氯乙基亚硝基脲导致DNA股间交联的荧光光谱法研究

氯乙基亚硝基脲(CENUs)是重要的临床抗癌药物,其抗癌作用机制与导致DNA股间交联密切相关。使用荧光光谱法对其导致的DNA股间交联进行定量分析。结果表明,交联率随着反应时间的延长逐渐增加,司莫司汀(Me-CCNU)和卡莫司汀(BCNU)的交联率分别约在8 h和5 h达到最大值,且药物浓度越大,交联率增加越快。比较了Me-CCNU和BCNU对DNA交联作用的反应动力学,发现分解较慢的Me-CCNU与DNA的交联过程中存在一段明显的"诱导期";而分解较快的BCNU与DNA的交联反应则不存在"诱导期",且BCNU浓度过高或反应时间过长均会使交联率下降。该文为阐明CENUs导致DNA交联的反应动力学和反应机理提供了依据。     

毛细管电泳-电致化学发光法分离并测定盐酸倍他司汀

碱性介质中,盐酸倍他司汀对三联吡啶钌电致化学发光起增敏作用,联用毛细管电泳,建立了一种简便、快速、准确、灵敏的检测盐酸倍他司汀含量的新方法.在优化的条件下,盐酸倍他司汀迁移时间:180 s,线性范围:5×10-7～1.5×10-4mol/L(r=0.9996),检出限(S/N=3):5×10-8mol/L,迁移时间与峰高的相对标准偏差分别为0.4%和2.8%.     

Fe3O4磁性纳米材料萃取光度法测定苯磺酸倍他司汀

提出了基于表面活性剂磁性固相萃取光度法测定药片及血清中苯磺酸倍他司汀含量的方法。在表面活性剂十二烷基磺酸钠(SDS)的辅助下,磁性材料四氧化三铁可以高效的定量萃取苯磺酸倍他司汀。对实验条件如缓冲溶液pH及用量、磁性材料用量、表面活性剂用量、萃取时间、洗脱剂种类及用量、材料重复利用率等进行优化。在优化条件下,吸附率与洗脱率均>90%,磁性纳米材料可重复使用5次,方法的回收率为95.2%~105.9%。方法可用于测定人血清及药物中苯磺酸倍他司汀含量。     

超分子溶剂萃取/超高效液相色谱-串联质谱法测定血浆中他克莫司含量

该文建立了血浆中免疫抑制剂他克莫司（TAC）的超分子溶剂（SUPRAS）萃取/超高效液相色谱-串联质谱分析方法。通过单因素实验结合响应面设计对超分子溶剂组成、用量及涡旋萃取时间等关键因素进行优化后,血浆样本以正戊醇、四氢呋喃和水形成的超分子溶剂进行高效萃取。萃取液经Waters ACQUITY UPLC BEH C18(50 mm×2.1 mm,1.7μm)色谱柱分离后,在电喷雾质谱正离子模式下,以多反应监测（MRM）模式对他克莫司进行测定,内标法定量。结果表明,他克莫司在0.5～30 ng/mL质量浓度范围内的线性关系良好,相关系数（r）为0.998 6;方法检出限和定量下限分别为0.1、0.5 ng/mL;在低、中、高3个加标水平下,平均回收率（n=3）为91.9%～99.9%,相对标准偏差（RSD）为1.7%～5.7%。所建立的方法快速、灵敏、稳定,适用于血浆中他克莫司的准确测定。     

高效液相色谱法测定盐酸倍他司汀片的有关物质

采用Kromasil C18分析柱,水-乙腈(体积比为68：32)混合液为流动相,流速为1．0ml/min,检测波长为261nm,用高效液相色谱法测定盐酸倍他司汀片的有关物质。盐酸倍他司汀样品溶液中有关物质的色谱峰面积之和及2％盐酸倍他司汀线性对照溶液中主成分的色谱峰面积均与盐酸倍他司汀的浓度线性相关,相关系数分别为0．9991、0.9997。     

大环内酯类免疫抑制剂他克莫司的生物合成机制研究进展

他克莫司(FK506)是一种来源于土壤链霉菌的大环内酯类免疫抑制剂,由典型的聚酮合酶(PKS)-非核糖体肽合成酶(NRPS)杂合系统负责催化其生物合成.他克莫司的化学结构特殊,包括骨架环的哌啶单元、4-羟基-3-甲氧基环己基官能团,以及甲氧基和烯丙基侧链.近年来,关于他克莫司的生物合成机制,特别是其特殊前体的形成途径的研究发展迅速.对他克莫司生物合成的酶学基础进行了系统性地综述,重点总结了其前体形成机制的研究新进展.     

