反相离子对色谱法分析3′, 5′反向寡核苷酸

3′,5′反向寡核苷酸是碱基组成和长度完全相同、碱基顺序相反的两个寡核苷酸序列。以三乙胺为离子对试剂,研究了缓冲液浓度(0.025～0.15 mol/L)、pH (5.0～6.8)、柱温(25～45 ℃)、流速(0.3～0.7 mL/min)以及不同初始洗脱强度和洗脱梯度条件下,6个3′,5′反向寡核苷酸模拟样品保留和分离的变化特点。三组反向序列在缓冲液浓度为0.05 mol/L,pH 6.8和流速0.4 mL/min条件下获得最大分离,温度对分离的影响不大,而初始洗脱强度对反向序列的影响远大于洗脱梯度。实验结果表明3′,5′反向寡核苷酸的分离和保留趋势不完全一致,色谱条件的优化应有利于实现样品在柱上的弱保留。研究结果还显示寡核苷酸序列中5′末端的保留强于3′末端。     

反相离子对色谱法分析3′, 5′反向寡核苷酸

3′,5′反向寡核苷酸是碱基组成和长度完全相同、碱基顺序相反的两个寡核苷酸序列。以三乙胺为离子对试剂,研究了缓冲液浓度(0.025～0.15 mol/L)、pH (5.0～6.8)、柱温(25～45 ℃)、流速(0.3～0.7 mL/min)以及不同初始洗脱强度和洗脱梯度条件下,6个3′,5′反向寡核苷酸模拟样品保留和分离的变化特点。三组反向序列在缓冲液浓度为0.05 mol/L,pH 6.8和流速0.4 mL/min条件下获得最大分离,温度对分离的影响不大,而初始洗脱强度对反向序列的影响远大于洗脱梯度。实验结果表明3′,5′反向寡核苷酸的分离和保留趋势不完全一致,色谱条件的优化应有利于实现样品在柱上的弱保留。研究结果还显示寡核苷酸序列中5′末端的保留强于3′末端。     

混合离子对试剂体系下寡核苷酸的IP-RPLC保留行为

利用离子对反相液相色谱(IP-RPLC)对寡核苷酸在混合离子对试剂三乙胺/丙胺-乙酸盐(TEA/PAAA)体系下的保留行为进行了研究,并与经典离子对试剂三乙胺乙酸盐(TEAA)体系下的保留行为进行了对比.实验结果表明,相同离子对试剂浓度下,寡核苷酸在TEA/PA-AA体系下的保留均弱于TEAA体系下的保留,且寡核苷酸的保留均随着离子对试剂浓度(20~120 mmol/L)的增加而增强.同型寡核苷酸(dC)_n作为特例,当n10时,保留基本趋于稳定,这是由于(dC)_n随着离子对试剂浓度的增加保留增长较快,在较低的离子对试剂浓度下即可达到最大保留.同时发现,短链同型寡核苷酸(dT)_n和异型寡核苷酸在TEA/PA-AA体系下的分离均优于TEAA体系,而同型寡核苷酸(dA)_n和(dC)_n的分离则在TEAA体系下更优.通过研究流动相中离子对试剂总浓度c_p与寡核苷酸保留因子k之间的关系,推断出2种体系下寡核苷酸的保留机理均以离子对模型占主导地位.总体而言,分离相同长度的异型寡核苷酸时,TEA/PA-AA混合离子对体系尤其在中等离子对浓度下比TEAA体系具有明显优势,低的离子对试剂浓度可增加与后续电喷雾质谱(ESIMS)的兼容性,有利于寡核苷酸的定性分析.     

高效液相色谱分离-在线光化学衍生/荧光光谱法测定5种水溶性维生素的研究

建立了高效液相色谱分离-在线光化学衍生/荧光光谱法测定水溶性维生素烟酸(NIA)、烟酸胺(NIC)、B1、B12及B2的新方法.以含有0.018 mol/L三乙胺、0.002 mol/L庚烷磺酸钠的0.05 mol/L磷酸盐缓冲液(A相,pH 5.8)和甲醇(B相)为流动相(85:15),等度洗脱分离5种水溶性维生素;...     

