吖啶橙-罗丹明B二聚体能量转移体系在DNA测定中的应用及机理研究

首次研究了吖啶橙 罗丹明B二聚体能量转移体系作为荧光探针用于DNA的测定 ,并对其机理进行了探讨。用于鲱鱼精DNA和小牛胸腺DNA的测定 ,线性范围分别为 0 33～ 1 33mg·L- 1 ,0 33～ 3 33mg·L- 1 ,检测限分别为 1 6 3× 10 - 3mg·L- 1 ,1 5 2× 10 - 3mg·L- 1 。对 1 0 0mg·L- 1 鲱鱼精DNA和小牛胸腺DNA的测定 ,相对标准偏差分别为 2 4 %和 2 0 %。     

Direct immobilization and hybridization of DNA on group III nitride semiconductors

A key concern for group III-nitride high electron mobility transistor (HEMT) biosensors is the anchoring of specific capture molecules onto the gate surface. To this end, a direct immobilization strategy was developed to attach single-stranded DNA (ssDNA) to AlGaN surfaces using simple printing techniques without the need for cross-linking agents or complex surface pre-functionalization procedures. Immobilized DNA molecules were stably attached to the AlGaN surfaces and were able to withstand a range of pH and ionic strength conditions. The biological activity of surface-immobilized probe DNA was also retained, as demonstrated by sequence-specific hybridization experiments. Probe hybridization with target ssDNA could be detected by PicoGreen fluorescent dye labeling with a minimum detection limit of 2 nM. These experiments demonstrate a simple and effective immobilization approach for attaching nucleic acids to AlGaN surfaces which can further be used for the development of HEMT-based DNA biosensors.     

Models of DNA-dye-complexes: Energy transfer and molecular structures as evaluated by laser excitation

Energy transfer in DNA-dye-complexes after excitation in the UV down to 220 nm by means of a tunable laser was investigated. DNA of different base composition and polynucleotides were compared. The dyes proflavine, acridine orange and ethidium bromide were used. The energy transfer from DNA-bases to dye molecules was measured in dependence of the excitation wavelength and the base composition of the DNA. Models of the energy transfer from DNA to dye and of the molecular structures of the DNA-dye-complex (intercalation) could be deduced.     

Surface enhanced Raman scattering based molecule detection using self-assembled DNA nanostructures

A novel method of Surface Enhanced Raman Scattering (SERS) based molecular detection was developed using self-assembled DNA nanostructures. Molecule detection using rigid, shape-controllable DNA nanostructures provides significant advantages such as accurate control of the particle distance and geometrical programmability of the DNA nanostructure. We successfully detected the Cy3 dye, a Raman-active molecule, at 1461, 1590, and 1619 cm⁻¹ using a Ag-enhanced Au-DNA nanostructure. Consequently, DNA-guided SERS detection is expected to contribute significantly to molecular detecting systems in the near future.     

The frequency characteristics analysis of series photodetector frequency circuit system based on circuit parameters of quartz crystal and its application for bacterial DNA identification

The circuit parameters of quartz crystal were employed for frequency sensitivity analysis of series photodetector frequency circuit system. The influence of circuit parameters of quartz crystal on the oscillation frequency and response sensitivity were theoretically derived and experimentally verified. On the basis of optimal circuit parameters, the DNA probe detection limit 2 pmol/L can be measured by 49.4 MHz sensor system. In comparison with the conventional fluorescence technique, the frequency method showed that the detection limits of DNA probe AH642 with Cy5 fluorescence dye and DNA probe VA180 with Cy5 fluorescence dye were lower than the conventional fluorescence technique by 2–3 orders; meanwhile, through the feature of probe uniqueness, Aeromonas hydrophila DNA and fluorescence probe AH642-Cy5 can be successfully judged for hybridization reaction. Moreover, Vibro alginolyticus DNA and fluorescence probe VA180-Cy5 can be successfully judged for hybridization reaction.     

Selective laser excitation of bases in nucleic acids

Selective electronic excitation of single kinds of DNA bases were performed with dye lasers. Different DNA-dye-complexes were used and the selective excitation could be proved by the varying strength of the interaction between the bases and the dye molecules.     

