光纤与生物分子界面上固定FITC标记抗体的荧光光谱

采用化学方法把FITC标记羊抗人抗体IgG共价固定到三氨基三乙氧基硅烷和戊二醛(APTES-Glu)修饰的石英光纤纤芯表面。通过研究未经过任何修饰的纤芯表面的吸附以及共价固定的FITC标记羊抗人抗体的荧光光谱性质发现:共价结合到纤芯表面的FITC荧光光谱相对于溶液中FITC的荧光峰位红移约9nm,而物理吸附的FITC的荧光峰位移动约4nm,而且两者相对荧光强度相差6倍。而在固定的人血清蛋白进行免疫反应后,FITC的荧光峰位只移动3～4nm,除此之外,研究了抗体共价键固定的稳定性问题。结果表明:共价键结合抗体的数量要大于表面吸附,共价键固定的FITC标记羊抗人抗体与光纤表面间的相互作用较吸附于光纤表面的FITC标记羊抗人抗体间的相互作用更强。这对于建立一种通过荧光光谱识别固体表面与生物分子的吸附还是共价结合的判据提供了物理基础。     

一种环境友好型的PAEs荧光光谱衍生分子修饰方法

传统的PAEs荧光检测法主要是借助与具有荧光光谱特征的牛血清蛋白反应而进行的间接荧光检测。以六种被列入环境优先控制污染物的PAEs为例,对其苯环上4号位进行分子修饰,以期获得具有高荧光光谱强度的PAEs衍生物,利于直接荧光检测,同时利用分子对接的方法模拟PAEs分子与牛血清蛋白的结合,计算与牛血清蛋白结合后的PAEs分子荧光光谱强度,并将其与PAEs衍生物的荧光光谱强度进行比较,筛选荧光光谱显著增强的PAEs衍生物,为PAEs衍生物的检测提供理论支持。研究结果显示：共设计出30种PAEs衍生物,其中18种PAEs衍生物的荧光光谱强度增强显著（100%～1850%）,说明PAEs衍生物直接荧光检测的强度相较于PAEs分子间接荧光检测的强度具有显著增强作用;18种PAEs衍生物的功能特性（以稳定性、绝缘性作为代表）受到的影响较小,且PAEs衍生物的环境持久性均有所降低,生物富集性无明显变化,迁移性和毒性有不同程度的降低。此外,PAEs衍生物之间、与其他具有荧光特性的物质（多环芳烃）之间不存在干扰（最小波数差大于荧光光谱检测分辨率0.30 nm）,占用轨道能量及最正密立根氢原子电荷数是导致PAEs衍生物具有荧光光谱特性的主要控制因素。     

Au标蛋白自组装表面增强拉曼光谱的研究

采用免疫Au标技术,用尺寸大约在13 nm的Au胶体颗粒标记了人血清蛋白(Human IgG),然后将Au标蛋白复合体固定在通过三氨基三乙氧基硅烷和戊二醛自组装单膜的Si片上。这种方法在基底表面上不仅牢固地固定了单层Au纳米颗粒标记的蛋白分子复合体而且提高了复合体的表面覆盖度,保持了生物分子的结构。利用原子力显微镜(AFM)观察了Au标蛋白的自组装表面。实验结果表明Au标蛋白在Si片表面聚积形成一定的Au标蛋白分子的复合体“岛状”单层,并且在这些岛状单层上获得了很明显的标记蛋白的表面增强拉曼散射(SERS)信号。文章在Si表面自组装了Au标蛋白分子,获得了较好的蛋白分子的SERS信号,提供了一种研究蛋白分子的SERS活性基底。     

异硫氰酸荧光素标记抗体的固体基质室温磷光性质研究

研究了异硫氰酸荧光素（FITC）标记的羊抗人抗体（GAHAb-FITC)和兔抗羊抗体9RAGAb-FITC)，及其以三种免疫方式与人免疫球蛋白（IgG)反应所得免疫复合物在多种固体薄膜基质上发射室温磷光（RTP）的适宜条件及其光谱，强度和寿命等性质。研究发现，标记抗体及其免疫复合物保留了FITC优良的固体基质室温磷光性质，λ m0643em^(max)=525/650nm，其RTP强度与其浓度线性相关，并有很高的灵敏度，更为重要的是，免疫反应及其RTP检测能结合于同一基质，方便地相继完成，在聚酰胺膜（PM）上，以Pb(Ac)2为重原子微扰剂，稀释比为1：10（ψ）的GAHAb-FITC,RAGAb-FITC及其与人IgG形成的免疫复合物均能发射较强制 RTP信号并有较长的RTP寿命，本结果为建立相应的固体基质室温磷光免疫分析（SS-RTP-IA）新方法奠定了相应的实验基础。     

