Improved Fluorometric DNA Determination Based on the Interaction of the DNA/Polycation Complex with Hoechst 33258

When DNA is mixed with the cationic polyelectrolyte poly(diallyldimethyl ammonium chloride) (PDDA), the DNA/PDDA complex is formed instantaneously at room temperature. This complex is much more efficient in enhancing the fluorescence of Hoechst 33258 (H 33258) than DNA alone. Based on the interaction of H 33258 with the DNA/PDDA complex, a new fluorescence assay for DNA is described. At pH 7.3 in Tris-HCl buffered solution, the DNA/PDDA complex causes a sharp enhancement in fluorescence intensity of H 33258. Simultanously, the emission maximum wavelength of H 33258 blueshifts from 490nm to 450nm, while the excitation redshifts from 345 to 350nm. The calibration graphs for calf thymus DNA (ctDNA) and herring sperm DNA (hsDNA) are both linear up to 5.0µgmL^–1 when the concentration of H 33258 and PDDA are fixed at 1.5×10^–6 and 1.6×10^–5molL^–1, respectively. The method is specific for native DNA. The 3 detection limits for ctDNA and hsDNA are 1.8 and 5.6ngmL^–1, respectively, i.e. much lower than in the presence of H 33258 alone. Four synthetic samples were determined satisfactorily. This method can also be developed to investigate the formation and the nature of the complexes between DNA and polycations, which have recently been widely applied in some fields such as genetic engineering and gene therapy.     

Synthesis of a new Pt(II) complex containing valganciclovir drug and calf-thymus DNA interaction study using multispectroscopic methods

A new complex, [Pt(valcyte)(DMSO)Cl]Cl, in which valcyte (trade name) served as valganciclovir hydrochloride drug ([2-[(2-amino-6-oxo-3H-purin-9-yl)methoxy]-3-hydroxypropyl](2S)-2-amino-3-methylbutanoate), was synthesized and characterized by different physicochemical methods. Binding interaction of this complex with calf-thymus DNA (ct-DNA) has been investigated by multispectroscopic techniques. The complex displays significant binding properties with ct-DNA. The results of fluorescence and UV–vis absorption spectroscopy indicated that this complex interacted with ct-DNA in a groove-binding mode, and the binding constant was 3.8 × 10⁴ M^?1. Furthermore, the complex induced detectable changes in the CD spectrum of ct-DNA and slightly changed its viscosity which verified the groove-binding mode. Finally, all results indicated that Pt(II) complex interact with DNA via groove-binding mode.     

DNA电化学传感器在DNA片段识别与测定中的应用

本文用乙基－（３－二甲基丙基）碳二亚胺盐酸盐（ＥＤＣ）和羟基丁二酰亚胺（ＮＨＳ）活化已被氧化的石墨电极,然后将单链ＤＮＡ（ｓｓＤＮＡ－１）固定在石墨电极上。运用核酸杂交技术,使具有电化学活性的染料Ｈｏｅｃｈｓｔ ３３２５８ 嵌入双链ＤＮＡ 分子（ｄｓＤＮＡ）的碱基对中,在石墨电极表面形成ｄｓＤＮＡ－Ｈｏｅｃｈｓｔ ３３２５８ 层,通过伏安法测定嵌入Ｈｏｅｃｈｓｔ ３３２５８的氧化峰电流,可以识别和测定溶液中互补的ｓｓＤＮＡ－２ 片段,ｓｓＤＮＡ－２ 的浓度在４．８×１０－ ５～１．１×１０－ ７ ｍ ｇ／ｍ Ｌ范围内,有线性关系,检测限可达６．０×１０－ ８ ｍ ｇ／ｍ Ｌ。     

大鼠脑缺血再灌注后运动皮质细胞凋亡的体视学观察

用体视学方法分析大鼠脑缺血再灌注后大脑运动皮质细胞的凋亡情况。随机将24只SD大鼠分为缺血再灌注后6、12、24 h和对照组。采用结扎大鼠左侧颈总动脉和夹闭右侧颈总动脉15m in后放开的方法制备大鼠脑缺血再灌注模型。应用Hoechst33258荧光染色及免疫组化Bax蛋白表达来检测细     

Fluorescence spectroscopic study of dip coated sol-gel thin film internal environment using fluorescent probes Hoechst33258 and Pyranine

The characterization of physicochemical properties of the internal environment of sol-gel thin films is required for understanding and designing applications in optical biosensors. We have investigated the dip coated tetraethyl-orthosilicate (TEOS) derived sol-gel thin films deposited on microscopic glass cover slips using molar ratio (water or ethanol / TEOS) R=32 using fluorescence spectroscopic measurements (emission, lifetime and anisotropy) on entrapped fluorescent probes. The effect of water and/or ethanol was studied as a function of storage (60 days) using fluorescent probes Hoechst 33258 (H258) and Pyranine (PY). Distribution of fluorescent probes in thin film was studied using confocal microscope. Emission maxima of H258 entrapped thin films from sol prepared using water as solvent showed emission maximum at 503 nm indicating the presence of water like environment which did not change during storage. On the contrary, PY entrapped thin films depicted emission bands at 434 nm and 513 nm, characteristics of ethanol and water respectively, up to the first few weeks and then the band at 434 nm prevailed (60 days), suggesting heterogeneous internal environment. Thin films from sol prepared using ethanol as solvent showed presence of ethanol through out storage. Fluorescence lifetime data of these probes in both sol-gel and thin films also suggested presence of heterogeneous internal environment. Thin films prepared from sol-gel using water as solvent suggested release of ethanol in the pores during hydrolysis and condensation reaction, which were clearly indicated by PY. The effect of sodium phosphate buffer was also studied in sol-gel and thin films. The results of these measurements showed that both the probes H258 and PY could be used effectively in monitoring the physicochemical properties of internal environment of thin films and sol-gel as a function of storage.     

