Application of Lectin Microarrays for Biomarker Discovery

Many proteins in living organisms are glycosylated. As their glycan patterns exhibit protein-, cell-, and tissue-specific heterogeneity, changes in the glycosylation levels could serve as useful indicators of various pathological and physiological states. Thus, the identification of glycoprotein biomarkers from specific changes in the glycan profiles of glycoproteins is a trending field. Lectin microarrays provide a new glycan analysis platform, which enables rapid and sensitive analysis of complex glycans without requiring the release of glycans from the protein. Recent developments in lectin microarray technology enable high-throughput analysis of glycans in complex biological samples. In this review, we will discuss the basic concepts and recent progress in lectin microarray technology, the application of lectin microarrays in biomarker discovery, and the challenges and future development of this technology. Given the tremendous technical advancements that have been made, lectin microarrays will become an indispensable tool for the discovery of glycoprotein biomarkers.     

Recent developments in microarray-based enzyme assays: from functional annotation to substrate/inhibitor fingerprinting

Recent advances in proteomics have provided impetus towards the development of robust technologies for high-throughput studies of enzymes. The term “catalomics” defines an emerging ‘-omics’ field in which high-throughput studies of enzymes are carried out by using advanced chemical proteomics approaches. Of the various available methods, microarrays have emerged as a powerful and versatile platform to accelerate not only the functional annotation but also the substrate and inhibitor specificity (e.g. substrate and inhibitor fingerprinting, respectively) of enzymes. Herein, we review recent developments in the fabrication of various types of microarray technologies (protein-, peptide- and small-molecule-based microarrays) and their applications in high-throughput characterizations of enzymes.     

Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes

Carbohydrate microarrays have become very powerful tools to elucidate the molecular basis of carbohydrate-recognition events in a high-throughput manner. This microarray technology has been applied in the rapid analysis of the binding properties of a variety of binding partners such as lectins, antibodies, mammalian cells, pathogens and viruses. In this feature article, methods for the preparation of carbohydrate microarrays and their applications in biological and biomedical research are described.     

糖芯片的检测及应用

糖芯片技术具有样品少、通量高和特异性强等优点,是一种糖组学研究的新的技术平台和强大的分析工具,已经广泛用于糖和蛋白质的特异性作用、酶活性和抑制剂、病毒入侵机理、细菌检测和免疫反应等方面的研究.本文简要介绍了糖芯片的原理、制备和信号的检测技术(荧光标记法、质谱法、SPR法等),分析了糖芯片在各个领域的应用及其发展前景.     

A chip-based miniaturized format for protein-expression profiling: the exploitation of comprehensively produced antibodies

Numerous antibodies have been developed and validated in recent years, and show promise for use in novel functional protein assays. Such assays would be an alternative to pre-existing comprehensive assays, such as DNA microarrays. Antibody microarrays are thought to represent those functional protein assays. While a variety of attempts have been made to apply DNA microarray technology to antibody microarrays, a fully optimized protocol has not been established. We have been conducting a project to comprehensively produce antibodies against mouse KIAA ("KI" stands for "Kazusa DNA Research Institute" and "AA" are reference characters) proteins. Using our library of antibodies, we established a novel antibody microarray format that utilizes surface plasmon resonance (SPR) technology. A label-free real-time measurement of protein expression in crude cell lysates was achieved by direct readout of the bindings using SPR. Further refinement of the antibody microarray format enabled us to detect a smaller quantity of target proteins in the lysate without the bulk effect. In this review, we first summarize available antibody array formats and then describe the above-mentioned format utilizing updated SPR technology.     

Advanced Immobilization and Amplification for High Performance Protein Chips

With the success of high-throughput DNA microarrays, protein biochips have been intensively investigated and broadly used in bioscience research, clinic diagnosis, drug discovery, and other applications. However, there is great need to significantly improve the sensitivity of protein chips, especially in early diagnosis. A major challenge of improving sensitivity is that protein detection does not have an effective amplification method, such as PCR for DNA microarrays. Construction of unique biofilms for efficient immobilization of protein probes and innovation of new amplification schemes could play a critical role in performance improvement of protein biochips. With dramatic developments in microfabrication, nanotechnologies, and biotechnologies, enormous progress has been made, particularly in improving biosensing sensitivity. This article reviews new advances in protein biochip technologies with emphasis on novel approaches for efficient probe immobilization and nanomaterials-assisted signal amplification for high performance protein chips. Prominent progress in integration of protein microarrays with microfluidic platforms is briefly discussed. The major challenges and perspectives on the future of protein biochips are also addressed.     

