罗丹明B自组装膜对单、双链脱氧核糖核酸的荧光识别

通过静电吸引作用将罗丹明B(RB)组装在硅烷化的石英基底上, 构建了修饰有RB的双层自组装膜Quartz/APES/RB.在三羟甲基氨基甲烷-盐酸(Tris-HCl)缓冲液(pH 7.4)中, 利用荧光分光光度法研究了Quartz/APES/RB与单、双链DNA的相互作用.考察了自组装膜的组装条件、膜性质及对单、双链DNA的作用机理.实验表明, Quartz/APES/RB对单、双链DNA检出限分别为 2.4 ng/L和0.85 ng/L, 可应用于痕量单、双链DNA的荧光识别.     

胶体金和Taq DNA聚合酶的相互作用研究

聚合酶链反应（PCR）是体外扩增DNA序列的技术，据报道：一定浓度的胶体金可以提高PCR的特异性。本文研究了胶体金与Taq DNA聚合酶(Taq)的相互作用。紫外-可见光谱，X射线光电子能谱(XPS)和光子交叉相关光谱 (PCCS)的结果表明，当胶体金和Taq溶液混合时会生成Taq-金结合物。用透射电镜(TEM)进一步研究发现，Taq-金结合物颗粒仍然呈球形，并且具有很好的分散性。通过分析PCR扩增产量，发现胶体金的加入会抑制PCR反应，而添加聚合酶的用量可以明显地消除这种抑制现象，这表明胶体金和Taq的相互作用会导致Taq的活性下降。     

光谱法与共焦荧光成像法研究罗丹明B与ct-DNA的相互作用

采用吸收光谱、荧光光谱以及共焦荧光成像技术研究生理条件下罗丹明B(Rhodamine B,RB)与小牛胸腺DNA(ct-DNA)的相互作用以及相互作用模式。光谱研究结果显示,在450~650 nm的吸收光谱范围内,ct-DNA溶液对罗丹明B有减色作用;在560~660 nm荧光发射光谱范围内,ct-DNA对罗丹明B有荧光猝灭作用。同时,随着ct-DNA的加入,罗丹明B溶液的荧光偏振值发生变化,说明罗丹明B与ct-DNA能发生相互作用。在竞争性实验中,罗丹明B未能将亚甲基蓝(MB)从MB-ct-DNA复合体系中置换出来,说明罗丹明B与ct-DNA通过沟槽结合方式发生相互作用。共焦荧光成像结果显示,ct-DNA加入后,罗丹明B的荧光猝灭十分明显。利用罗丹明B和4',6-二脒基-2-苯基吲哚(DAPI)对He La细胞染色后进行共焦荧光成像,结果进一步证实罗丹明B与ct-DNA是通过沟槽结合作用将细胞核染成红色。     

分子光谱法研究漆黄素与DNA的相互作用

利用多种光谱技术及伏安技术研究了漆黄素与DNA之间的相互作用.由荧光光谱法可知,DNA对漆黄素的猝灭过程是静态猝灭,计算得到不同温度下二者的结合常数及结合位点数.ΔH,ΔS,ΔG的计算结果表明漆黄素与DNA的作用力为范德华力或氢键作用.循环伏安法进一步表明了DNA的加入使漆黄素的峰电位发生了移动,二者产生了有效结合.离子强度实验、黏度法、紫外光谱法、圆二色光谱法、DNA熔解温度实验和单双链DNA对比实验的结果均表明漆黄素与DNA的结合是嵌插结合模式.     

基于DNA双链取代策略免标记检测铅离子的研究

建立了一种基于DNA双链取代策略和SYBR GreenⅠ(SG)作为荧光染料插入剂进行免标记铅离子检测的荧光传感方法。SG作为一种染料分子,与单链DNA作用产生的荧光强度很弱,但可以插入双链DNA,使SG荧光强度明显增强。检测时铅离子适配体首先与其部分互补单链DNA杂交形成稳定的双链DNA结构,当溶液中存在铅离子时,铅离子与其适配体特异性结合,双链DNA的数量减少,加入SG可实现铅离子的免标记定量检测。此方法具有灵敏度高、特异性强、简便快速等优点。最低检测浓度为2 nmol/L,检出限(S/N=3)为1.6 nmol/L,实际样品检测结果良好。     

