Time-resolved enzymatic determination of glucose using a fluorescent europium probe for hydrogen peroxide

An enzymatic assay for glucose based on the use of the fluorescent probe for hydrogen peroxide, europium(III) tetracycline (EuTc), is described. The weakly fluorescent EuTc and enzymatically generated H₂O₂ form a strongly fluorescent complex (EuTc–H₂O₂) whose fluorescence decay profile is significantly different. Since the decay time of EuTc–H₂O₂ is in the microseconds time domain, fluorescence can be detected in the time-resolved mode, thus enabling substantial reduction of background fluorescence. The scheme represents the first H₂O₂-based time-resolved fluorescence assay for glucose not requiring the presence of a peroxidase. The time-resolved assay (with a delay time of 60 s and using endpoint detection) enables glucose to be determined at levels as low as 2.2 mol L^–1, with a dynamic range of 2.2–100 mol L^–1. The method also was adapted to a kinetic assay in order to cover higher glucose levels (mmol L^–1 range). The latter was validated by analyzing spiked serum samples and gave a good linear relationship for glucose levels from 2.5 to 55.5 mmol L^–1. Noteworthy features of the assay include easy accessibility of the probe, large Stokes shift, a line-like fluorescence peaking at 616 nm, stability towards oxygen, a working pH of approximately 7, and its suitability for both kinetic and endpoint determination.     

A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine

An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K _D ) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC₅₀ of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 μg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).     

Luminarin 9: A new fluorescent reagent for the precolumn derivatizatioin of main volatile and non-volatile N-nitrosamines in RP-HPLC

Summary A sensitive precolumn fluorescence derivatization method for low level detection of the, volatile (N-nitrosodimethylamine and N-nitrosopyrrolidine) and non-volatile N-nitrosamines (N-nitrosoproline and N-nitrosodiethanolamine) an high-performance liquid chromatography was developed. This method is based on the denitrosation of the compounds of interest by a mixture of hydrobromic acid and acetic acid to produce the corresponding secondary amines. These are, then, able to react with, a quinolizinocoumarin derivative (luminarin 9^®) to form highly fluorescent labelled N-nitrosamine derivatives. The structural elucidation of the luminarin 9^® derivatives of N-nitrosoproline and N-nitrosodimethylamine by way of example, were established by liquid chromatography-mass spectrometry (LC-MS) and by direct chemical ionization-mass spectrometry (CI-MS). The separation, derivatization and detection conditioins were optimized for all the studied compounds. The detection limits (signal to noise ratio=3) were between 0.4 and 1.0 pmol injected depending on the compound. The calibration graphs were linear for derivatized amounts in the range of 0.5–40 nmol for N-nitrosodimethylamine and N-nitrosopyrrolidine, 0.4–2- nmol for N-nitrosoproline and 1.0–40 nmol for N-nitrosodiethanolamine. The repeatability (RSD less than 3.5%, n=6) and reproducibility (RSD less than 4.8%, n-6) were satisfactory.     

Self-assembled luminescent CdSe-ZnS quantum dot bioconjugates prepared using engineered poly-histidine terminated proteins

We report a simple and versatile approach for the conjugation of luminescent CdSe-ZnS core-shell quantum dots (QDs) to proteins through coordination of engineered C-terminal oligohistidine sequences. Several histidine tail containing proteins were self-assembled onto the QD surface using this method. A recombinant antibody specific for the high explosive 2,4,6-trinitrotoluene (TNT) was conjugated to QDs through a carboxy terminal histidine tail and the bioconjugate used to detect TNT by competitive immunoassay. TNT was detected over the range of 10 μg/ml down to 41 ng/ml using the scFv conjugated to QDs. These results open up the possibility to conjugate luminescent QDs to a whole range of proteins to form QD bioconjugates that can be effectively used in bio-oriented applications, such as sensing, imaging, immunoassay and other diagnostics.     

Development of aryl-containing dipyrrolyldiketone difluoroboron complexes (BONEPYs): Tune the hydrogen bond o–CH···F for fluoride recognition

Dipyrrolyldiketone difluoroboron complexes (BONEPYs) were synthesized by condensation of the corresponding pyrroles and malonyl chloride followed by treatment with BF₃·OEt₂. The aryl-substituted pyrrole is introduced to form a cyclic system in order to investigate anion binding studies. In BONEPYs 1–3 the o-H of the aryl group forms hydrogen bonding with F⁻ to give a more table complex. In contrast, the intramolecular hydrogen-bonded BONEPY endo-4 is more stable than its exo isomer. While adding F⁻, the hydrogen bonds must be broken first to give 4·(3)F⁻. Owing to the electron-rich group (-OMe), the o-H of the phenyl group can hardly interact with F⁻ via hydrogen bonding to give the less stable complex 4·(5)F⁻. The energy differences between the different conformations were calculated using DFT methods, which is consistent to the experimental observations.     

