几种新型生物芯片的研究进展

随着生物芯片技术的迅速发展,一些新型生物芯片,如生物电子芯片、凝胶元件微阵列芯片、药物控释芯片、毛细管电泳或层析芯片、PCR芯片及生物传感芯片等应运而生,这些芯片不同于常规的分子微阵列芯片,而是以各种结构微阵列为基础,用于分子杂交与扩增,以检测突变、分析多态性及测序,通过电泳及层析分离生物样品,控制药物释放以治疗疾病,作为生物传感器检测分子行为等,具有分析速度快、效率高、样品消耗少等特点,将成为生命科学与医学领域的新工具.     

Modeling of DNA hybridization kinetics for spatially resolved biochips

The marriage of microfluidics with detection technologies that rely on highly selective nucleic acid hybridization will provide improvements in bioanalytical methods for purposes such as detection of pathogens or mutations and drug screening. The capability to deliver samples in a controlled manner across a two-dimensional hybridization detection platform represents a substantial technical challenge in the development of quantitative and reusable biochips. General theoretical and numerical models of heterogeneous hybridization kinetics are required in order to design and optimize such biochips and to develop a quantitative method for online interpretation of experimental results. In this work we propose a general kinetic model of heterogeneous hybridization and develop a technique for estimating the kinetic coefficients for the case of well-spaced, noninteracting surface-bound probes. The experimentally verified model is then incorporated into the BLOCS (biolab-on-a-chip simulation) 3D microfluidics finite element code and used to model the dynamic hybridization on a biochip surface in the presence of a temperature gradient. These simulations demonstrate how such a device can be used to discriminate between fully complementary and single-base-pair mismatched hybridization using fluorescence detection by interpretation of the unique spatially resolved intensity pattern. It is also shown how the dynamic transport of the targets is likely to affect the rate and location of hybridization as well as that, although nonspecific hybridization is present, the change in the concentration of hybridized targets over the sensor platform is sufficiently high to determine if a fully complementary match is present. Practical design information such as the optimum transport speed, target concentration, and channel height is presented. The results presented here will aid in the interpretation of results obtained with such a temperature-gradient biochip.     

Coomassie blue is sufficient for specific protein detection of aptamer-conjugated chips

An aptamer-based biochip for protein detection and quantitation which combines the recent biochip technology and the conventional staining methods, is described. Using a model system comprising His-tagged proteins as the analyte and single-stranded RNA aptamers specific for His-tagged proteins as immobilized ligands on chips, we could demonstrate that aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity, respectively. The sensor has the characteristics of good stability, reproducibility and reusability, with detection limit as low as 85 ng/mL His-tagged protein. It has been demonstrated that the sensor can be stored for at least 4 weeks and reused with reasonable reduction rate of staining intensity. In conclusion, we could show the suitability of nucleic acid aptamers as low molecular weight receptors on biochips for sensitive and specific protein detection and quantitation.     

Reading Biochips by Raman and Surface-Enhanced Raman Spectroscopies

Biochips are a rapidly increasing research field, driven by the versatility of sensing devices and the importance of their applications. The regular approaches for creating biochips and for reading them suffer from some limitations, motivating development of miniature biochips and label-free formats. To push forward these challenges, we have chosen to combine the methods of printing of droplets of synthetic receptors by pipettes or nanofountain pens with detection by Raman spectroscopy or its surface-assisted plasmon variant, namely, surface-enhanced Raman spectroscopy (SERS). The selected receptors included molecularly imprinted polymers (MIPs), produced by polymerization of functional and cross-linking monomers around a molecular template, the β-blocking drug propranolol. The measured Raman and SERS spectra of the MIP constituents enabled identification of the template presence and consequently chemical imaging of individual and multiple dots in an array. This concept, combining nanolithography techniques with SERS paves the road toward miniaturized arrayed MIP sensors with label-free, specific and quantitative molecular recognition.     