Time-Resolved Fluorescence Anisotropy Study of the Interaction Between DNA and a Peptide Truncated from the p53 Protein Core Domain

Time-resolved fluorescence anisotropy spectroscopy was applied to study the interaction between a peptide truncated from the binding site of tumor suppressor p53 protein and the DNAs covalently labeled with 6-carboxyfluorescein (FAM) dye. Fluorescence intensity quenching and changes of anisotropy decay lifetime were monitored when FAM labeled DNA formed complex with the peptide. The results demonstrated that the sequence of DNA could not define the binding specificity between the peptide and DNA. But the anisotropy decay of FAM can be used to examine the binding affinity of the peptide to DNA. The fluorescent dynamics of FAM can also be used to represent the rigidity of the complex formed between the peptide and DNA.     

Improvement of frequency sensitivity of series photodetector frequency circuit system and its application for Cy5 fluorescence detection

This study elucidated the frequency characteristics of series photodetector frequency circuit system for detection of DNA probe ET996 marked with fluorescence dye Cy5. We developed 48 MHz series photodetector frequency circuit system with good sensitivity for fluorescence detection. In accordance with the theory of series photodetector frequency circuit system, the frequency sensitivity can be improved by adjusting circuit parameters such as A (tan θ), C_q, C₀, and C_p. In this research of A adjustment, the capacitance parameter C_m of 48 MHz series photodetector frequency circuit system was adjusted to improve the frequency sensitivity for detection of fluorescence dye concentration; moreover, the bias of photodetector was also adjusted to improve the frequency sensitivity. In the optimal conditions of capacitance match and photodetector bias, the detection limit of ET996-Cy5 fluorescence dye concentration 2 pmol/L can be measured by 48 MHz sensor system. The results of fluorescence experiment also demonstrated that the frequency shift of 48 MHz sensor system was linearly related to the logarithm of fluorescence dye concentration from 200 nmol/L to 2 pmol/L. The frequency method can be applied simply and the detection limit of ET996-Cy5 fluorescence dye concentration was lower than the conventional fluorescence technique by 2 orders.     

荷叶中紫云英苷和DNA相互作用的光谱学研究

在pH 7.4 的Tris-HCl的缓冲溶液中,采用紫外及荧光光谱法研究了荷叶中紫云英苷(AST)与DNA之间的相互作用,探讨了离子强度和阴离子猝灭剂KI对紫云英苷及紫云英苷-DNA体系荧光强度的影响,同时考察了紫云英苷和中性红与DNA结合的竞争性。结果表明DNA通过静态猝灭作用机制猝灭紫云英苷的荧光,并测得其在298及308 K时的猝灭速率常数(K_q)分别为3.120 ×1012和2.630×1012 L·mol-1·s-1,结合常数(K_d)分别为3.412×104和1.762×104 L·mol-1,结合位点数(n)分别为1.007和0.962;DNA的存在使紫云英苷的紫外吸收光谱发生减色效应且吸收波长产生红移;发现离子强度的改变对紫云英苷及紫云英苷-DNA体系的荧光强度影响不大;KI对结合形式存在的紫云英苷的荧光猝灭效率明显小于自由形式存在的紫云英苷的荧光猝灭效率;紫云英苷可插入DNA中置换出与DNA结合的中性红。这些结果说明荷叶中紫云英苷以嵌插模式与DNA进行结合。     

基于近红外光谱实现心输出量无创检测的色素谱分析方法

针对当前临床上心输出量参数检测技术存在的有创伤、操作复杂和患者易受感染而死亡的问题,研究了一种基于近红外光谱原理,动态测量和分析作为指示剂的吲哚氰绿色素在患者动脉血液中的浓度变化情况,从而根据其特征参数实现心输出量连续测量的无创检测方法。将吲哚氰绿色素经肘静脉注入患者体内后,光电脉搏色素谱测量装置作为下位机,连续、同步采集和记录其指端处的805和940 nm两个特征波长点的脉搏波信号,并将数据上传至上位计算机,由后者绘制色素稀释和排泄的浓度衰减曲线,以及计算平均循环时间等关键参数,最后推导出心输出量的数值。将该方法与作为“金标准”的热稀释法的测量结果相比较,其最大相对误差为9.76%,而平均相对误差为4.39%。该试验结果表明,所提出的方法为临床上的心输出量检测,提供了一种操作简便、无创和连续测量的解决方案。     

Studies on the Spectral-Luminescent Properties of the Novel Homodimer Styryl Dyes in Complexes with DNA

Series of homodimer styryls containing on (p-dimethylaminostyryl) pyridinium residues that are connected with aliphatic linkage group was synthesized. Spectral luminescent properties of obtained dyes in free state and in nucleic acids presence were studied. It was shown that DNA binding affinity of the novel homodimers exceeds that of parent monomer (p-dimethylaminostyryl)pyridine iodide. For homodimers with the linkage 4–10 carbon atoms preference in binding to DNA than to RNA was observed. It could be concluded that parent monomer has different mechanisms of binding to nucleic acids than corresponding homodimer dye.     

