基于双抗体夹心传感膜的信号增强方法用于致癌基因c—myc蛋白的检测

采用层层自组装方法,制备了一种基于双抗体夹心层修饰金电极的信号增强电化学生物传感器,即先通过组装L-半胱氨酸、戊二醛,固定c—Myc（9E10）单克隆抗体（C．AbI）,形成C．Ab1单抗修饰电极,可识别致癌基因c—myc蛋白;再结合上第二抗体羊抗鼠免疫球蛋白G抗体（c．Ab2）,形成C．Ab1／c—myc／C—Ab2双抗夹心修饰电极,响应信号大幅度增强,传感性能优于C—A1单抗修饰电极．通过电化学阻抗和循环伏安行为探讨了双抗夹心法信号增强的机理,其阻抗值与c．myc浓度对数在0．043-430nM范围内成良好的线性关系,线性方程可拟合为Y=10046．10＋863．33墨线性相关系数为0．9904,c—myc的最低检测限也降低至25．76pM．该传感器制备简单,选择性、重现性、稳定性和再生性好,在鼠血清样品中测得c—myc的回收率在97．4％-103．7％之间,表明该方法可用于实际肿瘤样品中c—myc的检测,在生物医学领域具有潜在的应用价值．     

一种蛋白质阵列芯片新技术的研究

将微印刷技术与银增强法相结合,建立了一种蛋白质阵列芯片制备与检测的新方法。通过定量分析证明了蛋白质可以被均匀地转印至固相载体表面,采用银增强法对蛋白质的直接法检测和夹心法检测的最低检测量分别可达0.33和0.13 fmol。此新方法应用于蛋白质阵列上具有灵活、灵敏及成本低廉等特点。     

High density oligonucleotide and DNA probe arrays for the analysis of target DNA

The acquisition of sequence, expression and other information concerning genetic material constitutes a crucial component of the modern revolution in molecular biology. One important advance in this area is the development of high density oligonucleotide/DNA microarrays which allows the rapid sequence analysis of genomic target samples in addition to diagnostic possibilities with respect to genetic and infectious disease. In the present article we review protocols for the design of such microarrays and their principles of operation. Together with a look at some recent applications we include brief remarks as to the possibilities for the future.     

Optimization of processing parameters on the controlled growth of ZnO nanorod arrays for the performance improvement of solid-state dye-sensitized solar cells

High-transparency and high quality ZnO nanorod arrays were grown on the ITO substrates by a two-step chemical bath deposition (CBD) method. The effects of processing parameters including reaction temperature (25-95 °C) and solution concentration (0.01-0.1 M) on the crystal growth, alignment, optical and electrical properties were systematically investigated. It has been found that these process parameters are critical for the growth, orientation and aspect ratio of the nanorod arrays, showing different structural and optical properties. Experimental results reveal that the hexagonal ZnO nanorod arrays prepared under reaction temperature of 95 °C and solution concentration of 0.03 M possess highest aspect ratio of ∼21, and show the well-aligned orientation and optimum optical properties. Moreover the ZnO nanorod arrays based heterojunction electrodes and the solid-state dye-sensitized solar cells (SS-DSSCs) were fabricated with an improved optoelectrical performance.     

In situ synthesis of oligonucleotide arrays by using surface tension

This work describes the in situ synthesis of oligonucleotide arrays on glass surfaces. These arrays are composed of features defined and separated by differential surface tension (surface tension arrays). Specifically, photolithographic methods were used to create a series of spatially addressable, circular features containing an amino-terminated organosilane coupled to the glass through a siloxane linkage. Each feature is bounded by a perfluorosilanated surface. The differences in surface energies between the features and surrounding zones allow for chemical reactions to be readily localized within a defined site. The aminosilanation process was analyzed using contact angle, X-ray photoelectron spectroscopy (XPS), and time-of-flight/secondary ion mass spectroscopy (TOF-SIMS). The efficiency of phosphoramidite-based oligonucleotide synthesis on these surface tension arrays was measured by two methods. One method, termed step-yields-by-hybridization, indicates an average synthesis efficiency for all four (A,G,C,T) bases of 99.9 +/- 1.1%. Step yields measured for the individual amidite bases showed efficiencies of 98.8% (dT), 98.0% (dA), 97.0% (dC), and 97.6% (dG). The second method for determining the amidite coupling efficiencies was by capillary electrophoresis (CE) analysis. Homopolymers of dT (40- and 60mer), dA (40mer), and dC (40mer) were synthesized on an NH(4)OH labile linkage. After cleavage, the products were analyzed by CE. Synthesis efficiencies were calculated by comparison of the full-length product peak with the failure peaks. The calculated coupling efficiencies were 98.8% (dT), 96.8% (dA), and 96.7% (dC).     