姜黄素-La3+-CTMAB-核酸体系的荧光增强效应及其应用

研究了镧离子(La3+)-姜黄素(CU)-十六烷基三甲基溴化铵(CTMAB)-核酸荧光增强体系.建立了测定核酸的新方法.体系的最优条件为:六次甲基四胺(HMTA)-HCl缓冲溶液(pH 5.80)中,1.00× 10-3 mol/L阳离子表面活性剂CTMAB存在下,姜黄素浓度为2.00×10-5 mol/L,La3+的浓度为1.40×10-4 mol/L时,核酸能增强La3+ -CU络合物的荧光强度,而且体系荧光的增强程度与核酸的加入量在一定范围内呈线性关系.fsDNA,ctDNA和yRNA线性范围分别为7.00×10-4～10.00 mg/L,4.00×10-4～10.00 mg/L和7.00×10-4～10.00 mg/L;检出限分别为0.17,0.02和0.14 μg/L.与已报道的核酸的分析方法相比,本方法具有较宽的线性范围和较高的灵敏度.研究表明,核酸对体系荧光的增强源于DNA主链上PO3-4与CU之间的静电结合,以及通过氢键和疏水力进行的沟槽式结合,为探针分子提供了疏水性的微环境,降低了体系的非辐射能量损失,使体系的荧光强度增加.     

毛细管电泳-激光诱导荧光测定大鼠血浆内痕量氨基酸类神经递质

用菁色素衍生物 (Cy5 )作为荧光衍生试剂 ,毛细管电泳结合激光诱导荧光分离测定痕量氨基酸类神经递质甘氨酸和谷氨酸。讨论了Cy5浓度 ,缓冲液浓度 ,pH值和反应时间等实验条件对衍生和分离的影响。衍生物的荧光强度和氨基酸的浓度在 7.5× 1 0 - 8～ 3 .5× 1 0 - 6 mol L范围内呈现良好的线性 ,线性相关系数均达到 0 .999。甘氨酸和谷氨酸的最低检出限浓度分别为 3 .3× 1 0 - 9mol L和 1 .0×1 0 - 9mol L。测定了大鼠血浆中甘氨酸和谷氨酸的浓度 ,分别为 (62± 1 4 )× 1 0 - 6 mol L和 (1 9± 2 )× 1 0 - 6 mol L。与其它氨基标记荧光试剂 ,如FITC和TRITC相比 ,Cy5与一级胺反应时间短 ,选择性高     

吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒的制备及其性质研究

以蛋白质或多肽修饰的吲哚类菁染料Cy3为内核, 采用实验条件简单的油包水反相微乳液方法成核, 通过正硅酸乙酯水解形成的网状二氧化硅包壳的方法制备吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒. 考察了以不同等电点的蛋白质和多肽修饰的Cy3为内核材料对吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒制备的影响. 结果表明, 分别采用人免疫球蛋白(IgG)或多聚赖氨酸修饰的Cy3为内核材料, 都能制备荧光强度高、荧光稳定性强和染料泄漏极少的Cy3嵌入的核壳荧光纳米颗粒. 进一步对Cy3嵌入的核壳荧光纳米颗粒进行了表征, 并将基于这一新型的荧光纳米颗粒建立起来的生物标记方法初步应用于流感病毒DNA的检测, 其检测线性范围为3.18×10-10～1.27×10-9 mol/L, 检测下限为3.51×10-10 mol/L, 相关系数r为0.986 5.     

