从而可利用其外表面的功能性基团对人血清白蛋白进…