头孢噻肟与人血清白蛋白的相互作用机制

用光谱技术研究人血清蛋白(HSA)与头孢噻肟(CTX)分子间结合作用机制, 头孢噻肟与β-内酰胺酶的亲和力。由Lineweave-Burk双倒数作图法确定了该反应的解离常数K_d=1.22×10-4,用同步荧光技术考察头孢噻肟对人血清蛋白构象的影响。依据Frster非辐射能量转移机制,得到给体-受体间的结合距离和能量转移效率,确定了头孢噻肟与人血清白蛋白以疏水作用为主。认为头孢噻肟对β-内酰胺酶稳定性与药物结构有关;抗菌活性和抗生素效力与能量转移效率和解离常数有关。同步荧光技术考察头孢噻肟对人血清蛋白构象的影响。     

一种新型微通道分析系统——发光二极管诱导荧光检测器

报道了一种新型发光二极管诱导荧光检测器。该系统采用共线性光学结构、通过柱上检测可与微通道分析系统在线联用。以异硫氰酸荧光素(FITC)及其标记的氨基酸为标样、结合毛细管电泳和流动注射技术评价了该系统。对FITC标记的苯丙氨酸浓度检测限为10-8 mol·L-1(柱上检测,0.05 mm内径),在1×10-7～2×10-5 mol·L-1有很好的线性关系(r2 =0.999),6次连续进样所得峰高值相对标准偏差(RSD)小于3 %。     

光谱法研究3H-吲哚季铵盐探针分子与牛血清底的相互作用

合成了一种新型的荧光探针分子2-(对-十二烷基氨基)苯基-3, 3-二甲基-5-乙酯基-3H-吲哚基-甲基-二-十六烷基碘化铵, 并运用荧光光谱探讨它与牛血清蛋白的作用. 结果表明, 探针分子与牛血清蛋白(BSA)的结合作用可使3H-吲哚的荧光强度增强, 而对蛋白质却具有猝灭性质, 其结合常数和结合位点数分别为Ka=1.995×105 dm3*mol-1和n=1.12.     

全氟辛酸与血清蛋白分子间作用的紫外-荧光光谱分析法建立及理论模建研究

全氟辛酸(PFOA)与血清蛋白(SA)分子间作用分析法的建立及理论模建研究。荧光光谱结合紫外光谱法研究PFOA与血清蛋白(HSA)分子间作用,获得PFOA-HSA分子间作用的紫外-荧光特征谱,紫外-荧光特征谱表明PFOA规律性降低了HSA紫外吸收和荧光强度,HSA最大发射波长明显发生蓝移,表明PFOA与HSA发生了分子间作用,根据双对数回归曲线方程得到结合常数,定量说明PFOA与HSA之间存在中等强度的分子间作用力。Van’t Hoff方程分析光谱数据得到 HSA与PFOA分子间作用的热力学参数ΔH>0,ΔS>0,ΔG<0,依据Ross理论分析PFOA与HSA分子间作用,表明二者主要通过疏水作用力和氢键发生分子间作用,并且HSA与PFOA之间的分子间作用是自发过程。同步荧光光谱解析出PFOA与HSA的分子间作用导致血清蛋白分子的微区构象发生改变,使色氨酸残基位域构象发生改变。建议偏振因子M,通过荧光偏振光谱,定量表征PFOA与HSA分子间作用。基于光谱数据分析,建立了PFOA 与HSA分子间作用的理论模型,模建结果说明PFOA与HSA的分子间作用主要发生在HSA活性位点Sudlow’s sites I,HSA与PFOA分子间作用是自发过程。光谱实验与理论模建结果基本一致,可为全面了解生物大分子与全氟化合物之间的分子间作用以及研究微观毒理机制提供有益参考。     

长方体形CdS微粒的合成及光谱性质

采用溶胶法,以硫脲为表面修饰剂,合成了长方体形CdS微粒,并用X射线粉末衍射、透射电子显微镜、红外光谱以及荧光光谱等手段进行了表征。实验结果表明,硫脲分子中的硫原子与CdS纳米晶表面的Cd2+离子存在配位作用;硫脲分子表面修饰的CdS纳米晶为立方闪锌矿结构,具有较好的荧光性质;长方体形CdS微粒可能是由硫脲分子表面修饰的CdS初级纳米晶粒自组装组成。该研究结果为硫脲分子表面修饰的CdS初级纳米晶粒在分子组装及作为新型发光材料方面的进一步研究奠定了基础。     

铽标记人血清白蛋白的荧光光谱及应用

本文研究了由对-氨基水杨酸衍生的铽资合物及其标记人血清白蛋白的荧光光谱;测量了它们的激发态寿命.首次提出铽资合物标记蛋白质中蛋白质对Tb3+离子发光无影响.建立了液相中竞争性的人血清白蛋白的荧光免疫分析法.     