基于阳离子型共轭聚合物和核酸适体的腺苷检测新方法

建立了一种基于阳离子型共轭聚合物和核酸适体的腺苷检测新方法. 荧光素修饰的短链DNA与腺苷的核酸适体部分互补, 形成双链DNA; 阳离子型共轭聚合物通过静电作用与双链DNA结合, 发生高效率的荧光共振能量转移(FRET). 加入腺苷后, 腺苷与核酸适体发生特异性结合, 导致双链DNA分解成单链, 使静电吸引力下降, 能量转移效率降低. 通过阳离子型共轭聚合物对单双链DNA的高效识别, 可快速简易地检测出腺苷.     

碳量子点对基于能量转移电化学发光DNA传感体系影响的研究

实验发现在鲁米诺-结晶紫能量转移电化学发光体系中再加入碳量子点后,体系的灵敏度和重现性均提高.本实验中,探究出用能量转移电化学发光分析法区分单双链DNA的优化条件.在优化条件下,测定目标DNA的线性范围为1.0×10-11～1.0×10-7mol/L,相关系数为0.9881,检出限为4.0×10-12mol/L,并且讨论了DNA与碳量子点可能的作用方式.本法操作方便,灵敏度高.     

盐酸小檗碱与DNA作用的序列特异性研究

基于分子吸收和发射测量,研究了盐酸小檗碱与特定序列DNA间的相互作用.结果发现双链DNA中含有不配对 G 碱基时,其与盐酸小檗碱作用后产生的光谱特征将明显不同于完全互补的双链DNA.该特征可被用于检测1个错配碱基的双链DNA.利用 Hyperchem Professional 软件包计算了4种寡聚核苷酸的电荷分布情况,并在此基础上进一步探讨了盐酸小檗碱与寡聚核苷酸之间的作用机理.     

基于氧化石墨烯作为传感平台的DNA电化学阻抗谱传感器制备

制备了一种基于氧化石墨烯作为传感平台的无标记DNA传感器。探针DNA通过π-π堆积作用固定到氧化石墨烯表面,电化学交流阻抗法作为传感表征及检测技术。当传感器识别目标DNA时,由于形成双链结构,双链DNA离开氧化石墨烯表面,进入溶液,使得电化学阻抗值减小。因此,可以利用杂化前后DNA传感器所展现出阻抗值的差异,实现对目标DNA的定量检测。结果表明:目标DNA浓度在1.0×10-8~1.0×10-12mol/L范围内,阻抗值变化与目标DNA浓度的对数呈线性关系,检出限为3.5×10-13mol/L(S/N=3)。此外,传感器能区分互补单碱基错配、完全错配的DNA序列。     

测定蛋白质的罗丹明B自聚平衡体系

以罗丹明B单体和二聚体平衡转化为基础，提出了罗丹明B自聚平衡体系测定蛋白质方法；实验表明，罗丹明B在阴离子表面活性剂的作用下自聚成二聚体，体系荧光降低，但是蛋白质加入后体系荧光增强；该法灵敏度高，简便，快速，选择性好，实验结果与传统的溴甲酚绿方法一致。     

Label free DNA detection based on gold nanoparticles quenching fluorescence of Rhodamine B

A novel and sensitive label free DNA detection method using gold nanoparticles (GNPs) and Rhodamine B (RB) has been developed. The assay is based on the following two properties. One is the different adsorption properties of single-stranded and double-stranded DNA on GNPs in colloidal solution. The other is the different quenching ability of aggregated GNPs and dispersed GNPs on RB. Un-aggregated GNPs could effectively quench the fluorescence of RB. However, the quenching ability greatly decreases after GNPs aggregated. The hybridization of probe DNA and target DNA is monitored by the fluorescence detection after the RB is added to the solution. Under the optimal experimental conditions, the detection limit of this assay is 2.9×10(-13) mol L(-1).     