盐酸倍他司汀对鲁米诺-高碘酸钾体系后发光作用机理的探讨及其快速测定

实验发现盐酸倍他司汀对鲁米诺-高碘酸钾发光体系具有后化学发光作用，并对其作用机理进行了探讨。结合流动注射技术，优化了反应条件，并建立了测定盐酸倍他司汀的新方法。该方法简便、快速、准确，线性范围为2.0×10^-7－4.0×10^-4mol／L（r=0.9975），检出限为1.0×10^-7mol／L；对4.0×10^-5mol／L的盐酸倍他司汀进行了11次平行测定，相对标准偏差为2.9％。方法成功地用于盐酸倍他司汀片中盐酸倍他司汀的测定。     

刺激响应双硒交联聚乙烯亚胺基因微载体

以丙酸二硒醚为交联剂,通过调节交联剂的用量及反应时间,制备了4种双硒交联聚乙烯亚胺(PEISeSe),研究了其对脱氧核糖核酸(DNA)缔合能力、质子缓冲能力和转染效率的影响.结果表明,随着交联程度的增大,质子缓冲能力降低.PEISeSe能有效诱导DNA的缔合,当聚合物与DNA的质量比≥8时,PEISeSe/DNA组装体可形成150 nm的粒子.在模拟细胞内的还原性环境下,双硒键能有效断裂,显示出很好的响应特性.将氯喹与PEISeSe/DNA组装体同时加入到HEK293T细胞中,氯喹的存在有利于PEISeSe/DNA组装体逃离溶酶体.在细胞内高浓度还原型谷胱甘肽的作用下,PEISeSe交联聚合物可被降解为低分子量的PEI片段,有利于释放出DNA并进攻细胞核,提高转染率并降低毒性.     

亚硝基脲对DNA损伤作用的研究进展

亚硝基脲是重要的抗癌烷化剂,应用于多种恶性肿瘤的临床治疗。亚硝基脲分解生成活泼亲电中间体而与生物大分子作用,导致DNA碱基烷化、互补碱基对横向交联等损伤,最终诱导癌细胞凋亡而发挥其抗癌作用。由于亚硝基脲在动物实验和临床应用中表现出的复杂生理活性,有关亚硝基脲对DNA损伤作用机制的研究,已成为该类药物设计开发以及癌症防治领域中的热点问题。本文对近年来国内外有关亚硝基脲对DNA损伤作用机制的研究进行了综述。     

N-亚硝基化合物导致DNA股间交联的HPLC-ESI-MS/MS分析

N-亚硝基化合物(NNCs)是一种重要的环境致癌物,它们能够导致DNA股间交联从而诱发癌症.研究表明NNCs经过代谢活化或分解后,最终以乙撑的形式导致DNA互补碱基对成对负性原子之间的共价交联.     

DNA interstrand crosslinks are induced in cells prelabelled with 5-bromo-2'-deoxyuridine and exposed to UVC radiation

It has been observed previously that 5-bromo-2'-deoxyuridine (BrdU) potentiates the effect of UVC radiation on the level of sister chromatid exchanges. It is not known which type of DNA damage is responsible for this enhancing effect and we have proposed this to be the DNA interstrand crosslink (ICL) which, theoretically, may arise in cells that are labelled with BrdU for one round of replication and exposed to UVC radiation. The aim of the present investigation was to verify if ICLs are indeed formed during this irradiation scenario. CHO-K1 cells were prelabelled with BrdU and exposed to UVC. ICLs were detected by a modified version of the comet assay that relies on the reduction of induced DNA migration in the agarose gel. Carboplatin was used as a positive control. We found that BrdU+UVC treatment indeed results in a reduction of the damage induced by gamma-radiation. Furthermore, we observed that CL-V4B cells exposed to BrdU+UVC, but not to UVC alone, showed a very high level of chromosomal damage. These cells have a deficient Rad51C paralog that renders them extremely sensitive towards ICLs. Interestingly, the cytogenetic results did not correlate with cell survival, where it was found that the CL-V4B cells tolerate BrdU+UVC better than the wild type cells. The possible reasons are discussed. Taken together our results indicate that ICLs are formed in DNA that was prelabelled with BrdU and exposed to UVC radiation.     