Studies of Fluorescence Immunosensor Using Eggshell Membrane as Immobilization Matrix

A novel immunosensor using eggshell membrane for determining the human immunoglobulin M (HIgM) in serum was developed. The immunosensor was fabricated by immobilizing goat anti-human IgM antibody on the eggshell membrane with glutaraldehyde. Based on the immunoreactions of goat anti-human IgM (primary antibody), HIgM (target antigen) and the goat anti-human IgM (secondary antibody), the sandwich complex were formed on the eggshell membrane and fluorescein isothiocyanate (FITC) labeling secondary antibody could be employed to detect the target antigen. Under the optimized conditions, the linear range for determining HIgM is 5-60 ng mL(-1) and the detection limit is 4.3 ng mL(-1), which are comparable with the results obtained by general immunonephelometric method. Meanwhile, this proposed sensor also exhibited remarkable storage stability, permeability and highly biocompatibility. The effects of temperature and pH value on eggshell membrane were investigated. Therefore, the proposed immunosensor, by using eggshell membrane as immobilization platform of antibody, offers an excellent fluorescence response to HIgM. The immunosesor provided a new alternative to determine antigens and other bioactive molecules.     

Silanization and antibody immobilization on SU-8

SU-8, an epoxy based negative photoresist, has emerged as a structural material for microfabricated sensors due to its attractive mechanical properties like low Young's modulus and chemical properties like inertness to various chemicals used in microfabrication. It can be used to fabricate MEMS structures of high aspect ratio. However, the use of SU-8 in BioMEMS application has been limited by the fact that immobilization of biomolecules on SU-8 surfaces has not been reported. In this study, the epoxy groups on the SU-8 surface were hydrolyzed in the presence of sulphochromic solution. Following this, the surface was treated with [3-(2-aminoethyl) aminopropyl]-trimethoxysilane (AEAPS). The silanized SU-8 surface was used to incubate human immunoglobulin (HIgG). The immobilization of HIgG was proved by allowing FITC tagged goat anti-human IgG to react with HIgG. This process of antibody immobilization was used to immobilize HIgG on microfabricated SU-8 cantilevers.     

Characterization of immobilization methods of antiviral antibodies in serum for electrochemical biosensors

In this paper, we describes different methods to immobilize Japanese encephalitis virus (JEV) antibodies in human serum onto the interdigitated surface of a microelectrode sensor for optimizing electrochemical detection: (1) direct covalent binding to the silanized surface, (2) binding to the silanized surface via a cross-linker of glutaraldehyde (GA), (3) binding to glutaraldehyde/silanized surface via goat anti-human IgG polyclonal antibody and (4) binding to glutaraldehyde/silanized surface via protein A (PrA). Field emission scanning electron microscopy, Fourier transform infrared spectrometry, and fluorescence microscopy are used to verify the characteristics of antibodies on the interdigitated surface after the serum antibodies immobilization. The analyzed results indicate that the use of protein A is an effective choice for immobilization and orientation of antibodies in serum for electrochemical biosensors. This study provides an advantageous immobilization method of serum containing antiviral antibodies to develop electrochemical biosensors for preliminary screening of viruses in clinical samples from outbreaks.     

Preparation and Fluoroimmunoassay Application of New Red-Region Fluorescent Silica Nanoparticles

This is the first report on the preparation and utilization of a novel red-region fluorescent dye (tetracarboxy aluminum phthalocyanine) doped silica nanoparticles. In these nanoparticles, the tetracarboxy aluminum phthalocyanine molecules were covalently bound to silica matrix to protect the dye leaking from nanoparticles in bio-applications. The surface of the nanoparticles was modified by amino groups and easily bioconjugated with goat anti-human IgG antibody. By employing these nanoparticles as fluorescent probe, a sensitive fluoroimmunoassay method has been developed for the determination of trace level of human IgG. The calibration graph for human IgG was linear over the range of 0–500 ng mL⁻¹ with a detection limit of 1.6 ng mL⁻¹. Compared with the corresponding system using free AlC₄Pc as a probe for determining human IgG, the sensitivity of the proposed system was notably increased. The method was applied to the analysis of human IgG in human sera with satisfactory results.     

