基于丙酮酸/还原型辅酶I/乳酸脱氢酶/氧化型辅酶I/乳酸正逆反应同时测定丙酮酸/乳酸的酶荧光毛细管分析

利用丙酮酸(PA)/还原型辅酶I(NADH)/乳酸脱氢酶(LDH)/氧化型辅酶I(NAD+)/乳酸(LA)荧光反应体系的正逆反应,建立了一种可直接用于临床检验、能同时测定血清中微量PA/LA的酶荧光毛细管分析法.本方法可在常规荧光光度计上,用普通玻璃毛细管同时实现了PA/LA的高灵敏分析,每次分析试剂和样品的用量仅9 ...     

新型乳酸固定化酶荧光毛细生物传感器

对长45 mm、内径0.9 mm的医用毛细管进行γ-氨丙基三乙氧基硅烷氨基化和戊二醛醛基化后,再将乳酸脱氢酶(LDH)的氨基与戊二醛的醛基结合,使其固定在毛细管内壁,构成一种新型固定化酶乳酸荧光毛细生物传感器(IE-LFCBS),实现了对乳酸的微量、快速测定.IE-LFCBS吸入辅酶Ⅰ与乳酸的混合液,在固定化酶催化下使乳酸与辅酶Ⅰ反应,生成荧光物质还原型辅酶Ⅰ;激发波长353 nm、发射波长466 nm.适用于IE-LFCBS的优化条件为:辅酶Ⅰ浓度4 mmol/L、用于固定化的LDH浓度60 kU/L、反应时间15 min、反应温度38 ℃、测定范围为1.0～5.0 mmol/L、回收率95%～98%,IE-LFCBS的相对标准偏差为RSD<1.5%(n=11),检出限为0.45 mmol/L.IE-LFCBS的试液用量极少(18 μL),并能重复使用,可望用于发酵食品、药品、血液标本等各类样品中乳酸的快速检测.     

尿中磺酸化胆汁酸的微量荧光分析法

检测尿中磺酸化胆汁酸(SBA)的浓度可判断人体肝胆系统是否正常。基于双酶催化反应和微量荧光毛细管分析技术,建立了一种新的用于尿中SBA测定的灵敏、准确、简便的微量荧光分析法。方法基本原理为:SBA在胆汁酸磺酸酯酶(BSS)催化作用下水解而脱磺酸基,生成3β-羟胆汁酸;后者在氧化型辅酶-βNAD 存在下,由3β-羟类固醇脱氢酶(3-βHSD)催化而转化为3-酮胆汁酸,同时-βNAD 被还原成NADH;通过NADH的荧光强度来定量SBA。本方法样品无需预处理,测定全过程仅需10 min。荧光强度与SBA浓度呈良好的线性关系,线性范围3.0~50μmol/L,方法检出限为2.2μmol/L,相对标准偏差(RSD)为3%。     

乳酸的液态酶荧光毛细分析法研究

基于乳酸脱氢酶(LDH)和烟酰胺腺嘌呤二核苷酸(NADH)氧化还原体系,提出了乳酸的液态酶荧光毛细分析方法(LE-FCA).在激发波长350nm、发射波长460nm奈件下,用LE-FCA法对乳酸进行了测定;其线性范围为0.2～1.0 mmol/L,r>0.9932,检出限为0.022 mmol/L,RSD<4.2%.LE-FCA能节省贵重的酶试剂,样品用量仅为18μL;可用于医药、卫生、工业、食品等含乳酸样品的测定.     

微波消解-原子荧光光谱法测定化妆品中的砷

建立了微波消解-原子荧光光谱法测定化妆品中微量砷的方法.砷浓度在0～50.00μg/L范围内与荧光强度呈线性关系,线性方程为,If=56.5486c,相关系数r=0.9993,砷的检出限为0.084μg/L.砷测定结果的相对标准偏差为1.49%(n=6),加标回收率为90.7%～107.5%.用该法对环境标准样品进行测定,结果与标准值相吻合.     