Carbohydrate microarrays: an advanced technology for functional studies of glycans

The biological significance of glycans in the post-genomic era requires the development of new technologies to enable functional studies of carbohydrates in a high-throughput manner. Recently, carbohydrate microarrays have been exploited as an advanced technology for this purpose. Efficient immobilization methods for carbohydrate probes on the proper surface are essential for the successful fabrication of carbohydrate microarrays. Up to date, several techniques have been developed to attach simple or complex carbohydrates to a solid surface. The developed glycan microarrays have been applied for functional glycomics, drug discovery, and diagnosis. In this concept article, we discuss the progress of immobilization methods of carbohydrates on solid surfaces, their potential uses for biological research and biomedical applications, and possible solutions for some remaining challenges to improve this new technology.     

Microarray: a versatile platform for high-throughput functional proteomics

The advent of microarray technologies has dramatically accelerated the functional study of proteins, including enzymes (catalomics) in a proteome. Herein, we review recent advances and exciting new developments of microarrays in high-throughput functional proteomics.     

化学生物学中光敏分子微阵列的表面构建与应用

分子微阵列是有机合成（特别是组合化学合成）方法应用于生物和医学研究而发展起来的高科技集成技术,通过把微电子、微加工技术和有机化学合成反应相结合,在固体基质（如硅片、玻片、瓷片等）表面构建微型的生物有机化学分子系统,以实现对细胞、蛋白质、核酸及其他生物组分进行快速、敏感、高效地处理．近年来,随着表面化学构建策略研究的不断深入和迅猛发展,分子微阵列技术的应用领域不断拓展,已从最初用于核酸分子的杂交测序延伸到基因组功能研究的各个方面．本文着重综述了光敏分子微阵列的表面化学构建策略研究及其在化学生物学分析中应用的最新进展,并展望了其发展的未来趋势．内容主要包括：小分子与多肽分子微阵列、蛋白质分子微阵列、核酸分子微阵列和糖分子微阵列等．     

分子阵列研究进展

小分子化合物可以调节生物学过程,是研究活性生物大分子（特别是蛋白质）以及药物的重要工具,而高通量筛选是发现活性分子的重要方法。分子阵列是近年来新出现的一种高通量筛选技术,上面含有成千上万种组合合成的化合物以及天然产物,可以用于发现新的先导化合物,以及筛选已有的先导化合物。现在分子阵列已经成功应用于蛋白分析、先导化合物的发现等许多领域。本文综述了近年来分子阵列的构建过程,原位合成、非原位合成等各种固定化策略以及荧光免疫检测,表面等离子体共振成像技术等检测手段,并介绍了化学分子印刷阵列方法,最后总结了分子阵列的应用,并对分子阵列在我国中药发展等有方面将起到的潜在作用作了展望。     

Microarray platforms for enzymatic and cell-based assays

This tutorial review introduces the uninitiated to the world of microarrays (or so-called chips) and covers a number of basic concepts such as substrates and surfaces, printing and analysis. It then moves on to look at some newer applications of microarray technology, which include enzyme analysis (notably kinases and proteases) as well as the growing enchantment with so-called cell-based microarrays that offer a unique approach to high-throughput cellular analysis. Finally, it looks forwards and highlights future possible trends and directions in the microarray arena.     

Protein microarrays and multiplexed sandwich immunoassays: what beats the beads?

Protein microarray technology allows the simultaneous determination of a large variety of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are currently applied for the identification, quantitation and functional analysis of proteins. Protein microarray technology is of major interest for proteomic research in basic and applied biology as well as for diagnostic applications. Miniaturized and parallelized assay systems have reached adequate sensitivity and hence have the potential to replace singleplex analysis systems. However, robustness and automation needs to be demonstrated before this technology will finally prove suitable for high-throughput applications. Miniaturized and parallelized sandwich immunoassays are the most advanced assays formats among the different protein microarray applications. Multiplexed sandwich immunoassays can be used for the identification of biomarkers and the validation of potential target molecules. In this review an overview will be given on the current stage of protein microarray technology with a special focus on miniaturized multiplexed sandwich immunoassays.     

DNA microarrays: tools for the 21st Century

Profiling of gene expression patterns with microarray technology is widely used in both basic and applied research. DNA microarrays have also shown great promise in clinical medicine and are paving the way toward effective pharmaceutical drug discovery and individualized drug regimens. With growing utilization of this high-throughput technology, new applications are making headlines on a regular basis. This review aims to outline the pros and cons of this methodology and direct the reader towards available useful resources. Various major array formats such as high-density oligonucleotide arrays and spotted cDNA arrays are examined, and advantages and options for using each format are presented. Factors important for the design and analysis of microarray experiments are also discussed.     