Whole-mount sections displaying microvascular and glandular structures in human uterus using multiphoton excitation microscopy

There has been considerable interest over many years in the precise structural relationships between microvessels and secretory glands in human endometrium. However, microcirculatory networks have rarely been studied in three-dimensions (3D) using modern computerised technologies, this has been partly due to the late arrival of suitable endothelial cell markers. This study was designed to develop a technique to visualize and to reveal the relationships between microvessels, their glandular environment and epithelial boundaries in 3D, using endometrium from human hysterectomy biopsies. Specimens were carefully selected from women with conditions unlikely to affect the microvascular networks. Monoclonal antibodies (mouse anti-human CD 34 and goat anti-mouse fluorescein (FITC)) were used to visualize the microvessels, and polyclonal antibodies (rabbit anti-human keratin and goat anti-rabbit tetramethylrhodamine (TRITC)) were used to visualize the glandular structures. The samples were studied with a Leica multiphoton system using a titanium–sapphire laser (excitation 800 nm with pulses in the 200 fs range) to obtain a stack of two-dimensional (2D) images to a minimal focus depth of 120 μm. The initial data sets acquired were volume rendered using the integrated software of the Leica system to produce 3D images. This software allowed for the acquisition of data sets from the microscope and for an observational morphological assessment to be made, but was limited in preparing the data for any quantitative analysis. The additional use of ImarisBasic 3.1 visualization software allowed for an observational morphological assessment but also included numerous tools for data manipulation.     

Accurate Sensitivity of Quantum Dots for Detection of HER2 Expression in Breast Cancer Cells and Tissues

Here we introduce novel optical properties and accurate sensitivity of Quantum dot (QD)-based detection system for tracking the breast cancer marker, HER2. QD525 was used to detect HER2 using home-made HER2-specific monoclonal antibodies in fixed and living HER2⁺ SKBR-3 cell line and breast cancer tissues. Additionally, we compared fluorescence intensity (FI), photostability and staining index (SI) of QD525 signals at different exposure times and two excitation wavelengths with those of the conventional organic dye, FITC. Labeling signals of QD525 in both fixed and living breast cancer cells and tissue preparations were found to be significantly higher than those of FITC at 460–495 nm excitation wavelengths. Interestingly, when excited at 330–385 nm, the superiority of QD525 was more highlighted with at least 4–5 fold higher FI and SI compared to FITC. Moreover, QDs exhibited exceptional photostability during continuous illumination of cancerous cells and tissues, while FITC signal faded very quickly. QDs can be used as sensitive reporters for in situ detection of tumor markers which in turn could be viewed as a novel approach for early detection of cancers. To take comprehensive advantage of QDs, it is necessary that their optimal excitation wavelength is employed.     

IgA免疫复合物微粒的共振散射光谱研究及其分析应用

在pH 6.2的Na₂HPO₄-柠檬酸缓冲溶液中及聚乙二醇4 000存在下,免疫球蛋白A(IgA)与羊抗人免疫球蛋白A通过库力引力、范德华力、氢键结合力、疏水等作用力发生特异性结合形成抗原-抗体免疫复合物微粒。激光散射法测得该微粒的平均粒径约为1 100 nm;而且该微粒在340,390,420,450,470,520 nm有6个共振散射峰。考察了pH值、不同分子量聚乙二醇、羊抗人IgA血清用量、温度及反应时间对共振散射光谱测定IgA的影响。在最佳实验条件下,IgA浓度在0.133～4.67 μg·mL-1范围内与340和470 nm处的共振散射强度均呈线性关系,其回归方程分别为ΔI_{340 nm}=18.61 c_IgA+3.19,ΔI_{470 nm}=18.57 c_IgA+6.51,相关系数分别为0.998 5和0.998 7,检出限分别为0.068和0.072 μg·mL-1。该法用于人血清IgA的测定,相对标准偏差在2.2％～4.2％,并与免疫比浊法测定结果作线性回归分析,其斜率、截距和相关系数分别为1.064,-0.213和0.929 9,结果令人满意。     

Optimization of sonochemical method of functionalizing Amino-Silane on superparamagnetic iron oxide nanoparticles using Central Composite Design

Optimization of sonochemical method of functionalizing a Silane coupling agent, Amino-Silane on Superparamagnetic Iron Oxide Nanoparticles (SPION) using Central Composite Design is reported. The Amino-Silane is grafted on the SPION in an iced bath environment using a Vibra-Cell 20 kHz ultrasonic irradiator with 13 mm diameter horn. Throughout the experiment amplitude of the ultrasonic device is maintained at 47%. The percentage atomic compositions of various APTES elements which bind to the SPION due to the ultrasonic irradiation were determined using X-ray photoelectron spectrometer (XPS). The influence of ultrasonic irradiation time and amount of APTES required for facile, rapid and effective functionalization of Organo-metallic compound on SPION are optimized. The optimized sonication time and amount of APTES are 8.49 min and 3.40 ml, respectively. The predicted results were validated with experimental data. Using the optimized values APTES were functionalized on the SPION experimentally and the results were compared. The experimental results validate the predicted data. Results show that very minimum sonication time is required for effective grafting of APTES on SPION.