Efficient All-Perovskite Tandem Solar Cells with Low-Optical-Loss Carbazolyl Interconnecting Layers

Combining wide-band gap (WBG) and narrow-band gap (NBG) perovskites with interconnecting layers (ICLs) to construct monolithic all-perovskite tandem solar cell is an effective way to achieve high power conversion efficiency (PCE). However, optical losses from ICLs need to be further reduced to leverage the full potential of all-perovskite tandem solar cells. Here, metal oxide nanocrystal layers anchored with carbazolyl hole-selective-molecules (CHs), which exhibit much lower optical loss, is employed to replace poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT : PSS) as the hole transporting layers (HTLs) in lead-tin (Pb-Sn) perovskite sub-cells and ICLs in all-perovskite tandem solar cells. Optically transparent indium tin oxide nanocrystals (ITO NCs) layers are employed to enhance anchoring of CHs, while a mixture of two CHs is adopted to tune the surface energy-levels of ITO NCs. The optimized mixed Pb-Sn NBG perovskite solar cells demonstrate a high PCE of 23.2 %, with a high short-circuit current density (J_sc) of 33.5 mA cm⁻². A high PCE of 28.1 % is further obtained in all-perovskite tandem solar cells, with the highest J_sc of 16.7 mA cm⁻² to date. Encapsulated tandem solar cells maintain 90 % of their reference point after 500 h of operation at the maximum power point (MPP) under 1-Sun illumination.     

A novel method for time-resolved fluorimetric determination and imaging of the activity of peroxidase, and its application to an enzyme-linked immunosorbent assay

A new type of fluorescence assay for the determination of peroxidase (POx) activity is presented. The assay is based on the indication of the enzymatic consumption of H(2)O(2) (HP), using a fluorescent europium-tetracycline (Eu(3)TC) complex as indicator. On addition of HP, this complex forms a highly fluorescent adduct (Eu(3)TC-HP), which is decomposed in the presence of POx to form the weakly fluorescent europium-tetracycline (Eu(3)TC). Hence, the activity of the enzyme can be directly determined by means of the luminescent Eu(3)TC complex as indicator. The POx assay demonstrated herein was elaborated starting from a spectral characterization of the complex systems involved. Due to the long lifetime of lanthanide luminescence, both steady-state and time-resolved luminescence assays can easily be performed. The time-resolved assay can quantify POx in the range from 4.0 x 10(-5) to 5.9 x 10(-3) U mL(-1), with a limit of detection of 1.0 x 10(-5) U mL(-1). The effects of POx inhibitors such as cyanide, hydroxylamine, and azide have also been studied. In addition, a time-resolved fluorescent detection method for a POx-based enzyme-linked immunosorbent assay (ELISA) has been developed, which is demonstrated in a sandwich model assay with bovine IgG serving as analyte. Furthermore, a time-resolved fluorescent imaging method is demonstrated that makes use of a straightforward imaging set-up adjusted to the optical properties of the europium reagent.     

Fluorescent nanoparticle adhesion assay: a novel method for surface pKa determination of self-assembled monolayers on silicon surfaces