纳米孔测序技术应用于食品微生物检测的可行性分析

近些年来DNA测序技术发展迅速,已经从第一代生化测序发展到第三代单分子测序。作为第三代测序技术中的一种不同于当前流行的其他测序技术,纳米孔测序技术是基于电信号的一种物理方法测序。许多研究者通常将高通量测序技术应用于食品微生物的研究,但是将纳米孔测序技术应用于食品中微生物的检测却鲜有报道。Oxford Nanopore Technologies(牛津纳米孔科技公司)研发的DNA测序仪MinION,是世界首例用于商业测序的纳米孔测序仪,经过不断完善,近年来MinION在DNA测序中被广泛应用。MinION 测序一次需要的DNA量约1μg,其标准识别速度为一秒钟识别250个碱基,平均读长可至13kb~20kb,测序准确率可以达到98%。纳米孔测序的高识别速度和高准确率,完全满足快速检测的要求,将其应用于食品中微生物检测是完全可行的。     

DNA芯片制作原理及其杂交信号检测方法

文章讨论了DNA芯片的制作原理和杂交信号的检测方法。依其结构,DNA芯片可分为两种形式,DNA阵列和寡核苷酸微芯片。DNA芯片的制作方法主要有光导原位合成法和自动化点样法。DNA芯片与标记的探针或DNA样品杂交,并通过探测杂交信号谱型来实现DNA序列或基因表达的分析。适应于DNA芯片的发展,同时出现了许多新型的杂交信号检测方法。主要有激光荧光扫描显微镜、激光扫描共焦显微镜、结合使用CCD相机的荧光显微镜、光纤生物传感器、化学发生法、光激发磷光物质存储屏法、光散射法等。     

固态纳米孔:下一代DNA测序技术——原理、工艺与挑战

固态纳米孔测序技术作为新兴的第四代DNA测序技术,具有低成本、高读长、易集成等优势.如今,随着半导体工艺技术的飞速发展,小型化、高速度、大通量的纳米孔测序芯片的实现成为可能.相比传统的测序技术,固态纳米孔测序技术在成本、速度等方面有着十分巨大的优势.然而,作为一种新兴的测序技术,固态纳米孔在制造、测序、集成等方面也存在着诸多挑战.本文主要介绍了纳米孔测序技术的原理、制备工艺和面临的挑战,并展望了未来纳米孔测序技术的发展前景.     

Biophysical methods for biochip analysis. Use of wide-field digital fluorescence microscopy

This paper discusses the development of biophysical methods for biochip analysis. A scheme and construction of a biochip analyzer based on wide-field digital fluorescence microscopy are described. The analyzer is designed to register images of biological microchips labeled with fluorescent dyes. The device developed is useful for high-sensitivity throughput recording of analyses with biochips after interaction of immobilized probes with fluorescently labeled sample molecules as well as it provides a higher rate of the analysis compared with laser scanning devices. With this analyzer, the scope where biological microchips can be applied becomes wider, development of new protocols of the analyses is possible and standard analyses run faster with the use of biochips, the expenses for performing routine analyses can be reduced.     

Biophysical methods for biochip analysis. Use of wide field digital fluorescent microscopy

This paper discusses an issue on the development of biophysical methods for biochip analysis. A scheme and construction of a biochip analyzer based on wide-field digital fluorescence microscopy are described. The analyzer is designed to register images of biological microchips labeled with fluorescent dyes. The device developed is useful for high-sensitive throughput recording analyses by biochips after interaction of immobilized probes with fluorescently labeled sample molecules as well as it provides the higher rate of the analysis compared to laser scanning devices. With this analyzer a scope where biological microchips can be applied becomes wider, the development of new protocols of the analyses is possible and standard analyses run faster with the use of biochips, the expenses for the analysis performance can be reduced.     

Perspectives of DNA microarray and next-generation DNA sequencing technologies

DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation DNA sequencing method was first introduced by the 454 Company in 2003, immediately followed by the establishment of the Solexa and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology, with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research. Supported by the National High-Tech Research Program of China (Grant No.2006AA020704) and Shanghai Science and Technology Commission (Grant No. 05DZ22201)     

Evaluation of biochips for the rifampin resistance detection of M. tuberculosis in strains isolated at the Novosibirsk and Tomsk regions