Enhancement of the Efficacy of Photodynamic Inactivation of Candida albicans with the Use of Biogenic Gold Nanoparticles

This study reports on successful photodynamic inactivation of planktonic and biofilm cells of Candida albicans using Rose Bengal (RB) in combination with biogenic gold nanoparticles synthesized by the cell‐free filtrate of Penicillium funiculosum BL1 strain. Monodispersed colloidal gold nanoparticles coated with proteins were characterized by a number of techniques including SEM–EDS, TEM, UV–Vis absorption and fluorescence spectroscopy, as well as Fourier transform infrared spectroscopy to be 24 ± 3 nm spheres. A Xe lamp (output power of 20mW, delivering intensity of 53 mW cm^?2) was used as a light source to study the effects of RB alone, the gold nanoparticles alone and the RB‐gold nanoparticle mixture on the viability of C. albicans cells. The most effective reduction in the number of planktonic cells was found after 30 min of Xe lamp light irradiation (95.4 J cm^?2) and was 4.89 log₁₀ that is 99.99% kill for the mixture of RB with gold nanoparticles compared with 2.19 log₁₀ or 99.37% for RB alone. The biofilm cells were more resistant to photodynamic inactivation, and the highest effective reduction in the number of cells was found after 30 min of irradiation in the presence of the RB–gold nanoparticles mixture and was 1.53 log₁₀, that is 97.04% kill compared with 0.6 log₁₀ or 74.73% for RB. The probable mechanism of enhancement of RB‐mediated photodynamic fungicidal efficacy against C. albicans in the presence of biogenic gold nanoparticles is discussed leading to the conclusion that this process may have a multifaceted character.     

Investigation on the interaction between colloidal gold and human complement factor 4 at different pH by spectral methods

The interaction between colloidal gold and human complement factor 4 (human C4) at different pH was investigated by spectral methods, including absorption and resonance light-scattering spectrometry. According to the changes of color and absorption spectra of colloidal gold solution in presence of human C4, the interaction between colloidal gold and human C4 was quantitatively investigated using a semi-empirical "flocculation parameter". At the same time, the changes of resonance light-scattering spectra and transmission electron microscopy (TEM) images indicate that the aggregation of colloidal gold happens by electrostatic interaction in presence of human C4 in the pH range 5-6. However, the colloidal gold solution remains stable at pH >6 and pH <5 due to the repulsive electrostatic interaction between colloidal gold and human C4. The flocculation parameter is directly proportional to the concentration of human C4 in the range from 9.7 to 233.0 microgl(-1). In addition, the interactions between the colloidal gold and bovine serum albumin (BSA) as well as human serum albumin (HSA) were also investigated using the same methods. It was found that there was no aggregation of colloidal gold in presence of BSA and HSA in the pH range 5-6. However, when the pH of solution is 4, the aggregation of colloidal gold happens. Because BSA and HSA have different structure, the intensity of aggregation of colloidal gold in presence of BSA is greater than that in presence of HSA at pH 4.     

Riboflavin and UV-Light Based Pathogen Reduction: Extent and Consequence of DNA Damage at the Molecular Level

We are developing a technology based on the combined application of riboflavin (RB) and light for inactivating pathogens in blood products while retaining the biological functions of the treated cells and proteins. Virus and bacteria reduction measured by tissue culture infectivity or colony formation with UV light alone and in combination with RB yield equivalent results. The effects of RB as a sensitizing agent on DNA in white cells, bacteria and viruses in combination with UV light exposure have been evaluated. UV-mediated DNA degradation in Jurkat T cells and leukocytes in plasma as measured by the FlowTACS assay was significantly increased in the presence of RB. Agarose gel electrophoretic analysis of DNA in Escherichia coli and leukocytes in plasma demonstrated enhanced DNA degradation in the presence of RB. UV light in combination with RB prevented the reactivation of lambda phage compared with samples irradiated in the absence of RB. UV-mediated oxidative damage in calf thymus DNA was also enhanced in the presence of RB. These observations clearly demonstrate that the presence of RB and UV light selectively enhances damage to the guanine bases in DNA. These data also suggest that the type and extent of damage to DNA for virus in the presence of RB and light make it less likely to be repaired by normal repair pathways available in host cells.     

