A novel phosphoramidite method for automated synthesis of oligonucleotides on glass supports for biosensor development

Two protocols for functionalization of glass supports with hexaethylene glycol (HEG)-linked oligonucleotides were developed. The first method (standard amidite protocol) made use of the 2-cyanoethyl-phosphoramidite derivative of 4,4′-dimethoxytrityl-protected HEG. This was first coupled to the support by standard solid-phase phosphoramidite chemistry followed by extension with a thymidylic acid icosanucleotide. Stepwise addition of the linker phosphoramidite graduated at 1% (relative to the total sites available) perstep at 50°C resulted in an optimal yield of immobilized oligonucleotides at a density of 2.24 × 10¹⁰ strands/mm². This observed loading maximum lies well below the theoretical maximum loading owing to nonspecific adsorption of HEG on the glass and subsequent blocking of reactive sites. Surface loadings as high as 3.73 × 10¹⁰/mm² and of excellent sequence quality were achieved with a reverse amidite protocol. The support was first modified into a 2-cyanoethyl-N,N-diisopropylphosphoramidite analog followed by coupling with 4,4′-dimethoxytrityl-protected HEG. This protocol is conveniently available when using a conventional DNA synthesizer. The reverse amidite protocol allowed for control of the surface loading at values suitable for subsequent analytical applications that make use of immobilized oligonucleotides as probes for selective hybridization of sample nucleic acids of unknown sequence and concentration.     

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.     

In vitro study of experimental factors affecting the microdialysis results

The aim of the present study was to determine the influence of a series of experimental conditions (probe, perfusate flow rate and the method used to ascertain recovery) as well as the pharmacokinetic variables (concentration and time) on the estimation of the recovery coefficient of microdialysis probes. Two in vitro pharmacokinetic assays were also carried out to compare the results provided by microdialysis and those obtained with traditional sampling. The probes used were made in our laboratory and ciprofloxacin was used as a model compound. The results revealed that in all cases recovery was dependent on the probe and independent of time for a 80 min sampling time period. The effects of concentration on recovery depend on the flow rate; this was not statistically significant for a flow rate of 2 μl/min but an increase in flow rate to 6 μl/min transformed this parameter into a concentration-dependent variable. A decrease in recovery parallel to the increase in flow rate was found, with an exponential relationship between the two variables. Statistically significant differences were also found between the recovery values obtained for direct dialysis (18.44±1.61) and retrodialysis by loss of the analyte of interest (16.79±3.42). The values of the protein binding of ciprofloxacin as calculated by equilibrium dialysis and by microdialysis were similar. Characterization of the in vitro kinetic profile revealed no statistically significant differences for coefficients and exponents obtained by traditional sampling and microdialysis, although the confidence intervals of the curves were wider for microdialysis.     

A statistical method to detect chromosomal regions with DNA copy number alterations using SNP-array-based CGH data

Single nucleotide polymorphism (SNP) arrays were used to detect chromosomal regions with DNA copy number alterations. Current statistical methods for microarray-based comparative genomic hybridization (array-CGH) analysis generally assume certain relationships among adjacent markers on the same chromosome, and these assumptions may be questionable. For an SNP-array-based CGH study, multiple normal reference SNP arrays were collected. In order to utilize these normal reference SNP arrays, we derived an empirical distribution of signal ratios for each SNP marker. With an assumed threshold value for the overall error rate control and the defined signal ratio ranges for chromosomal amplification and deletion, we proposed a procedure to identify chromosomal alteration regions based on several bootstrapped one-sample t-tests and the false discovery rate control. When we have multiple arrays for different individuals with the same disease, our method can also be used to detect SNP markers for chromosomal alteration regions that are common among these individuals. We applied our method to a published SNP array data set for breast carcinoma cell lines. For an individual with breast cancer, numerous chromosomal alteration regions were identified. Compared to results of previous studies, our method identified more chromosomal alteration regions, with some being implicated in the literature to harbor genes associated with breast cancer. For multiple cancer arrays, our results suggested the existence of common chromosomal alteration regions. However, a high proportion of false positives also indicated that genetic variations among different individuals with breast cancer can be present.     

