液体环境单分子免疫球蛋白G的原子力显微镜高分辨成像

原子力显微镜技术( AFM)具有纳米级高分辨成像能力,是研究生物大分子结构和功能的重要工具之一。制备合适的样品是获取高分辨成像的关键要素。本研究结合DNA折纸技术,将抗原分子修饰在DNA折纸上,通过分子识别作用,抗体分子与抗原分子特异性结合,形成由DNA折纸和抗原抗体复合物构成的纳米结构。利用DNA折纸在云母表面上的吸附特点,使得抗体分子选择性地吸附在衬底表面上,由此获得了液体环境中的单个地高辛抗体免疫球蛋白G( IgG)分子的“Y”超微结构形貌。本方法简单、方便,为AFM在单分子水平上检测和表征生物分子结构和功能提供帮助。     

Detection of DNA and Protein Molecules Using an FET‐Type Biosensor with Gold as a Gate Metal

In this study, a field effect transistor (FET)‐type biosensor based on 0.5 μm standard complementary metal oxide semiconductor (CMOS) technology is proposed and its feasibility for detecting deoxyribonucleic acid (DNA) and protein molecules is investigated. Au, which has a chemical affinity with thiol by forming a self‐assembled monolayer (SAM), was used as the gate metal in order to immobilize DNA and protein molecules. A Pt pseudo‐reference electrode was employed for the detection of biomolecules. The sensor was fabricated as a p‐channel (P)MOSFET‐type because PMOSFET with positive surface potential is useful for detecting negatively charged biomolecules from the view point of its high sensitivity and fast response time. DNA and protein molecules were detected by measuring the variation of the drain current due to the variation of biomolecular charge and capacitance. DNA and protein molecules used in the experiment were 15mer–oligonucleotide probe and streptavidin‐biotin protein complexes, respectively. DNA was detected by both in situ and ex situ measurements. Additionally, to verify the interactions among SAM, streptavidin, and biotin, surface plasmon resonance (SPR) measurement was performed.     

Total internal reflection ellipsometry as a label-free assessment method for optimization of the reactive surface of bioassay devices based on a functionalized cycloolefin polymer

We report a label-free optical detection technique, called total internal reflection ellipsometry (TIRE), which can be applied to study the interactions between biomolecules and a functionalized polymer surface. Zeonor (ZR), a cycloolefin polymer with low autofluorescence, high optical transmittance and excellent chemical resistance, is a highly suitable material for optical biosensor platforms owing to the ease of fabrication. It can also be modified with a range of reactive chemical groups for surface functionalization. We demonstrate the applications of TIRE in monitoring DNA hybridization assays and human chorionic gonadotrophin sandwich immunoassays on the ZR surface functionalized with carboxyl groups. The Ψ and Δ spectra obtained after the binding of each layer of analyte have been fitted to a four-layer ellipsometric model to quantitatively determine the amount of analytes bound specifically to the functionalized ZR surface. Our proposed TIRE technique with its very low analyte consumption and its microfluidic array format could be a useful tool for evaluating several crucial parameters in immunoassays, DNA interactions, adsorption of biomolecules to solid surfaces, or assessment of the reactivity of a functionalized polymer surface towards a specific analyte.     

DNA发夹结构自组装信号放大策略应用的研究进展

DNA发夹结构自组装因具有无酶参与、等温以及识别序列能力强等优点,在生物分子和金属离子检测方面展现了良好的发展前景。该文梳理了DNA发夹结构自组装信号放大策略的类型,综述了近年来该策略在致病菌、核酸肿瘤标记物、蛋白质、无机金属离子,以及生物小分子检测中应用的研究进展,并对其未来发展趋势进行了展望,旨在为基于DNA发夹结构自组装检测生物分子提供一定的参考。     

Electrochemical Treatment of Glassy Carbon for Label‐Free Detection of DNA Bases and Neurotransmitters

Electrochemically reduced glassy carbon (r‐GC) showed a superior electrochemical sensing performance, compared to oxidized GC (ox‐GC) and untreated GC for the oxidation of 4 DNA bases and neurotransmitters (epinephrine, norepinephrine and serotonin). r‐GC exhibited not only the largest current intensities of all redox biomolecules, but also displayed an excellent selectivity in detecting coexisting redox biomolecules. The enhanced performance of r‐GC was attributed to the improved surface cleanliness of electrode and its catalytic surface functional groups. The results presented herein imply that simple electrochemical treatments are a viable method to produce sensitive and selective electrodes for label‐free biosensing.     