裂解加热-荧光毛细管法快速测定血液红细胞内丙酮酸

在常规红细胞处理方法中,用生理盐水对红/白细胞清洗分离会导致细胞内丙酮酸外渗,造成丙酮酸测定值不能反映细胞内丙酮酸的真实浓度。本研究设计了"氯化铵裂解液特异性裂解红细胞-离心分离白细胞/血小板-加热去除蛋白-丙酮酸酶荧光毛细管分析"方法。最优化的血样预处理条件是:以16000 r/min离心血液1 min,获得红细胞;裂解液裂解红细胞后在100℃下加热5 min。对比实验发现,本方法优于其它红细胞处理法。本研究中丙酮酸测定方法的线性范围为10～120 mmol/L;检出限为0.98 mmol/L;灵敏度为5.64 F L/mmol,高于文献方法60倍以上;测定红细胞内丙酮酸值的相对标准偏差小于2.8%(n=11),回收率在98.3%～104.1%之间。     

氢化物发生-原子荧光光谱法测定牡蛎中的镉

利用HNO3–HClO4混合酸消解样品,采用氢化物发生–原子荧光光谱法测定牡蛎中镉的含量。在优化的仪器工作条件下,镉的质量浓度在0.20～1.50μg/L范围内与荧光强度呈良好的线性关系,线性相关系数为0.9992,检出限为0.10μg/L,测定结果的相对标准偏差为4.48%(n=12),加标回收率为94.3%～106.1%。测定了标准物质贻贝GB 08571中镉的含量,测定结果与标准值一致。该方法可满足牡蛎中微量镉的分析测定。     

毛细管胶束电动色谱在线高盐堆积技术检测3-硝基酪氨酸

利用毛细管胶束电动色谱在线高盐堆积技术研究NaNO2-H2O2-氯高铁原卟啉(hemin)-酪氨酸(tyro-sine)体系中3-硝基酪氨酸(3-NT)的测定,建立了一种新的测定生物样品中微量3-NT的方法。最佳分离条件为:含SDS(30mmol/L)的Na2B4O7(36mmol/L)缓冲液(pH9.3),进样压力为3.4kPa,进样时间为30s,毛细管柱温度25℃,分离电压20kV,样品中NaCl100mmol/L。在此条件下,3-NT检出限为0.1μmol/L(S/N=3);线性范围为0.78～50μmol/L;线性相关系数为0.9953。保留时间日内与日间的RSD(n=5)分别为1.6%和2.8%,加标回收率为95.2%～103.1%;样品不同浓度添加水平的日内和日间的RSD(n=5)均小于3%。本方法简单、快速、灵敏度高,为直接分析生物样品中的微量3-NT提供了方法。     

葡萄糖氧化酶-辣根过氧化物酶-葡萄糖-愈创木酚-荧光反应体系测定血糖

利用愈创木酚的荧光特性,将其导入葡萄糖-葡萄糖氧化酶(GOD)-辣根过氧化物酶(HRP)的荧光猝灭反应体系,并与荧光毛细分析技术结合,开发了一种能测定微量葡萄糖的新方法.本方法的的最佳测定条件为:反应时间15 min,反应温度30C,GOD和HRP的浓度分别为4500U/L、2500U/L,愈创木酚浓度为150μmol...     

酶荧光毛细分析法测定葡萄糖

基于酶催化和荧光毛细分析法(FCA)开发了一种微量、快速测定葡萄糖的新方法(GE-FCA)。优选的实验条件为:反应时间15min;反应温度30℃;磷酸盐缓冲液(pH6.0),HRP浓度和GOD浓度分别是200和150U/L;线性范围0.1～4mg/L;检出限0.070mg/L。GE-FCA对血液中葡萄糖进行测定,其回收率在98.9%～103.8%之间。与其它方法相比,GE-FCA法操作简单,而且试样用量少(仅为18μL),节省了酶试剂用量,实验成本低,易于普及推广。     

Spectrophotometric determination of formaldehyde by using formaldehyde dehydrogenase