比率检测高/半胱氨酸荧光染料的合成及活细胞成像研究

经一步简单反应获得了一例长波长尼罗蓝衍生物荧光染料RB-S. 该染料分子RB-S对高/半胱氨酸具有明显的荧光响应,且随着高/半胱氨酸浓度（0.03～0.33 μmol/L）逐渐增加,染料荧光强度在685 nm处减弱,相应地在755 nm处增强,在685与755 nm处的荧光变化具有良好线性关系的比率荧光变化. 通过高效液相色谱-质谱联用技术以及核磁滴定氢谱研究了染料分子检测高/半胱氨酸的机理. 试验结果表明：染料分子中的醛基与高/半胱氨酸发生成环反应生成噻唑烷. 在体外溶液测试中（pH=7.4）,染料分子RB-S对高/半胱氨酸的检出限为0.025 μmol/L,且不存在其他物质干扰,成功用于血清样品中内高/半胱氨酸荧光比率检测. 染料分子RB-S具有良好的活细胞膜通透性,使其能够对活细胞内高/半胱氨酸荧光比率成像.     

荧光探针法研究疏水改性聚丙烯酰胺溶液的疏水缔合行为

采用胶束共聚 共水解方法合成疏水改性水溶性聚合物聚(丙烯酰胺/丙烯酸钠/N 辛基丙烯酰胺)[P(AM/NaAA/C8AM)],并以芘为荧光探针,应用稳态荧光光谱法研究了它的疏水缔合行为。结果表明,随聚合物浓度、疏水单体摩尔分数、疏水侧链长和温度的增加,疏水缔合作用增强;不同疏水单体含量的P(AM/NaAA/C8AM)的临界缔合浓度为1.5~3.0 g/L;表面活性剂十二烷基硫酸钠(SDS)与P(AM/NaAA/C8AM)发生了强烈的疏水相互作用,形成混合胶束,得到SDS的临界胶束浓度(CMC)为8×10-3 mol/L;由于聚合物链上羧基的存在,使其具有良好的 pH敏感性,随 pH值的增大,P(AM/NaAA/C8AM)的疏水缔合作用呈现先减弱后恒定再增强的变化。     

重氮树脂接枝罗丹明B聚合物对酸、碱的响应性

利用重氮树脂(DR)中N2+的光解特性,通过罗丹明(B(RhB))对重氮树脂的接枝反应,得到了染料功能化的接枝聚合物DR-g-RhB,并由傅里叶红外光谱、1H-NMR、紫外-可见吸收光谱和荧光光谱等分析方法确认了产物结构。结果表明DR-g-RhB具有良好的热稳定性,失重5%所对应的温度超过360°C。通过溶液涂敷法形成的薄膜在不同酸碱度溶液中的UV-Vis吸收光谱会随浸入时间产生规律性变化:在pH=7.0的水中,吸光值基本不随时间变化;而在pH=12.0的肼水溶液中,吸光值随时间先下降、再增加;但在酸中则持续增加,而且对盐酸(pH=2.0)的响应比对醋酸(pH=2.3)更灵敏。     

Comparison of A+T-rich oligonucleotides with and without self-complementary sequence using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry

Both A+T-rich oligonucleotides with and without self-complementary sequences were analyzed using ion- pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) by tryethylammonium acetate (TEAA) and hexafluoroisopropanol (HFIP) buffer systems to study the characteristics of their retention behavior and electrospray ionization tandem mass spectrometry (ESI-MS/MS) response. The results indicated that the chain length had the same effect on the retention of A+T-rich oligonucleotides in both of TEAA and HFIP buffer systems but the sequence had a different impact on the retention in the two buffer systems. A+T- rich oligonucleotides with a self-complementary sequence were much shorter than those without in the TEAA buffer system whereas a slight difference was observed in the HFIP buffer system. Similar total ion current (TIC) intensity was observed both in oligonucleotides with or without self-complementary sequence. The opposite trend of a change in the TIC intensities with increasing chain length were observed in both the TEAA and HFIP buffer systems. A lower charge state was predominant in the TEAA buffer system whereas a higher charge state was mainly distributed in the HFIP buffer system. The oligonucleotides without self-complementary sequences had a higher charge state than those with self-complementary sequences. A- and T- are more esily formed at a higher charge state whereas the sequence fragments will be formed more easily at a lower charge state in both A+T-rich oligonucleotides with and without self-complementary sequences.     