Specificity of Cyanine Dye L-21 Aggregation in Solutions with Nucleic Acids

Optical spectroscopy experiments were used to study the features of cyanine dye 3,3′-dimethyl-9-(2-thienyl)-thiacarbocyanine iodide (L-21) aggregation in binary solutions DMF:Tris–HCl buffer (pH = 8) containing nucleic acids (DNA or RNA). The appearance of absorption and luminescence bands associated with J-aggregates and dimers that are formed within the minor groove of DNA has been observed. The model of L-21 J-aggregate structure is proposed. It has been found that dimers are the building blocks of L-21 J-aggregates. Disorientation in dimers caused by the minor groove curvature is reason of observation of Davydov splitting in absorption spectrum of L-21 J-aggregates. In the solution containing DNA the absorption and luminescence bands of L-21 J-aggregates exhibit the specific properties that allows the dye L-21 to be used as a fluorescent probe for DNA detection.     

Detection of PCR products amplified from DNA of epizootic pathogens using magnetic nanoparticles and SERS

Here we present a novel approach using surface‐enhanced Raman scattering (SERS) spectroscopy for the sequence‐specific detection of DNA utilizing magnetic nanoparticles (MNPs) for the enrichment of the target molecules. To achieve fast and efficient binding of longer DNA strands, e.g. PCR products, the hybridization procedure is performed in solution. To further purify and enrich the DNA strands of interest, MNPs are used for their separation. Following the binding of the target DNA, a dye‐modified, short synthetic ssDNA is hybridized, which serves as label for the SERS detection. The SERS spectra are used to identify the bound molecules. The applicability of this approach was first tested with short synthetic oligonucleotides to evaluate its specificity. Afterward, the system was applied to detect PCR products amplified from DNA of specific agents of epizootic diseases. Sequences of the bacterium Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), causing contagious bovine pleuropneumonia (CBPP) were used as PCR targets. To demonstrate the multiplexing capability of SERS, the simultaneous detection of three different PCR products labeled with three dyes was performed. Copyright © 2010 John Wiley & Sons, Ltd.     

激光波长对荧光激发谱分布影响研究

激光器是DNA测序仪中的重要部件,寻求合适波长的激光器可有效提高DNA测序仪性能。分析了DNA检测所用的四种荧光染料FAM、JOE、TAMRA和ROX的激发谱和发射谱,设计实验技术方案利用488nm、505nm和515nm三种不同波长的激光器分别对其进行激发。在相同的积分时间和相同的功率下,488nm激光器对于荧光染料ROX的激发强度太低,515nm激光器对于荧光染料FAM的激发强度太低,而505nm激光器对于四种荧光染料的激发强度均比较强。实验结果表明在三种不同波长中,505nm激光器对四种荧光染料的激发效果最佳,可以替代目前大多数DNA测序仪中的氩离子激光器。     

Surface‐enhanced Raman scattering study of the red dye laccaic acid

FT‐Raman and surface‐enhanced Raman scattering (SERS) spectroscopy were applied to the study of lac dye, a highly fluorescent anthraquinone red dye. The SERS spectra were obtained at different pH values, on Ag nanoparticles prepared by chemical reduction with citrate and hydroxylamine, and at several excitation wavelengths, in order to find the best experimental conditions for the detection of the lac dye. The lower detection limit was achieved using nanoparticles prepared by reduction with hydroxylamine, excitation at 514.5 nm, and slightly acidic pH conditions, thus exploiting a combination of factors including lower electrostatic repulsion between dye and nanoparticles and resonance Raman enhancement. A comparison between the adsorption of laccaic acid (LA) and carminic acid (CA), another anthraquinone red dye, was also done, based on the SERS spectra of both dyes. Copyright © 2007 John Wiley & Sons, Ltd.     