Non-covalent monolayer-piercing anchoring of lipophilic nucleic acids: preparation, characterization, and sensing applications

Functional interfaces of biomolecules and inorganic substrates like semiconductor materials are of utmost importance for the development of highly sensitive biosensors and microarray technology. However, there is still a lot of room for improving the techniques for immobilization of biomolecules, in particular nucleic acids and proteins. Conventional anchoring strategies rely on attaching biomacromolecules via complementary functional groups, appropriate bifunctional linker molecules, or non-covalent immobilization via electrostatic interactions. In this work, we demonstrate a facile, new, and general method for the reversible non-covalent attachment of amphiphilic DNA probes containing hydrophobic units attached to the nucleobases (lipid-DNA) onto SAM-modified gold electrodes, silicon semiconductor surfaces, and glass substrates. We show the anchoring of well-defined amounts of lipid-DNA onto the surface by insertion of their lipid tails into the hydrophobic monolayer structure. The surface coverage of DNA molecules can be conveniently controlled by modulating the initial concentration and incubation time. Further control over the DNA layer is afforded by the additional external stimulus of temperature. Heating the DNA-modified surfaces at temperatures >80 °C leads to the release of the lipid-DNA structures from the surface without harming the integrity of the hydrophobic SAMs. These supramolecular DNA layers can be further tuned by anchoring onto a mixed SAM containing hydrophobic molecules of different lengths, rather than a homogeneous SAM. Immobilization of lipid-DNA on such SAMs has revealed that the surface density of DNA probes is highly dependent on the composition of the surface layer and the structure of the lipid-DNA. The formation of the lipid-DNA sensing layers was monitored and characterized by numerous techniques including X-ray photoelectron spectroscopy, quartz crystal microbalance, ellipsometry, contact angle measurements, atomic force microscopy, and confocal fluorescence imaging. Finally, this new DNA modification strategy was applied for the sensing of target DNAs using silicon-nanowire field-effect transistor device arrays, showing a high degree of specificity toward the complementary DNA target, as well as single-base mismatch selectivity.     

Electrochemical characteristics of the immobilization of calf thymus DNA molecules on multi-walled carbon nanotubes

Immobilization of DNA on carbon nanotubes plays an important role in the development of new types of miniature DNA biosensors. Electrochemical characteristics of the immobilization of calf thymus DNA molecules on the surfaces of multi-walled carbon nanotubes (MWNTs) have been investigated by cyclic voltammetry and electrochemical impedance analysis. The peak currents for Fe(CN)(6)(3-)/Fe(CN)(6)(4-) redox couple observed in the cyclic voltammograms decrease and the electron-transfer resistance (R(et)) obtained from the Nyquist plots increase due to the immobilization of DNA molecules (dsDNA or ssDNA) on the surfaces of MWNTs. Most of calf thymus DNA are covalently immobilized on MWNTs via diimide-activated amidation between the carboxylic acid groups on the carbon nanotubes and the amino groups on DNA bases, though the direct adsorption of the DNA molecules on MWNTs can be observed. Additionally, the interaction between DNA molecules immobilized on MWNTs and small biomolecules (ethidium bromide) can be observed obviously by cyclic voltammetry and electrochemical impedance analysis. This implies that the DNA molecules immobilized at the surface of MWNTs, with little structure change, still has the ability to interact with small biomolecules.     

Multicomponent submicron features of biomolecules created by voltage controlled deposition from a nanopipet

We have used a nanopipet as a nanopen to locally and controllably deposit complex biomolecules, including antibodies and DNA, onto a surface to create multicomponent and functional submicron features. Key advantages of this method are that the biomolecules are always in solution and the applied voltage provides fine control of the delivery down to the single molecule level. Two consecutive cycles of deposition, to produce spatially varying features with different biological properties, were demonstrated with fluorescently labeled antibodies or biotin. This approach combines "top-down" fabrication, using the nanopen for local application, and "bottom-up" fabrication, using molecular recognition for self-assembly at defined positions, and opens up new possibilities in nanotechnology.     

A Self‐Reporting Tetrazole‐Based Linker for the Biofunctionalization of Gold Nanorods

A photochemical approach based on nitrile imine‐mediated tetrazole‐ene cycloaddition is introduced to functionalize gold nanorods with biomolecules. For this purpose, a bifunctional, photoreactive linker containing thioctic acid as the Au anchoring group and a tetrazole moiety for the light‐induced reaction with maleimide‐capped DNA was prepared. The tetrazole‐based reaction on the nanoparticles’ surface results in a fluorescent pyrazoline product allowing for the spectroscopic monitoring of the reaction. This first example of nitrile imine‐mediated tetrazole‐ene cycloaddition (NITEC)‐mediated biofunctionalization of Au nanorods paves the way for the attachment of sensitive biomolecules, such as antibodies and other proteins, under mild conditions and expands the toolbox for the tailoring of nanomaterials.     