稀溶液中环型聚苯乙烯的激基缔合物荧光效应

采用乙酸乙酯为环型和线型聚苯乙烯的非良溶剂, 确定了Mark-Houwink方程分别为[η]_r=1.91×10~(-2)·M~(0.607)和[η]_l=4.06×10~(-2)·M~(0.572)。研究了环型和线型聚苯乙烯的激基缔合物的荧光效应。通过稀溶液中环型和线型高分子线团在柔顺性上存在着差异, 解释了二者之间在荧光效应方面的差异, 并根据实验结果,确定了(I_E/I_M)c→0～M关系式分别为(I_E/I_M)r,c→0=1.91+3.40×10~(-6)M和(I_E/I_M)l,c→0=2.15+3.05×10~(-6)M。     

Development of ELISA and immunochromatographic assay for ofloxacin

Two rapid,sensitive and reliable immunoassay methods,namely competitive indirect enzyme-linked immunosorbent assay(CI- ELISA)and colloidal gold-based immunochromatographic assay(CGIA),were developed to detect ofloxacin(OFL).The linear range of the CI-ELISAwas from 0.5 to 128 ng/mL with a limit of detection(LOD)of 0.35 ng/mL.Good recoveries were obtained in analyzing simulated swine urine samples.The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min,and test results were read visually without any instrument.     

Development of an indirect competitive ELISA for the determination of papaverine

Papaverine (1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, PAP) is a member of the benzylisoquinoline sub-group of the opium alkaloids. It has been widely used for treating diseases like pulmonary arterial embolism and renal or biliary colic. In this paper, a specific conjugate of mono-demethylated papaverine-O-carboxylmethyl ether (MDMPAP-O-CME) and bovine serum albumin (BSA) was synthesized and used as the complete antigen (PAP-BSA), with which we successfully obtained a high-titer anti-PAP polyclonal antibody (pAb) by immunization of rabbits. The anti-PAP pAb showed high affinity to papaverine with an affinity constant (K_aff) of 7.3 × 10⁷ L/mol. With this antibody, we established a sensitive immunochemical method for the determination of papaverine based on indirect competitive enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of the coated antigen (PAP-OVA) and purified pAb used in the ELISA were 5 and 1.2 μg/mL, respectively. The cross reactivity of other benzylisoquinoline derived substances, including 1-(3,4-dihydroxybenzyl)-7-hydroxy-6-methoxy-isoquinoline (6-methoxy-papaveroline, MPAPO), morphine (MP) and codeine (CD) were all lower than 1%. The linear range of the calibration curve was 0.1-1000 ng/mL. Normal human serum samples were spiked with known amount of papaverine and measured by the ELISA. Recoveries were between 102% and 105%. Papaverine content in a commercial papaverine hydrochloride injection sample was also determined using the established ELISA. Compared with the results given by the control experiment of HPLC, the recoveries of ELISA to detect injection samples were 102-110%. The limits of detection for synthetic serum samples and injection samples of papaverine hydrochloride were 0.25 and 0.06 ng/mL, respectively.     

2-(苯并噻唑-2-基)-5-(N,N-二乙基氨基)苯酚衍生物的合成及其对H2S的识别

以4-N,N-二乙基氨基水杨醛为原料,制备了2-(苯并噻唑-2-基)-5-(N,N-二乙基氨基)苯酚衍生物(探针L),并对其结构进行了表征。在DMSO/PBS(体积比3∶7,pH=7.4)溶液中,探针L具有高选择性并可荧光"关-开"识别H_2S,在365nm紫外灯照射下,由无荧光变成蓝色荧光。实验表明,探针L识别H_2S的检测限为2.05×10~(-6)mol/L,pH适用范围为6～9,可用于检测实际水样中的H_2S。     

荧光探针法研究PEO-PPO嵌段共聚物胶束的特性

利用十六烷基罗丹明Ｂ作为主要荧光探针，研究了水溶性嵌段共聚物Ｐｌｕｒｏｎｉｃ Ｆ－６８在水溶液中的胶束化行为，研究表明，Ｐｌｕｒｏｎｉｃ－Ｆ－６８形成胶束的监界胶束浓度与温度有极大关系，随温度的升高，其ＣＭＣ值急剧下降，并且温度对胶束的性质也有很大，影响温度升高可导致胶束的微观粘度增大，表现出很强的负粘－温效应，并发现引起这种负粘－温效应的主要原因是Ｐｌｕｒｏｎｉｃ Ｆ－６８分子中聚环氧乙烷（ＰＥ