Recently in Russia biochips for rifampin resistance detection of M. tuberculosis were developed. To investigate the conformity between rifampin resistance results determined both by the routinely used absolute concentration method and USING the biochips, 272 DNA samples of M. tuberculosis isolated from TB patients at Novosibirsk and Tomsk regions in 2000-2005 were analyzed. The biochip can detect 30 mutations in rpoB gene. The mutations were also tested using the single stranded conformational polymorphism method (SSCP). In addition, 60 DNAs were randomly sampled and sequenced. The results of rifampin resistance detection using biochip and absolute concentration methods were congruent in 86% cases, and were different when analyzed samples consisted of the susceptible and resistant strains of M. tuberculosis mixture. The most frequent mutations in the rpoB gene were S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). In 94% of rifampin resistant strains, there was also resistance to isoniazid. Therefore, in Siberia the rifampin resistance is the reliable marker for MDR strains of M. tuberculosis, and biochips can be used also for their detection. To hybridize with biochip the fluorescent-labeled single-stranded DNAs were routinely synthesized by two PCR, and intermediary product after the first PCR should be transferred into another tube. The last stage included high risk of cross-contamination. To exclude the risk, primer concentrations and temperature-time profile of PCR reactions were improved, and both PCR were combined in one tube. The two methods were congruent in 100%. The one tube method would be especially attractive for the routine PCR laboratory.     

Magnetoresistive-based biosensors and biochips

Over the past five years, magnetoelectronics has emerged as a promising new platform technology for biosensor and biochip development. The techniques are based on the detection of the magnetic fringe field of a magnetically labeled biomolecule interacting with a complementary biomolecule bound to a magnetic-field sensor. Magnetoresistive-based sensors, conventionally used as read heads in hard disk drives, have been used in combination with biologically functionalized magnetic labels to demonstrate the detection of molecular recognition. Real-world bio-applications are now being investigated, enabling tailored device design, based on sensor and label characteristics. This detection platform provides a robust, inexpensive sensing technique with high sensitivity and considerable scope for quantitative signal data, enabling magnetoresistive biochips to meet specific diagnostic needs that are not met by existing technologies.     

Optical Biochip with Multichannels for Detecting Biotin–Streptavidin Based on Localized Surface Plasmon Resonance

A rapid and accurate detection of molecular binding of antigen-antibody signaling in high throughput is of great importance for biosensing technology. We proposed a novel optical biochip with multichannels for the purpose of detection of biotin–streptavidin on the basis of localized surface plasmon resonance. The optical biochip was fabricated using photolithography to form the microarrays functioning with multichannels on glass substrate. There are different nanostructures in each microarray. Dry etching and nanosphere lithography techniques were applied to fabricate Ag nanostructures such as hemispheres, nanocylindricals, triangular, and rhombic nanostructures. We demonstrated that 100-nM target molecule (streptavidin) on these optical biochips can be easily detected by a UV-visible spectrometer. It indicated that period and shape of the nanostructures significantly affect the optical performance of the nanostructures with different shapes and geometrical parameters. Our experimental results demonstrated that the optical biochips with the multichannels can detect the target molecule using the microarrays structured with different shapes and periods simultaneously. Batch processing of immunoassay for different biomolecular through the different channels embedded on the same chip can be realized accordingly.     

生物芯片技术及其在基础生物科学研究中的应用

回顾了生物芯片的发展历史,重点介绍了生物芯片技术的两大技术基础：分子生物技术和微细加工生物技术;阐述了生物芯片技术的核心内容,总结了生物芯片的三大类型,并对生物芯片技术在生命科学基础研究中的应用进行了深入探讨和展望。     

Protein micro- and macroarrays: digitizing the proteome

The early applications of microarrays and detection technologies have been centered on DNA-based applications. The application of array technologies to proteomics is now occurring at a rapid rate. Numerous researchers have begun to develop technologies for the creation of microarrays of protein-based screening tools. The stability of antibody molecules when bound to surfaces has made antibody arrays a starting point for proteomic microarray technology. To minimize disadvantages due to size and availability, some researchers have instead opted for antibody fragments, antibody mimics or phage display technology to create libraries for protein chips. Even further removed from antibodies are libraries of aptamers, which are single-stranded oligonucleotides that express high affinity for protein molecules. A variation on the theme of protein chips arrayed with antibody mimics or other protein capture ligand is that of affinity MS where the protein chips are directly placed in a mass spectrometer for detection. Other approaches include the creation of intact protein microarrays directly on glass slides or chips. Although many of the proteins may likely be denatured, successful screening has been demonstrated. The investigation of protein-protein interactions has formed the basis of a technique called yeast two-hybrid. In this method, yeast "bait" proteins can be probed with other yeast "prey" proteins fused to DNA binding domains. Although the current interpretation of protein arrays emphasizes microarray grids of proteins or ligands on glass slides or chips, 2-D gels are technically macroarrays of authentic proteins. In an innovative departure from the traditional concept of protein chips, some researchers are implementing microfluidic printing of arrayed chemistries on individual protein spots blotted onto membranes. Other researchers are using in-jet printing technology to create protein microarrays on chips. The rapid growth of proteomics and the active climate for new technology is driving a new generation of companies and academic efforts that are developing novel protein microarray techniques for the future.     