Characterization of G-rich and T-rich oligonucleotides using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry

Characteristics of G-rich and T-rich oligonucleotides were investigated to compare their retention time, total ion current (TIC) intensity, charge-state distribution and product ion using ion-pair reversed-phase high- performance liquid chromatography/tandem electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) at room temperature. Three commonly used mobile phases for the analysis of oligonucleotides, triethylammonium acetate (TEAA), triethylammonium bicarbonate (TEAB) and triethylammonium hexafluoroisopropanol (HFIP) have been utilized. Retention time of G-rich and T-rich oligonucleotides was significantly different in TEAA and TEAB buffer systems, while in the HFIP buffer system it was affected more by the length of oligonucleotides. On the other hand, the ESI-MS ion abundance in the HFIP buffer system was higher than that in both TEAA and TEAB buffers. The TIC intensity of T-rich oligonucleotides was much higher than that of G-rich oligonucleotides in all mobile phases. In addition, much higher charge-state fragments were observed in HFIP buffer system than that in the case of TEAA and TEAB buffer systems. Product ions of both G-rich and T-rich oligonucleotides were affected by charge state of parent ions and collision energy.     

Protein-labeling effects in confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) is being increasingly used for observing protein uptake in porous chromatography resins. Recent CLSM studies have revealed the possible existence of a nondiffusive protein transport mechanism. Observing protein uptake with CLSM requires labeling the protein with a fluorescent probe. This study examines the effect of the probe identity on the subsequent CLSM adsorption profiles. The adsorption of lysozyme conjugated with different fluorescent probes (Cy5, BODIPY FL, Atto 635, and Atto 520) on SP Sepharose Fast Flow was measured using CLSM and zonal chromatography experiments. Results from zonal chromatography show that the retention time of lysozyme-dye conjugates differ significantly from unlabeled lysozyme. The change in retention of lysozyme upon conjugation with a fluorescent probe is consistent with the difference in net charge between the lysozyme-dye conjugate and unlabeled lysozyme. The adsorption profiles measured by CLSM show significantly different behavior depending upon whether the lysozyme-dye conjugate is retained longer or shorter than the unlabeled lysozyme. These results strongly suggest that the lysozyme concentration overshoot observed in previous CLSM experiments is the result of displacement of weaker binding labeled lysozyme by stronger binding unlabeled lysozyme.     

High-performance liquid chromatographic separation of detritylated oligonucleotides on highly cross-linked poly-(styrene-divinylbenzene) particles.

Detritylated oligonucleotides were separated by reversed-phase high-performance liquid chromatography on highly cross-linked polystyrene-based particles having a mean particle diameter of 2.3 microns. The addition of poly(vinyl alcohol) during polymerization, which resulted in the presence of hydroxyl groups on the surface of the poly(styrene-divinylbenzene) beads, was necessary to obtain baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases. The impact of temperature was investigated and optimum resolution was achieved at 40 degrees C. At pH 7.0, the retention times of oligonucleotides were found to depend on the ratio of bases and to increase in the order of C less than G less than A less than T. Under the same conditions, it was possible to separate phosphorylated from dephosphorylated oligonucleotides, the former being eluted earlier. Recoveries ranged from 92 to 100%.     

Enhanced mass detection of oligonucleotides using reverse-phase high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry

A new method providing enhanced sensitivity for the analysis of oligonucleotides using an on-line coupled system of reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization ion-trap mass spectrometry (ESI-MS) has been developed. The presented method allows the use of the standard gradient elution of 0.1 M triethylammonium acetate (TEAA) buffer (adjusted to pH 7.0 with acetic acid) and acetonitrile that is typically used for the separation of oligonucleotides in RP-HPLC. An added feature of this method is the ability to combine and mix additional 0.1 M imidazole in acetonitrile after the separation column for improved ESI-MS performance. This is similar to the post-column reaction method in liquid chromatography (LC) and the liquid sheath flow method in LC/ESI-MS, both of which offer the advantage of not compromising the chromatographic separation conditions. The application of this new method is demonstrated to afford improved sensitivity for the analysis of oligonucleotides (20-50 mer) via on-line coupled HPLC/ESI-MS analysis and purification systems.     