Protein micropatterning using surfaces modified by self-assembled polystyrene microspheres

A technique for micropatterning of proteins on a nonplanar surface to improve the coverage and functionality of biomolecules is demonstrated. A nonplanar microstructure is created by the self-assembly of polystyrene microspheres into an array of microwells on a silicon wafer to allow the integration of a nonplanar spot on a planar chip. After the microspheres were deposited into the microwells, they were conjugated with proteins. The curve surfaces of the microspheres present more surface area for attaching biomolecules which will increase the density of biomolecules and, hence, the sensitivity for detection. Moreover, proteins immobilized on a curved surface can retain their native structures and function better than on a planar surface because of a smaller area of interaction between the protein and the substrate. Patterning of biomolecules was tested with two model fluorescent proteins. The results show that precise patterning of biomolecules on a nonplanar spot can be achieved with this technique.     

Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies

Monoliths are considered as the fourth-generation chromatography material. Their use for preparative separation of biomolecules has been evolved over the past decade. Monolithic columns up to 8L in size are already commercially available for separation of large biomolecules such as proteins, protein aggregates, plasmid DNA, and viruses. These applications leverage monoliths' inherent properties, such as fast operation and high capacity for large biomolecules. The height equivalent to a theoretical plate (HETP) and dynamic binding capacity do not change with velocity. This is explained by the convective transport through the channels with a diameter of above 1000 nm and has been experimentally verified and also supported by theoretical analyses. Despite low absolute surface area, these large channels provide enough area for adsorption of these large biomolecules, which cannot penetrate into conventional chromatography media designed for protein separation. Monoliths for preparative separations are mainly cast as polymethacrylate or polyacrylamide blocks and have been functionalized as ion exchangers or hydrophobic interaction chromatography media. So-called cryogels have channels more than 30 microm wide, enabling efficient processing of suspensions or even cell-chromatography. This review discusses the pressure drop characteristics, mass transfer properties, scale-up, and applications of monoliths in the context of conventional chromatography media.     

Fabrication of a Biocompatible Mica/Gold Surface for Tip-Enhanced Raman Spectroscopy

Tip-enhanced Raman spectroscopy (TERS) is a promising technique for structural studies of biological systems and biomolecules, owing to its ability to provide a chemical fingerprint with sub-diffraction-limit spatial resolution. This application of TERS has thus far been limited, due to difficulties in generating high field enhancements while maintaining biocompatibility. The high sensitivity achievable through TERS arises from the excitation of a localized surface plasmon resonance in a noble metal atomic force microscope (AFM) tip, which in combination with a metallic surface can produce huge enhancements in the local optical field. However, metals have poor biocompatibility, potentially introducing difficulties in characterizing native structure and conformation in biomolecules, whereas biocompatible surfaces have weak optical field enhancements. Herein, a novel, biocompatible, highly enhancing surface is designed and fabricated based on few-monolayer mica flakes, mechanically exfoliated on a metal surface. These surfaces allow the formation of coupled plasmon enhancements for TERS imaging, while maintaining the biocompatibility and atomic flatness of the mica surface for high resolution AFM. The capability of these substrates for TERS is confirmed numerically and experimentally. We demonstrate up to five orders of magnitude improvement in TERS signals over conventional mica surfaces, expanding the sensitivity of TERS to a wide range of non-resonant biomolecules with weak Raman cross-sections. The increase in sensitivity obtained through this approach also enables the collection of nanoscale spectra with short integration times, improving hyperspectral mapping for these applications. These mica/metal surfaces therefore have the potential to revolutionize spectromicroscopy of complex, heterogeneous biological systems such as DNA and protein complexes.     

Micropatterning of polystyrene nanoparticles and its bioapplications

Micropatterning of biomolecules forms the basis of cell culture, biosensor and microarray technology. Currently, the most widely used techniques are photoresist lithography, soft lithography or using robots which all involve multi-step surface modification directly on a planar substrate. Here we report a method to pattern biomolecules through self-assembling polystyrene nanoparticles in arrayed microwells on a solid surface to form well-ordered patterning, followed by attaching biomolecules to the assembled nanoparticles. The formation of colloidal patterns depends on capillary force, surface wettability and physical confinement. This method can be used for micropatterning a variety of biomolecules such as protein and antibody.     

SPE and purification of DNA using magnetic particles

Superparamagnetic particles have been attractive for molecular diagnostics and analytical chemistry applications due to their unique magnetic properties and their ability to interact with various biomolecules of interest. This paper presents a critical overview of magnetic nano ‐ and microparticles used as a solid phase for extraction and purification of DNAs. The mechanisms of DNA binding to the surface of functionalised magnetic particles are described. The most widely used materials including silica supports, organic polymers and other materials, mostly containing magnetite or paramagnetic metallic elements are reviewed. The main application areas of magnetic particles for DNA separation are briefly described.     