Studies on the interaction of colloidal gold and serum albumins by spectral methods

The interactions of colloidal gold and serum albumins, including bovine serum albumin (BSA) and human serum albumin (HSA), were studied by fluorescence and absorption spectrometry. Fluorescence quenching spectrometry was applied to study the interactions between colloidal gold and serum albumins. At pH 7.4 phosphate-buffered saline (PBS), the intensity of fluorescence emission spectrum of serum albumins decreased in the presence of colloidal gold, which indicated that colloidal gold quenched the fluorescence of serum albumins. Experimental results indicated that the combination reactions of colloidal gold and serum albumins were static quenching processes. Based on the effect of colloidal gold on fluorescence intensity, the binding constants, the numbers of binding sites and the acting forces between colloidal gold and serum albumins were found.     

DNA dissociation and degradation at gold nanoparticle surfaces

The instability of DNA-functionalized gold nanoparticles (Au-DNA conjugates) upon exposure to high temperatures is characterized using fluorescence spectroscopy, gel electrophoresis and ion-exchange chromatography. Above 70 °C, aqueous Au-DNA conjugates decompose within hours due to both desorption of thiol-terminated DNA from the gold nanoparticle surface and chemical degradation of DNA in the presence of colloidal gold. Although the chemical mechanism for DNA degradation was not identified in this study, the gold surface participates directly in the cleavage reaction. These results have important implications for the use of Au-DNA conjugates in biotechnological and clinical applications that require high temperatures, such as polymerase chain reaction (PCR).     

Dynamic investigation of gold nanocrystal assembly using in situ grazing-incidence small-angle X-ray scattering

Here we investigate the dynamic self-assembly pathway of ordered gold nanocrystal arrays during the self-assembly of gold nanocrystal micelles, with and without the presence of colloidal silica precursors, using grazing-incidence X-ray scattering performed at a synchrotron source. With silica precursors present, a lattice with rhombohedral symmetry is formed from the partial collapse of a face-centered cubic structure. In the absence of silica, a transient body-centered orthorhombic phase appears, which rapidly collapses into a glassy nanocrystal film. The appearance of face-centered and body-centered structures is consistent with a phase diagram for charged colloidal particles with assembly modulated via Coulomb screening.     

Study on the Interaction of Colloidal Gold with Taq DNA Polymerase

The interaction of colloidal gold with Taq DNA polymerase （Taq） was investigated in this study. Taq-gold conjugate was formed by adding the enzyme to the colloidal gold solution, as evidenced by UV-Vis spectroscopy, X-ray photoelectron spectroscopy, and photon cross correlation spectroscopy measurements. The conjugate was further characterized by transmission electron microscopy. It was found that the Taq-gold conjugate particles were still spherical and well-dispersed. The influence of gold nanoparticles on the bioactivity of Taq was studied by analyzing the yield of the polymerase chain reaction amplification. Results indicated that the enzymatic activity of Taq decreased after interaction with the colloidal gold.     

Characterization of single- and double-stranded DNA on gold surfaces

Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5'-C(6)H(12)SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS). The formation of dsDNA on the surface was identified by PM-IRRAS by a dsDNA IR signature at approximately 1678 cm(-)(1) that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 degrees C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au-S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.     

Electrostatic-assembly metallized nanoparticles network by DNA template

Eighteen-nanometer gold and 3.5-nm silver colloidal particles closely packed by cetyltrimethylammonium bromide (CTAB) to form its positively charged shell. The DNA network was formed on a mica substrate firstly. Later, CTAB-capped gold or silver colloidal solutions were cast onto DNA network surface. It was found that the gold or silver nanoparticles metallized networks were formed owing to the electrostatic-driven template assembling of positive charge of CTAB-capped gold and silver particles on the negatively charged phosphate groups of DNA molecules by the characterizations of AFM, XPS and UV-vis. This method may provide a novel and simple way to studying nanoparticles assembly conjugating DNA molecules and offer some potential promising applications in nanocatalysis, nanoelectronics, and nanosensor on the basis of the fabricated metal nanoparticles network.