A new method for the determination of formaldehyde by using formaldehyde dehydrogenase is described. The method is based on the quantitative oxidation of formaldehyde with oxidized nicotinamide adenine dinucleotide (NAD⁺), in the presence of formaldehyde dehydrogenase, to form the reduced dinucleotide (NADH). This enzyme does not require glutathione as a co-factor and the NADH produced, which is directly proportional to the concentration of formaldehyde in the assay solution, is then measured spectrophotometrically at 340 nm. Formaldehyde can be determined in the range 0.3–8.0 μg ml^?1 (1.0×10^?5–2.7× 10^?4 M) with a sensitivity of 0.216 absorbance/ μg ml^?1 (0.0065 absorbance/μM). Optimal conditions and the selectivity of this enzyme toward formaldehyde are described.     

Electrochemical Immunosensor for Lactate Dehydrogenase Detection Through Analyte-driven Catalytic Reaction on Multi-walled Carbon Nanotubes and Gold Nanoparticle Modified Carbon Electrode

A novel electrochemical immunosensor for lactate dehydrogenase (LDH) detection was proposed based on analyte-driven catalytic reaction by attaching LDH antibodies on multi-walled carbon nanotubes (MWCNTs) and gold nanoparticles (AuNPs) modified glassy carbon electrodes (GCE). As LDH was captured by the antibodies on electrode surface, it catalyzed the formation of pyruvate and the reduced form of nicotinamide adenine dinucleotide (NADH), thus a sensitive electrochemical signal obtained from the above redox reaction was recorded by differential pulse voltammetry (DPV). Under optimum conditions, the developed immunosensor exhibits high sensitivity for LDH quantification ranging from 0.001 μg/mL to 0.5 μg/mL with a low detection limit at 0.39 ng/mL. This developed immunosensor reveals ideal accuracy and feasibility for LDH detection in Streptococcus uberis (S. uberis) induced bovine mammary epithelial cells (MECs) samples by comparison with conventional commercial kit, which shows remarkably application potential in diseases diagnosis.     

Voltammetric measurement of reduced nicotinamide-adenine nucleotides and application to amperometric measurement of enzyme reactions.

The electrochemical oxidations of reduced nicotinamide-adenine dinucleotide and reduced nicotinamide-adenine dinucleotide phosphate on platinum and carbon electrodes are described. Well defined voltammetric anodic waves are observed on carbon electrodes, with a linear relationship between peak height and concentration for 0–0.5mM NADH and NADPH. Amperometric methods for NAD oxidoreductase analyses by direct electrochemical oxidation of the reduced nucleotide have been developed for lactic dehydrogenase and ethanol in serum.     

Regeneration of NADH and ketone hydrogenation by hydrogen with the combination of hydrogenase and alcohol dehydrogenase

Applied Biochemistry and Biotechnology - The regeneration of nicotinamide-adenine dinucleotide (reduced form, NADH) by the reaction of NAD with hydrogen gas was carried out in the presence of the...     

Gold nanoparticles: catalyst for the oxidation of NADH to NAD(+)

Nicotinamide adenine dinucleotide is an important coenzyme involved in the production of ATP, the fuel of energy, in every cell. It alternates between the oxidized form NAD(+) and the reduced form dihydronicotinamide adenine dinucleotide (NADH) and serves as a hydrogen and electron carrier in the cellular respiratory processes. In the present work, the catalytic effect of gold nanoparticles on the oxidization of NADH to NAD(+) was investigated. The addition of gold nanoparticles was found to quench the NADH fluorescence intensities but had no effect on the fluorescence lifetime. This suggested that the fluorescence quenching was not due to coupling with the excited state, but due to changing the ground state of NADH. The intensity of the 340 nm absorption band of NADH was found to decrease while that of the 260 nm band of NAD(+) was found to increase as the concentration of gold nanoparticles increased. This conversion reaction was further supported by nuclear magnetic resonance and mass spectroscopy. The effect of the addition of NADH was found to slightly red shift and increase the intensity of the surface plasmon absorption band of gold nanoparticles at 520 nm. This gives a strong support that the conversion of NADH to NAD(+) is occurring on the surface of the gold nanoparticles, i.e. NADH is surface catalyzed by the gold nanoparticles. The catalytic property of this important reaction might have important future applications in biological and medical fields.     