Since the computer industry enables us to generate smaller and smaller structures, silicon surface chemistry is becoming increasingly important for (bio-)analytical and biological applications. For controlling the binding of charged biomacromolecules such as DNA and proteins on modified silicon surfaces, the surface pK(a) is an important factor. Here we present a fluorescent nanoparticle adhesion assay as a novel method to determine the surface pK(a) of silicon surfaces modified with weak acids or bases. This method is based upon electrostatic interactions between the modified silicon surface and fluorescent nanoparticles with an opposite charge. Silicon slides were modified with 3-aminopropyltriethoxysilane (APTES) and were further derivatized with succinic anhydride. Layer thickness of these surfaces was determined by ellipsometry. After incubating the surfaces with an amine-reactive fluorescent dye, fluorescence microscopy revealed that the silicon surfaces were successfully modified with amine- and carboxyl-groups. Two surface pK(a) values were found for APTES surfaces by the fluorescent nanoparticle adhesion assay. The first surface pK(a) (6.55 ± 0.73) was comparable with the surface pK(a) obtained by contact angle titration (7.3 ± 0.8), and the second surface pK(a) (9.94 ± 0.19) was only found by using the fluorescent nanoparticle adhesion assay. The surface pK(a) of the carboxyl-modified surface by the fluorescent nanoparticle adhesion assay (4.37 ± 0.59) did not significantly differ from that found by contact angle titration (5.7 ± 1.4). In conclusion, we have developed a novel method to determine the surface pK(a) of modified silicon surfaces: the fluorescent nanoparticle adhesion assay. This method may provide a useful tool for designing pH-dependent electrostatic protein and particle binding/release and to design surfaces with a pH-dependent surface charge for (bio-)analytical lab-on-a-chip devices or drug delivery purposes.     

Application of europium(III) chelates-bonded silica nanoparticle in time-resolved immunofluorometric detection assay for human thyroid stimulating hormone

Eu(III) chelate-bonded silica nanoparticle was used as a fluorescent label to develop a highly sensitive time-resolved immunofluorometric assay (TrIFA) for human thyroid stimulating hormone (hTSH). The limit of detection of the assay calculated according to the 2SD method was 0.0007 mIU L⁻¹ and became 0.003 mIU L⁻¹ when serum-based matrix was used for calibrators, indicating that this TrIFA is comparable with the most sensitive assays. The linear range was from 0.005 to 100 mIU L⁻¹ of hTSH with coefficient of variation between 1.9% and 8.3%. The correlation study using 204 blood spot samples from newborns showed that the results from this new method were coincident with that of the commercial dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) system, with a correlation coefficient of 0.938. The fluorescent nanoparticle label allows directly reading the fluorescent signal, omitting the signal development step required for the DELFIA system, and the whole procedure of this assay is fulfilled within 2 h. Thus, we developed a novel, sensitive, quantitative and simple nanoparticle label-based TrIFA assay, suitable for routine application in hTSH screening of neonatal hypothyroidism.     

A density functional theory investigation on the formation mechanisms of DNA interstrand crosslinks induced by chloroethylnitrosoureas

Chloroethylnitrosoureas (CENUs) are an important family of alkylating agents used in the clinical treatment of cancer. Their anticancer mechanism primarily involves the formation of DNA interstrand crosslinks (ICLs) induced by the chloroethyldiazonium ion derived from the decomposition of CENUs. In this work, the mechanism for the formation of ICLs was investigated by density functional theory (DFT) with B3LYP, wB97XD, and M062X functinoals using conductor‐like polarizable continuum model solvent model. Three pathways leading to the formation of three types of G–C crosslinks were compared. G(N1)–C(N3) crosslink is predicted to be the dominant crosslinking product other than G(O⁶)–C(N⁴) and G(N²)–C(O²) crosslinks, which is consistent with the previous results obtained from QM/MM computations. The results indicate that the formation of the G(N1)–C(N3) crosslink via pathway A is the most favorable mechanism from both kinetic and thermodynamic standpoints. In this pathway, the chloroethyldiazonium ion alkylates guanine on the O⁶ site followed by intramolecular cyclization to form O⁶,N1‐ethanoguanine ( 4 ). The cytosine then reacts with intermediate 4 on the C^α atom to yield the G(N1)–C(N3) crosslink. This work provides reasonable explanations for the supposed mechanism of CENUs‐induced ICLs formation obtained from experimental investigations. © 2012 Wiley Periodicals, Inc.     

Microbial detection in microfluidic devices through dual staining of quantum dots-labeled immunoassay and RNA hybridization

This paper reported the development of a microfludic device for the rapid detection of viable and nonviable microbial cells through dual labeling by fluorescent in situ hybridization (FISH) and quantum dots (QDs)-labeled immunofluorescent assay (IFA). The coin sized device consists of a microchannel and filtering pillars (gap = 1-2 μm) and was demonstrated to effectively trap and concentrate microbial cells (i.e. Giardia lamblia). After sample injection, FISH probe solution and QDs-labeled antibody solution were sequentially pumped into the device to accelerate the fluorescent labeling reactions at optimized flow rates (i.e. 1 and 20 μL/min, respectively). After 2 min washing for each assay, the whole process could be finished within 30 min, with minimum consumption of labeling reagents and superior fluorescent signal intensity. The choice of QDs 525 for IFA resulted in bright and stable fluorescent signal, with minimum interference with the Cy3 signal from FISH detection.     