Microfluidic devices for fluidic circulation and mixing improve hybridization signal intensity on DNA arrays

Reactions of biomolecules with surface mounted materials on microscope slides are often limited by slow diffusion kinetics, especially in low volumes where diffusion is the only means of mixing. This is a particular problem for reactions where only small amounts of analyte are available and the required reaction volume limits the analyte concentration. A low volume microfluidic device consisting of two interconnected 9 mm x 37.5 mm reaction chambers was developed to allow mixing and closed loop fluidic circulation over most of the surface of a microscope slide. Fluid samples are moved from one reaction chamber to the other by the rotation of a magnetic stirring bar that is driven by a standard magnetic stirrer. We demonstrate that circulation and mixing of different reagents can be efficiently accomplished by this closed loop device with solutions varying in viscosity from 1 to 16.2 centipoise. We also show by example of a microarray hybridization that the reaction efficiency can be enhanced 2-5 fold through fluid mixing under conditions where diffusion is rate limiting. For comparison, similar results were achieved with a disposable commercial device that covers only half of the reaction area of the closed loop device.     

Micelles for signal enhancement and novel selectivity of dansyl amino acids

Fluorescence intensity of various chemical species is enhanced in the microenvironment provided by micelles. Parameters which affect fluorescence intensities are examined by using dansyl (Dns) amino acids as the probe. The retention behavior of Dns-amino acids in micellar LC is examined by using ion-exchange-induced stationary phases. The type and concentration of micellar agent and modifier ion as well as concentration of acetonitrile in the mobile phase affect the retention and signal intensity of Dns-amino acids. The order of elution of Dns-amino acids obtained with the micellar mobile phase is very different from that observed in conventional reversed-phase LC. Fluorescence intensities of Dns-amino acids are enhanced by the micellar mobile phase.     

微孔板蛋白芯片可视化检测方法的研究

将微孔板生物芯片制备技术与银增强显色方法相结合,建立了一种高通量蛋白免疫分析新方法。研究采用夹心法反应原理,将人IgG捕获抗体以微阵列形式点样于96孔板底部制备成蛋白微阵列,并依次与人IgG、生物素标记二抗及链亲和素胶体金反应,最后用银增强法显色。采用平板式扫描仪对微孔板显色结果进行快速扫描成像,图像采用luxscan 3.0软件处理。结果表明:微孔板蛋白芯片的最低检测量可达0.6 ng/mL,线性范围10 ng/mL~100μg/mL,相关系数为0.98,重复性较好(CV10%)。     

Enzymatic fabrication of DNA nanostructures: extension of a self-assembled oligonucleotide monolayer on gold arrays

Nucleic acid nanostructures are useful as templates for bionanofabrication of composite molecular nanostructures in materials science, molecular electronics, and biosensing. Here, we demonstrate that terminal deoxynucleotidyl transferase, which repetitively adds mononucleotides to the 3' end of a short DNA initiator, can be used to rapidly fabricate DNA nanostructures up to 121 nm high with lateral dimensions from 0.1 to 4 mum in 2 h. These programmable scaffolds can potentially be employed to build more complex nanostructures consisting of natural or unnatural nucleotides with selective docking sites along the single-stranded DNA.     

Data and signal processing using photochromic molecules

Photochromes are chromophores that are reversibly isomerized between two metastable forms using light, or light and heat. When photochromes are covalently linked to other chromophores, they can act as molecular photonic analogues of electronic transistors. As bistable switches, they can be incorporated into the design of molecules capable of binary arithmetic and both combinatorial and sequential digital logic operations. Small ensembles of such molecules can perform analogue signal modulation similar to that carried out by transistor amplifiers. Examples of molecules that perform multiple logic functions, act as control elements for fluorescent reporters, and mimic natural photoregulatory functions are presented.     