Synthesis of three fluorescent boronic acid sensors for tumor marker Sialyl Lewis X in cancer diagnosis

Sialyl Lewis X (sLex) is a carbohydrate that is considered not only a marker for cancer, but also an antigen associated with the malignant behavior of cancerous cells. We have synthesized three fluorescent boronic acid sensors as potential sensors for sLex. Photoinduced electron transfer by a fluorescence analyzer was used to assess sensor-sLex antigen binding. The reaction was carried out in mixed aqueous solution, and Sensor 3 was identified as showing the strongest fluorescence enhancement upon binding to sLex at 10 nM. In cell-line culture experiments, Sensor 1 was shown to label sLex expression positively for HepG2, Colo 205, and COS-7 cells, and negatively for MDA-MB-231 cells; Sensor 2 did so positively for HepG2, PLC/PRF/5, and Colo 205 cells, and negatively for MDA-MB-231 and COS7 cells; and Sensor 3 did so positively for PLC/PRF/5 and HepG2 cells, and negatively for MDA-MD-231 and COS7 cells. MTT cytotoxicity experiment results showed that the three sensors are nontoxic, and Hoechst 33258 experiments showed that no apoptosis occurred.     

Cucurbit[8]uril-improved recognition using a fluorescent sensor for different metal cations

ABSTRACT

Metal cations in aqueous solutions at different pHs (1.5, 4.5 and7.2) can be detected using the known inclusion complex H33258@Q8 as a fluorescent probe. Indeed, the fluorescence intensity of H33258 increases rapidly upon increasing the amount of Q[8] up to a Q[8]:H33258 ratio of 2:1, even at pH 1.5. The experimental results reveal that the probe not only exhibited selective recognition of metal cations at different pH but also showed different recognition for metal cations at different pHs. At pH 7.2, the probe responded to quite a variety metal cations, including one of the third main group elements, Al³⁺, four transition metal cations, Hg²⁺, Fe³⁺, Fe²⁺, and Cr³⁺ and three lanthanide cations Eu³⁺, Tm³⁺ and Yb³⁺; At pH 4.5, the probe responded to only two transition metal cations, Hg²⁺ and Fe³⁺. Unexpectedly, the probe lost its recognition properties at pH 1.5.     

Quenching of nucleotide-derived radicals by bis benzimidazole derivative Hoechst-33258 in aqueous solution

The pulse radiolysis technique has been employed to investigate the reaction of DNA-minor-groove ligand bisbenzimidazole Hoechst 33258 with pyrimidine and purine nucleotide-derived radicals. Formation of an N-centred Hoechst-33258 radical is observed. Bimolecular rate constants and the yields of Hoechst-33258 radical have been evaluated. While the rate constant for the reaction of pyrimidine-derived radicals with Hoechst-33258 remained the same (1–2) × 10⁹ dm³ mol⁻¹ s⁻¹, the yields of the Hoechst-33258 radical varied from 25% (5′-cytidine monophosphate) to 75% (5′-guanosine monophosphate) under anoxic conditions. The rate constant values for the reaction of purine-derived radicals with Hoechst-33258, under oxic and anoxic conditions, remained the same whereas with pyrimidine-derived radicals, the rate constant value under oxic conditions was about two orders of magnitude lower than under anoxic conditions. The difference in the yields of Hoechst-33258 radical with various nucleotide-derived radicals suggest the formation of different types of radicals and that the reaction mainly occurs by electron transfer from Hoechst-33258 to the nucleotide radicals.     

An ab initio study of the conformational preferences of Hoechst 33258 in gas-phase and aqueous solution environments

A quantum mechanical study of the conformational preferences of Hoechst 33258, a synthetic minor groove-binding drug, has been performed in both gas-phase and aqueous solution. Gas-phase calculations were performed at the HF/6-31G(d) and MP2/6-31G(d) levels of theory, whereas calculations in the aqueous solution phase were performed using the PCM model with the 6-31G(d) basis set. The molecule was divided into three fragments, which were submitted to a systematic and detailed conformational study. The results clearly indicate that Hoechst 33258 does not adopt a planar conformation in either the gas-phase or aqueous solution. Thus, a folded conformation is not induced by binding of the molecule to DNA, but is an intrinsic property of the compound. Received: 3 March 1998 / Accepted: 29 May 1998 / Published online 19 August 1998     

脱氧核糖核酸与双苯甲亚胺相互作用的荧光特性研究

采用荧光光谱法对脱氧核糖核酸（ＤＮＡ）与双苯甲亚胺（Ｈｏｃｅｈｓｔ ３３２５８）相互作用的方式及其作用机理进行了研究,证实了Ｈｏｅｃｈｓｔ ３３２５８与ＤＮＡ的相互作用除嵌入作用时,还存在持异性的静电作用;Ｈｏｅｃｈｓｔ３３２５８与ＤＮＡ主要作用在Ａ－Ｔ匹配,同时,在磷酸根的存在下可以减弱Ｈｏｅｃｈｓｔ３３２５８与ＤＮＡ的非特异性作用。另外,根据荧光峰增强与Ｈｏｅｃｈｓｔ３３２５８浓度的线性关系,