High-throughput Screening:Synthesis of a Novel Fluorescent Microspheres

As one of efficient analytes, fluorescent microspheres have shown much usability on many biochemical and biomedical processes. Recent applications with fluorescent microspheres have included cytokine quantitation, single nucleotide polymorphism genotyping, phosphorylated protein detection, and characterization of the molecular interaction of nuclear receptors. These,coupled with the rapid advances in molecular biology and synthesis techniques of drugs, have presented a basis for drug screening in a high-throughput format. Based on fluorescent microspheres,earlier assay formats of HTS relied mainly on proximity-dependent energy transfer including scintillation proximity assay (SPA) (Amersham Pharmacia Biotech) and FlashPlatesTM (NEN Life Science Products, Boston, MA). Indeed, drug screening-based such fluorescent emission is still accounting for about 20～50% of current content of high-throughput screening (HTS). Now, SPA is almost a standard technique in common HTS-lab. In literature, SPA microspheres is generally prepared from inorganic scintillators such as yttrium silicate and hydrophobic polymers such as polyvinyl toluene. However, in HTS research, such microspheres often show the disadvantages of strong hydrophobicity and low quantum efficiency. The strong hydrophobicity is mainly attributed to the hydrophobic monomer, vinyl toluene. The low quantum efficiency can be as a result of low transparence of the polymer, polyvinyl toluene. Thus, the subsequent treatments for such microspheres, so as coat a polyhydroxy film to decrease the hydrophobicity, are actually considerably complicated.It has been well known that poly(methyl methacrylate) (PMMA), a good biocompatible polymer with not only adequate mechanical strength but also excellent transparence, can be regarded as an ideal candidate material for fluorescent matrix. In present study, methyl methacrylate as monomer and 2,5-diphenyloxazole (DPO) as fluorescent dye were used to the fluorescent microspheres. In guaranteeing the hydrophilicity of microsphere surfaces, dispersion polymerization was in common use (stabilizer, Polyvinyl pyrrolidone). As is apparent, with such a method in hand, one would normally find that almost no subsequent treatment for microspheres can be involved. Also such a novel fluorescent microspheres is a more suitable and the method used in present research is more practical method in comparison to common method. As the original work, the purposes of this article are to synthesize such fluorescent microspheres and probe the probable roles of synthesis conditions on microsphere synthesis. The effects of stabilizers, initiators, dispersion mediums, monomer content, and reaction temperature on the synthesis process and the particle size, as well as its panicle distribution have been shown. Also the probable role of fluorescent dye in the polymerization has been thermodynamicaily discussed.     

A novel microplate reader-based high-throughput assay for estrogen receptor binding

Coumestrol is a well-known ligand for the estrogen receptor (ER). The compound itself is fluorescent, and its fluorescence intensity at 408?nm increases upon binding to the ER. Here we describe a novel binding assay in 96-well plate format for estrogenic compounds, based on the competition between fluorescent coumestrol and estrogenic compounds for binding to the ligand binding domain (LBD) of the ER-alpha. Displacement of coumestrol was measured as a decrease in fluorescence intensity using a Victor² 1420 multilabel reader. Competitive binding curves for the well-known estrogenic compounds, 17β-estradiol (E₂), ethinylestradiol, 4-nonylphenol, 4-octylphenol, genistein, bisphenol A, tamoxifen and diethylstilbestrol were constructed by using 7–10 different concentrations of the compounds and a fixed concentration of ER-α-LBD (14?nmol) and coumestrol (100?nmol). IC₅₀ values and relative potencies (compared to E₂) of the estrogenic compounds were determined. The assay was validated by comparing the relative potencies to those from standard radioligand binding assays in the literature. Within day and between day variations were determined and the performance of the assay was assessed by determining the coefficients of variation and Z′ values. The present fluorescent binding assay has proven to be fast and easy, and allows accurately quantifying the binding affinity of estrogenic ligands. The method is also suitable as a high-throughput screening assay for ER ligands.