Applications of protein microarray technology

Protein microarrays, an emerging class of proteomic technologies, are quickly becoming essential tools for large-scale and high throughput biochemistry and molecular biology. Recent progress has been made in all the key steps of protein microarray fabrication and application, such as the large-scale cloning of expression-ready prokaryotic and eukaryotic ORFs, high throughput protein purification, surface chemistry, protein delivery systems, and detection methods. Two classes of protein microarrays are currently available: analytical and functional protein microarrays. In the case of analytical protein microarrays, well-characterized molecules with specific activity, such as antibodies, peptide-MHC complexes, or lectins, are used as immobilized probes. These arrays have become one of the most powerful multiplexed detection platforms. Functional protein microarrays are being increasingly applied to many areas of biological discovery, including drug target identification/validation and studies of protein interaction, biochemical activity, and immune responses. Great progress has been achieved in both classes of protein microarrays in terms of sensitivity and specificity, and new protein microarray technologies are continuing to emerge. Finally, protein microarrays have found novel applications in both scientific research and clinical diagnostics.     

Optical technologies for the read out and quality control of DNA and protein microarrays

Microarray formats have become an important tool for parallel (or multiplexed) monitoring of biomolecular interactions. Surface-immobilized probes like oligonucleotides, cDNA, proteins, or antibodies can be used for the screening of their complementary targets, covering different applications like gene or protein expression profiling, analysis of point mutations, or immunodiagnostics. Numerous reviews have appeared on this topic in recent years, documenting the intriguing progress of these miniaturized assay formats. Most of them highlight all aspects of microarray preparation, surface chemistry, and patterning, and try to give a systematic survey of the different kinds of applications of this new technique. This review places the emphasis on optical technologies for microarray analysis. As the fluorescent read out of microarrays is dominating the field, this topic will be the focus of the review. Basic principles of labeling and signal amplification techniques will be introduced. Recent developments in total internal reflection fluorescence, resonance energy transfer assays, and time-resolved imaging are addressed, as well as non-fluorescent imaging methods. Finally, some label-free detection modes are discussed, such as surface plasmon microscopy or ellipsometry, since these are particularly interesting for microarray development and quality control purposes.     

Carbohydrate microarrays for assaying galactosyltransferase activity

[reaction: see text] Carbohydrate microarrays have been used recently for the rapid analysis of glycan-protein or glycan-cell interactions and for the detection of pathogens. As a demonstration of its significance and versatility, the microarray technology has been applied in this effort to assay glycosyltransferase activities. In addition, carbohydrate microarray based methods have been employed to quantitatively determine binding affinities between lectins and carbohydrates.     

An improved gel-based DNA microarray method for detecting single nucleotide mismatch

3-D polyacrylamide gel-based DNA microarray platforms provide a high capacity for nucleic acids immobilization and a solution-mimicking environment for hybridization. However, several technological bottlenecks still remain in these platforms, such as difficult microarray preparation and high fluorescent background, which limit their application. In this study, two new approaches have been developed to improve the convenience in microarray preparation and to reduce the background after hybridization. To control the polymerization process, solutions containing acrylamide-modified oligonucleotide, acrylamide, glycerol and ammonium persulfate are spotted onto a functionalized glass slide, and then the slide is transferred to a vacuum chamber with TEMED, so that TEMED is vaporized and diffused into the spots to induce polymerization. By applying an electric field across a hybridized microarray to remove the nonspecifically bound labeled targets, this approach can solve the problem of high fluorescent background of the gel-based microarray after hybridization. Experimental results show that our immobilization method can be used to construct high quality microarrays and exhibits good reproducibility. Moreover, the polymerization is not affected by PCR medium, so that PCR products can be used for microarray construction without being treated by commercial purification cartridges. Electrophoresis can improve the signal-to-noise significantly and has the ability to differentiate single nucleotide variation between two homozygotes and a heterozygote. Our results demonstrated that this is a reliable novel method for high-throughput mutation analysis and disease diagnosis.     

Fluorescence Analysis in Microarray Technology

Fluorescence has been the preferred choice for data quantification in biomedical microarray formats since their earliest days. As much as the formats have grown and evolved over the years, the methods in optical analysis have become ever more sophisticated and complex in order to produce more and better output. This review will provide an insight into the most common methods and the state-of-the-art of all areas in microarray fluorescence analysis. Starting with an overview on microarray formats with a focus on their demands on the readout, the most common and useful organic fluorescent stains are discussed before proceeding on to other approaches; the use of semiconductor nanocrystals (quantum dots), polymer and silica nanoparticles and fluorescent proteins. Ways to enhance the intrinsically low signal on biochips have become increasingly important as they offer a sound approach towards the detection of low concentration sample content. The three main categories are presented: amplification using DNA, enzymes, and dendrimers. As much diversity as on the microarrays themselves can be found at the detection device. Standard optical microarray detectors, and non-standard methods using fluorescence anisotropy, fluorescence lifetime imaging (FLIM) and fluorescence resonance energy transfer (FRET), and their advantages and disadvantages are discussed.