Molecular recognition and signal processing in biosensors

The specificity of molecular recognition is reflected to a high degree by the selectivity of the respective biosensor response. Therefore, the application of highly specific enzymes offers advantages for analytical purposes. Using lactate monooxygenase in combination with lactate dehydrogenase and pyruvate kinase, sequentially acting enzyme electrodes for lactate, pyruvate, ADP and enzyme activities, e.g. lactate dehydrogenase, creatine kinase and aminotransferases were developed. A high sensitivity was achieved based on the cycling enzyme pairs hexokinase/pyruvate kinase for ATP and ADP, and lactate monooxygenase/malate dehydrogenase for malate and oxaloacetate, respectively. On the other hand, in a sensor for a large group of substances, the unspecific microsomal cytochrome P-450 system was applied.     

DNA-templated self-assembly of protein and nanoparticle linear arrays

Self-assembling DNA tiling lattices represent a versatile system for nanoscale construction. Self-assembled DNA arrays provide an excellent template for spatially positioning other molecules with increased relative precision and programmability. Here we report an experiment using a linear array of DNA triple crossover tiles to controllably template the self-assembly of single-layer or double-layer linear arrays of streptavidin molecules and streptavidin-conjugated nanogold particles through biotin-streptavidin interaction. The organization of streptavidin and its conjugated gold nanoparticles into periodic arrays was visualized by atomic force microscopy and scanning electron microscopy.     

Self-assembled, mesoporous polymeric networks for patterned protein arrays

A facile, self-assembly approach to the fabrication of a robust, mesoporous, biocompatible polymeric network for the spatial organization of proteins is described. Surface-deposited poly(styrene) (PS) beads that assemble into a two-dimensional (2-D) hexagonal array are used to template cross-linked poly(vinyl alcohol) (PVA), yielding an inverse opal structure. The porous, water insoluble network is used to entrain a model, soluble protein, green fluorescent protein (GFP). The polymeric network is characterized by atomic force microscopy (AFM) and optical microscopy, and the spatial localization of the incorporated GFP is determined by fluorescence microscopy. The results demonstrate that this system may constitute a versatile platform for the lateral organization of biomolecules.     

Analysis of Variance (ANOVA) for Optimizing the Nano-SiO2 Content of High Performance Epoxy Nanocomposites

Epoxy based nanocomposite samples containing SiO₂ nanoparticles (0.0–3.0 %w) were prepared for physical and mechanical evaluation. Some thermomechanical and physical properties of samples were investigated using dynamic mechanical analysis (DMA), tensile strength, hardness and abrasion tests. The main aim of experimentation was to realize the optimum amount of nano-SiO₂ which would demonstrate the best improving effect on mechanical and physical properties of nanocomposite samples and finding how significant a factor is for improving in physical and mechanical properties. Analysis of variance (ANOVA) was applied for optimization of SiO₂ content in epoxy based nanocomposites.     

A self-assembling protein template for constrained synthesis and patterning of nanoparticle arrays

Self-assembling biomolecules that form highly ordered structures have attracted interest as potential alternatives to conventional lithographic processes for patterning materials. Here, we introduce a general technique for patterning nanoparticle arrays using two-dimensional crystals of genetically modified hollow protein structures called chaperonins. Constrained chemical synthesis of transition metal nanoparticles is initiated using templates functionalized with polyhistidine sequences. These nanoparticles are ordered into arrays because the template-driven synthesis is constrained by the nanoscale structure of the crystallized protein. We anticipate that this system may be used to pattern different classes of nanoparticles based on the growing library of sequences shown to specifically bind or direct the growth of materials.     

Bisdioxaborine polymethines with large third-order nonlinearities for all-optical signal processing

Organic materials with large third-order nonlinearities in the near-infrared spectral regime are critical in the development of photonic devices to be utilized in all-optical signal processing. We have developed polymethine materials, specifically bisdioxaborine-terminated polymethine dyes, which possess large ultrafast third-order nonlinearities and low nonlinear loss all in the near-infrared spectral regime. An extended bisdioxaborine polymethine anion exhibited the largest value of gamma (third-order microscopic nonlinearity) at 1.3 mum (|gamma| = 5.7 x 10-32 esu) and showed no characteristics of symmetry breaking, unlike other polymethines of similar lengths. A neat film of this molecule maintained relatively low linear loss in the near-infrared and showed a large third-order macroscopic nonlinearity at 1.3 mum (|chi(3)| = 3.6 x 10-10 esu), with a temporal response of less than 8 ps. Furthermore, the real part of chi(3) was nearly an order-of-magnitude larger than the imaginary component. Consequently, this material exhibited good figures of merit for all-optical signal processing throughout the entire telecommunications band.