DNA sequencing: an overview of solid-state and biological nanopore-based methods

The field of sequencing is a topic of significant interest since its emergence and has become increasingly important over time. Impressive achievements have been obtained in this field, especially in relations to DNA and RNA sequencing. Since the first achievements by Sanger and colleagues in the 1950s, many sequencing techniques have been developed, while others have disappeared. DNA sequencing has undergone three generations of major evolution. Each generation has its own specifications that are mentioned briefly. Among these generations, nanopore sequencing has its own exciting characteristics that have been given more attention here. Among pioneer technologies being used by the third-generation techniques, nanopores, either biological or solid-state, have been experimentally or theoretically extensively studied. All sequencing technologies have their own advantages and disadvantages, so nanopores are not free from this general rule. It is also generally pointed out what research has been done to overcome the obstacles. In this review, biological and solid-state nanopores are elaborated on, and applications of them are also discussed briefly.     

DNA芯片技术研究进展

DNA芯片技术是近年来发展迅速的生物高技术 .其基本过程是采用寡核苷酸原位合成或显微打印手段 ,将大量探针片段有序地固化于支持物如硅芯片的表面 ,然后与扩增、标记的生物样品杂交 ,通过对杂交信号的检测分析 ,即可得出样品的遗传信息 .该技术不仅可以对遗传信息进行定性、定量分析 ,而且扩展到基因组研究和基因诊断等方面的应用 .尽管目前在硬件和软件上还面临一些困难 ,但其发展和应用的前景广阔 .     

纳米孔测序信号处理及其在DNA数据存储的应用

随着高通量测序技术的不断更新,可以在单个分子水平读取核苷酸序列的第三代测序技术迅速发展,纳米孔测序技术是其具有代表性的单分子测序技术,该技术通过检测DNA单链分子穿过纳米孔时引起的跨膜电流信号的变化,实现碱基识别.纳米孔测序仪在便携性、碱基读取速度、测序读段长度等方面较传统的第一代与第二代测序技术都有明显优势.随着纳米...     

表观遗传学研究方法进展

表观遗传调控是基因表达调控的重要组成部分,已成为当前研究的热点.目前其研究主要集中在DNA甲基化和组蛋白修饰.针对这两种表观修饰,其研究方法也取得了较太进展,一方面方法的是敏度和特异性都在不断提高;另一方面表现修饰的检测正在逐步从定性检测向定量分析方向发展,从个别位点向高通量检测发展.此外,新一代测序技术的应用特大大推动表观遗传研究的发展,包括单分子实时测序法、单分子纳米孔科序法等.综述目前常用的DNA甲基化、组蛋白修饰研究方法以及最新的单分子测序技术,并对它们在表观遗传修饰检测中的应用作了简要对比分析.     

基因芯片技术和蛋白质组技术在神经科学中的应用及其研究进展

基因芯片技术和蛋白质组技术是最近发展起来的高通量技术,二者的出现使同时分析神经系统的大量基因的表达和基因产物蛋白质及其相互作用网络成为可能。它们在神经科学中的应用为了解脑功能提供了前所未有的机会。一个典型的基因芯片实验包括：芯片的准备或购买、靶DNA和探针的准备或标记、标记探针与靶DNA的杂交、芯片扫描和影象信息的数据分析。蛋白质组技术较为复杂,包括蛋白质分离、鉴定和信息分析三方面的内容。其中,分离技术多种多样。若分离技术以二维电泳为基础,则该实验通常由以下步骤组成：蛋白质样品的准备、电泳分离、染胶、分离蛋白点的切除、蛋白质的酶解(常用胰蛋白酶)、质谱分析(鉴定)和数据的信息处理。本文综述这两项技术的内容和实验步骤,然后着重叙述它们在神经科学中的应用,讨论其优缺点和面临的挑战,展望其发展前景。