DNA microarray scanner with a DVD pick-up head

A low-cost highly sensitive DNA microarray scanner for fluorescent detection is developed based on the pick-up head of a commercially available optical storage device, DVD. A laser beam of 650 nm, generated by a DVD laser diode, is used for dynamic auto-focusing as well as the excitation of Cy5 fluorescent dye. The fluorescence intensity emitted from Cy5 dye is measured by a photomultiplier tube (PMT). In contrast to other microarray scanners, the DVD-based scanner offers the auto-focusing function using the focus error signal (FES) and a voice coil motor (VCM), and this enables fast response, high accuracy and compact size. The fluorescence-detecting performance of the scanner is inspected by using a commercial BAC (bacterial artificial chromosome) oligonucleotide chip and a scanner evaluation microarray (DS01). Experiments have shown that the DVD-based scanner meets the limit of detection, ensuring the feasibility of a low-cost, highly sensitive DNA microarray scanner.     

Protein adsorption drastically reduces surface‐enhanced Raman signal of dye molecules

There is an increasing interest in developing surface enhancement Raman spectroscopy methods for intracellular biomolecule and for in vitro protein detection that involve dye or protein–dye conjugates. In this work, we have demonstrated that protein adsorption on silver nanoparticle (AgNP) can significantly attenuate the surface‐enhanced Raman spectroscopy (SERS) signal of dye molecules in both protein/dye mixtures and protein/dye conjugates. SERS spectra of 12 protein/dye mixtures were acquired using 4 proteins [bovine serum albumin (BSA), lysozyme, trypsin, and concanavalin A] and three dyes [Rhodamine 6G, adenine, and fluorescein isothiocyanate (FITC)]. Besides the protein/dye mixtures, spectra were also obtained for the free dyes and four FITC‐conjugated proteins. While no SERS signal was observed in protein/FITC mixtures or conjugates, a significantly reduced SERS intensity (up to 3 orders of magnitude) was observed for both R6G and adenine in their respective protein mixtures. Quantitative estimation of the number of dye molecules absorbed onto AgNP implied that the degree of R6G SERS signal reduction in the R6G/BSA sample is 2 to 3 orders of magnitude higher than what could be accounted for by the difference in the amount of the absorbed dyes. This finding has significant implications for both intracellular SERS analyses and in vitro protein detection using SERS tagging strategies that rely on Raman dyes as reporter molecules. Copyright © 2009 John Wiley & Sons, Ltd.     

基于近红外量子点的荧光共振能量转移生物探针构建及应用

本论文构建了基于近红外量子点In P/Zn S和Cy7(C45H44K3N3O16S4)的荧光共振能量转移(FRET)体系,完成了不同p H值和不同浓度下的FRET体系转换效率的检测。检测结果显示:当量子点浓度保持不变时,随着染料浓度的增加,体系转换效率也随之增加,当In P/Zn S量子点与Cy7浓度比为1∶250时,转换效率高达68%。细胞测试结果表明,FRET体系对p H值有较高敏感度,对细胞微环境p H值的检测精度可达0.1,该体系可以作为敏感型FRET探针用于生物微环境检测。     

阴离子染料百里酚蓝与纳克级核酸的共振光散射光谱特性及其分析应用

在 p H6 .0 0— 7.0 0和离子强度低于 0 .0 5 0 mol/ L的条件下 ,研究了百里酚蓝 (TB) -十六烷基三甲基溴化铵 (CTMAB) -核酸体系的共振光散射光谱 (RLS) ,影响因素及最佳反应条件。在最佳条件下 ,体系的ΔIRL S与 y DNA、ct DNA和 fs DNA在一定的浓度范围内呈线性关系。检出限可达 1.30 ng/ m L。该方法简便、快速 ,具有较高的灵敏度和准确度 ,应用于合成样品中核酸的测定 ,结果令人满意     

基于修正脉搏色素谱的循环血量检测方法

循环血量(CBV)作为主要的血流动力学参数,在心血管疾病的病情评估和手术监护中具有重要的临床应用价值。将吲哚菁绿色素(ICG)作为示踪剂的脉搏色素谱法,通过建立ICG稀释排泄的色素谱曲线,实现CBV的在体无创测量。在实际临床应用中,由于受到血氧波动和环境背景光等干扰因素的影响,脉搏色素谱法测量CBV的准确度低于预期值。为解决这一问题,研究了一种基于修正脉搏色素谱的循环血量检测方法。具体操作是,在患者的肘静脉处注入吲哚菁绿试剂,利用光电传感器分别采集特征波长点的透射光谱信号和背景光电信号,采用差分算法消除血氧波动和环境背景光的干扰影响,建立准确的ICG色素谱曲线,从而计算CBV等血流动力学参数。与131I同位素“金标准法”相比较的试验结果表明,该研究提出的基于修正脉搏色素谱的循环血量检测方法,将CBV测量的平均相对误差从6.85%降低为4.53%,显著提高了其测量准确度。