Bioconjugated nanoparticles for DNA protection from cleavage

We have developed a novel method to protect DNA from cleavage using bioconjugated nanoparticles. Positively charged amino-modified silica nanoparticles have been directly prepared using water-in-oil microemulsion. Plasmid DNA can be easily enriched onto the positively charged nanoparticle surface, and the DNA strands are well protected from enzymatic cleavage. When incubated with nuclease enzyme for enzymatic cleavage, free plasmid DNA strands are completely cleaved, while those on the nanoparticle surfaces are intact. Our results clearly demonstrate unique properties of nanomaterials when combined with biomolecules. Our simple bionanotechnology will be highly useful in DNA separation, manipulation, and detection, and possibly in genetic engineering and gene therapy, as plasmid DNA can be protected in cellular environments without any change in its property.     

Characterization of grafting density and binding efficiency of DNA and proteins on gold surfaces

The surface grafting density of biomolecules is an important factor for quantitative assays using a wide range of biological sensors. We use a fluorescent measurement technique to characterize the immobilization density of thiolated probe DNA on gold and hybridization efficiency of target DNA as a function of oligonucleotide length and salt concentration. The results indicate the dominance of osmotic and hydration forces in different regimes of salt concentration, which was used to validate previous simulations and to optimize the performance of surface-stress based microcantilever biosensors. The difference in hybridization density between complementary and mismatched target sequences was also measured to understand the response of these sensors in base-pair mismatch detection experiments. Finally, two different techniques for immobilizing proteins on gold were considered and the surface densities obtained in both cases were compared.     

Preservation of the biofunctionality of DNA and protein during microfabrication

Microfabrication processes, especially in silicon, are not compatible with biomolecules. Silicon and metal-based materials having crystalline structures are manipulated under harsh conditions with acids, bases, and organic solvents at high temperature. In comparison, organic biomolecules such as DNA and proteins have complex, three-dimensional structures and are sensitive to denaturation, oxidation, hydrolysis, and thermal destruction. Here, we report on the integration of DNA and the biotin-binding protein NeutrAvidin into microfabrication processes by using a novel approach based on a gold passivation mask. Our data show that this passivation method preserves approximately 84% of the biofunctionality of DNA and approximately 30% of that of NeutrAvidin under harsh process conditions. This novel technology enables the integration of DNA, proteins, and potentially other biological molecules into mass scalable microfabrication processes for biomedical devices, biochips, biosensors, and microelectromechanical systems with biomolecules (BioMEMS).     

Carbon-on-metal films for surface plasmon resonance detection of DNA arrays

Surface plasmon resonance (SPR) imaging affords label-free monitoring of biomolecule interactions in an array format. A surface plasmon conducting metal thin film is required for SPR measurements. Gold thin films are traditionally used in SPR experiments as they are readily functionalized with thiol-containing molecules through formation of a gold-sulfur bond. The lability of this gold-thiol linkage upon exposure to oxidizing conditions and ultraviolet light renders these surfaces incompatible with light-directed synthetic methods for fabricating DNA arrays. It is shown here that applying a thin carbon overlayer to the gold surface yields a chemically robust substrate that permits light-directed synthesis and also supports surface plasmons. DNA arrays fabricated on these carbon-metal substrates are used to analyze two classes of biomolecular interactions: DNA-DNA and DNA-protein. This new strategy allows the combinatorial study of binding interactions directly from native, unmodified biomolecules of interest and offers the possibility of discovering new ligands in complex mixtures such as cell lysates.     

Photoactivated capture molecule immobilization in plasmonic nanoapertures in the ultraviolet

We demonstrate a photoactivated surface coupling scheme for achieving spatial overlap between biomolecules of interest and optical near field excitation. Using aluminium nanoapertures, we obtained increased coupling efficiency of biotinylated capture probe oligos to the photoactivated surface due to ~3× nanoaperture enhancement of UV light. We further validate DNA sensor functionality via the hybridization of Cy-5 labeled target oligos, with up to 8× fluorescence enhancement obtained from a commercial microarray scanner. This generic photoimmobilization strategy is an essential step to realizing miniaturized plasmon enhanced detection arrays by virtue of localizing capture molecules to the region of plasmonic enhancement.     

氧化铁磁性纳米粒子的制备、表面修饰及在分离和分析中的应用

氧化铁磁性纳米粒子通过表面化学修饰得到无机、有机或聚合物壳包覆在其表面。其中的壳结构既具有生物适应性,又具有可键合生物分子如细胞、蛋白质、酶、抗体和核酸的活性基团,而核具有磁性特性。本文总结了氧化铁磁性纳米粒子的制备方法,介绍了其表面化学修饰及在分离和分析应用的最新进展。