In vivo monitoring the changes of interstitial pH and FAD/NADH ratio by fluorescence spectroscopy in healing skin wounds

The aim of our study was to evaluate the changes of interstitial pH and flavin adenine dinucleotide (FAD)/reduced nicotinamide adenine dinucleotide (NADH) ratio in healing skin wounds using fluorescence spectroscopy in Sprague Dawley rats. In the experiment, excisional and incisional models of wound healing were used. The florescein as the pH-sensitive probe using excitation spectra (lambda(Em) = 535 nm) was used for the measurement of pH changes, and synchronous fluorescence spectra (Deltalambda = 60 nm) for the monitoring of FAD/NADH ratio changes were measured from the surfaces of healing wounds. Increase of interstitial pH and FAD/NADH ratio was recorded during the time interval from the 15th to the 65th minute after surgery. The decrease of pH between the 48th and the 72nd hour after surgery as well as the increase of FAD/NADH ratio between the 72nd and the 96th hour of wound healing were recorded. The results indicate that the use of fluorescence spectroscopy may be considered as a valuable tool for noninvasive in vivo monitoring of selected redox parameters in the early phases of wound healing.     

密闭罐溶样–氢化物发生–原子荧光光谱法测定中药中的汞和砷

建立了密闭罐溶样–氢化物发生–原子荧光光谱法测定中药中痕量汞和砷的分析方法。采用密闭罐溶解复杂基体中药样品,进行易挥发元素分析的样品前处理,技术简单,快速,能耗低。汞和砷的质量浓度分别在0~10μg/L和0~200μg/L范围内与荧光强度成线性关系,线性相关系数均大于0.99。汞、砷的检出限分别为0.014,0.086μg/L;测定结果的相对标准偏差不大于4.67%(n=5);加标回收率分别为99.0%~106.4%,95.2%~101.7%。该方法操作简便,可用于中药中汞、砷元素的质量控制。     

高效液相色谱法测定血浆中舒必利浓度

讨论了测定舒必利血药浓度的反相高效液相色谱方法。选用美国Ｂｉｏ－Ｒａｄ７００型高效液相色谱仪,ＲＰ－３１８色谱柱,ＵＶ检测波长２９０ｎｍ,流动相为甲醇－水－冰醋酸（６０３０１）,内标物为胃复安。回收率为９７．９５％～９９．９６％,ＲＳＤ为２．６％～５．１％,最低检出浓度１．０ｍｇ／Ｌ,线性范围５～１００ｍｇ／Ｌ。测定３０例服药病人的血药浓度,得到了满意的结果。     

A flow-injection method for the amperometric determination of l-lactate with immobilized enzymes and a chemically modified electrode

A sensitive flow-injection system for l-lactate is described. Lactate dehydrogenase, LDH, and glutamic-pyruvic transaminase, GPT, were co-immobilized on a gluteraldehyde-activated porous silica support and used in a packed-bed enzyme reactor. l-lactate is oxidized to pyruvate in the presence of NAD⁺, an equivalent amount of NADH being produced. The equilibrium of the reaction is unfavourable, but by co-immobilizing LDH with GPT and adding l-glutamate, the pyruvate reacts and enough force is obtained to drive the oxidation of l-lactate totally to the product side. The NADH formed is then detected electrocatalytically at an electrode chemically modified with Meldola Blue, operated at 0 mV vs. Ag/AgCl. The system responds linearly to injected samples (25 μl) of l-lactate in the concentration range 10 μM–1.5 mM. The maximum sample throughput is 30 h^?1. The LDH/GPT reactor was stable for four weeks when used daily at optimal pH 8.8.