玻璃载体表面脱氧核糖核酸的固定及其化学发光检测

用硅烷化偶联剂把ＤＮＡ直接共价固定在载玻片表面，将辣根过氧化物酶标记的探针与之进行核酸杂交，杂交后用增强的化学发光检测。方法的检出限为７５ｐｇ。研究了ＤＮＡ分子固定在玻璃载体表面的各种条件，并建立了在玻璃载体表面进行核酸杂交的体系，为研究光纤ＤＮＡ生物传感器打下了基础。     

New approaches in radical carbonylation chemistry: fluorous applications and designed tandem processes by species-hybridization with anions and transition metal species

New approaches in radical carbonylation chemistry are described. We have successfully integrated tin mediated radical carbonylation chemistry into modern fluorous applications and separation techniques. We revealed that radical carbonylation reactions can be performed using fluorous tin mediators, such as fluorous tin hydride and fluorous allyltin reagents. Fine tuning of the reaction conditions resulted in a good efficiency equivalent to conventional tin mediators. The tedious procedure of removing organotin byproducts can be circumvented through the use of fluorous/organic liquid-liquid extraction or fluorous liquid-solid phase extraction with fluorous reverse phase silica (FRPS). Also described are newly developed tandem carbonylation reactions that are based on species hybridization approaches. Using a radical/anionic hybrid system based on zinc-induced one-electron reduction, we achieved a three-component coupling reaction consisting of 4-alkenyl iodides, carbon monoxide, and electron-deficient alkenes. We observed two types of annulations processes, namely [4 + 1](radical)/[3 + 2](anionic) and [5 + 1](radical)/[3 + 2](anionic), which lead to the production of bicyclo[3.3.0]octanols and bicyclo[3.2.1]octanols, respectively. We found a radical/palladium hybrid system to be useful in the construction of new cyclic systems that incorporate two or three molecules of carbon monoxide.     

Fluorescence Probe Studies of Ionic Micelles in Concentrated Salt Solutions

We have investigated the effect of salt concentration and temperature on the average aggregation number and micro-polarity of the interior of micelles of sodium dodecyl sulfate (SDS). sodium tetradecyl sulfate (STDS) and lithium dodecyl sulfate (LiDS). The transient fluorescence decay of micelle-solubilized pyrene has been measured and analyzed. An exponent weighted average aggregation number <n>_e was obtained by this technique. For SDS and STDS in NaCl solution, <n>_e increases as the temperature is lowered or salt concentration is increased <n>_e increased from ～ 50 to ～ 250 over [NaCl] = 0 to 0.8 M. Due to the strong counterion binding of lithium in the micellar solution, the LiDS micelle is much, smaller and does not increase appreciabily even at [LiCl] =0.8 M. From the fluorescence spectrum fine structure of pyrene and the fluorescence decay of the monomer and excimer, we can understand the local polarity and the water penetration to the interior of the micelle upon addition of salts and with changing temperature. The interior of the micelle becomes more nonpolar as the salt concentration is increased and the temperature is lowered. A complete kinetic analysis of the time–dependence of the fluorescence is given. The kinetic analysis is in agreement with the results reached by fluorescence spectral analysis.     

A novel fluorescent method for determination of peroxynitrite using folic acid as a probe

A novel method for the determination of peroxynitrite using folic acid as a fluorescent probe is described. The method is based on the oxidation of the reduced, low-fluorescent folic acid by peroxynitrite to produce a high-fluorescent emission product. The fluorescence increase is linearly related to the concentration of peroxynitrite in the range of 3 × 10⁻⁸ to 5.0 × 10⁻⁶ mol L⁻¹ with a correlation coefficient of 0.998, and the detection limit is 1 × 10⁻⁸ mol L⁻¹. Interferences from some metal ions normally seen in biological samples, and also some anions structurally similar to peroxynitrite were studied. The optimal conditions for the detection of peroxynitrite were evaluated.     

电化学石英晶体微天平研究界面电场对DNA杂交的影响

采用电化学石英晶体微天平, 现场监测不同界面电场下完全匹配的靶标DNA和不完全匹配的靶标DNA分别与寡聚核苷酸探针分子杂交的过程. 结果表明, 电极表面荷正电时DNA表观杂交效率比电极表面荷负电时高, 但假阳性比较显著; 而电极表面荷负电时能有效地抑制错配杂交. 探讨了引入界面电场后探针分子取向和微观作用力对DNA杂交的影响.     

Configuration of MFI-type zeolite interacting with the probe molecule P(CH<Subscript>3</Subscript>)<Subscript>3</Subscript> – a theoretical study

With P(CH₃)₃ as the probe molecule adsorbed on titanium silicalite (TS-1) zeolite, the special and important role of T12 site in MFI-type zeolite was clearly elucidated. There are altogether three active sites present in TS-1 zeolite with Ti at the T12 site. Owing to the preferential adsorption of probe molecules on the first Brönsted acidic site, the Ti12 center will probably fail to show Lewis acidity. The ionic [HP(CH₃)₃]⁺ species can be stabilized by the first or second Brönsted acidic site, with the former energetically favored. The latter was formed through the transfer of the ionic [HP(CH₃)₃]⁺ species from the first to the second Brönsted acidic site.