Surface-enhanced fluorescence of fluorescein-labeled oligonucleotides capped on silver nanoparticles

Tiopronin monolayer-protected silver nanoparticles with different core sizes (average diameter = 2, 5, 20 nm) were prepared by using different mole ratios of silver nitrate/tiopronin. Ligands on the silver particles were partially displaced by fluorescein-labeled thiolate single-stranded oligonucleotides or their complementary unlabeled oligonucleotides through ligand exchange. The fluorophores on silver particles showed a surface-enhanced fluorescence (SEF) dependent on the size of metallic cores. The particles could be coupled through hybridizations of oligonucleotides bound on the particles. The coupled particles were aggregated due to multiple displacements of oligonucleotides on each particle, resulting in stronger SEF. The dye-labeled oligonucleotides were assembled on the silver islands on the solid substrate, and the complementary oligonucleotide-displaced particles were coupled via oligonucleotide hybridization. The couplings between particles and islands resulted in an obvious fluorescence enhancement.     

The use of 6-carboxynaphthofluorescein phosphoramidite in the automated synthesis of quencher-dye oligonucleotide probes (QDOPs)

Real-time detection and quantitation of specific amplicons have been achieved using quencher and dye-labeled oligonucleotides such as molecular beacons. The molecular beacon molecule has a fluorescent reporter dye at the 5′-end and a quencher at the 3′-end. When the closed molecular beacon is excited by irradiation, the reporter fluorescence is greatly reduced by quenching through the process of fluorescence resonance energy transfer. When the molecular beacon hybridizes to the target, the stem loop opens making the fluorophore and quencher spatially distinct, thus increasing the reporter dye fluorescence intensity. Labeling of dyes to 5′-end of oligonucleotides has been done typically using manual methods, it is possible to do manual coupling at the milligram scale. Described here is the development of a scalable process for oligonucleotide labeling, which is robust, and has been achieved for 6-carboxynaphthofluorescein by connecting it to a corresponding phosphoramidite [Theisen, P.; McCollum, C.; Upadhya, K.; Jacobson. K.; Vu, H.; Andrus, A.; Tetrahedron Lett.1992, 33, 5033-5036].     

Hydrophobic labeling of amino acids: transient trapping-capillary/microchip electrophoresis

Transient trapping (tr-trapping) was developed as one of the on-line sample preconcentration techniques to improve a low concentration-sensitivity in microchip electrophoresis (MCE), providing highly effective preconcentration and separation based on the trap-and-release mechanism. However, a poor performance to hydrophilic analytes limited the applicability of tr-trapping. To overcome this drawback, tr-trapping was combined with a sample labeling using a hydrophobic reagent in CE. Three commercially available fluorescent dyes, fluorescein isothiocyanate, succinimidyl esters of Alexa Fluor 488 and BODIPY FL-X, were tested as derivatization reagents to increase the hydrophobicity of amino acids (AAs) that were undetectable due to no fluorescence/UV-absorbance. As a result, it was confirmed that BODIPY labeling allowed various AAs to be analyzed in tr-trapping-micellar electrokinetic chromatography (tr-trapping-MEKC) by the increase in the hydrophobicity. In tr-trapping-MEKC, both the improvement of the resolution and 106-125-fold enhancements of the detectability of labeled AAs were achieved relative to the conventional capillary zone electrophoresis. The limit of detection of labeled phenylalanine was improved from 800 to 5 pM by applying tr-trapping-MEKC. In tr-trapping-microchip MEKC, furthermore, an 80-160-fold enhancement of the peak intensity and a baseline separation was also achieved within 30 s. These results clearly demonstrate that the tr-trapping technique with hydrophobic labeling will make CE/MCE more sensitive for various analytes.