Fast and reliable protein microarray production by a new drop-in-drop technique

In contrast to DNA microarrays, production of protein microarrays is an immense technological challenge due to high complexity and diversity of proteins. In this paper we investigate three essential aspects of protein microarray fabrication based on the highly parallel and non-contact TopSpot technology: evaporation of probes during long lasting production times, optimization of protein immobilization and improvement of protein microarray reproducibility. Evaporation out of the printhead reservoirs was reduced to a minimum by sealing the reservoirs with gas permeable foils or PDMS frames. This led to dramatically lowered setup times through the possibility of long-term, ready-to-print storage of filled printheads. To optimize immobilization efficiency 128 printing buffers were tested by printing two different proteins onto seven different microarray slide types. This way we were able to reduce the CV of spot diameter on the microarray slide below 1.14%. To remarkably increase protein immobilization efficiency on microarray slides the commonly used EDC-NHS system (a laboratory method for immobilization of proteins) was miniaturized by using a new drop-in-drop printing technique. Additionally the very fast UV cross-linking was used to immobilize antibodies. The optimized system was used to produce antibody microarrays and with it microarray ELISA experiments were performed successfully.     

Three-dimensional micro-XRF investigations of paint layers with a tabletop setup

The non-destructive investigation of art and archaeological objects with depth sensitivity is now possible using 3D micro-XRF spectroscopy. More detailed answers for questions on painting techniques, on the pigment palette, on the production processes and indirectly on dating or provenancing of objects are accessible now. This was already illustrated by the investigation of Mughal miniatures with a confocal setup at the synchrotron source BESSY.In this paper we demonstrate the feasibility of 3D micro-XRF spectroscopy with a tabletop setup and discuss its sensitivity in comparison to the synchrotron-based setup. Investigated objects are glass standards and also prepared paint layers. Perspectives for other types of studies are proposed.     

Polymer microfluidic chip for online monitoring of microarray hybridizations

A disposable single use polymer microfluidics chip has been developed and manufactured by micro injection molding. The chip has the same outer dimensions as a standard microscope slide (25 x 76 x 1.1 mm) and is designed to be compatible with existing microscope slide handling equipment like microarray scanners. The chip contains an inlet, a 10 microL hybridization chamber capable of holding a 1000 spot array, a waste chamber and a vent to allow air to escape when sample is injected. The hybridization chamber ensures highly homogeneous hybridization conditions across the microarray. We describe the use of this chip in a flexible setup with fluorescence based detection, temperature control and liquid handling by computer controlled syringe pumps. The chip and the setup presented in this article provide a powerful tool for highly parallel studies of kinetics and thermodynamics of duplex formation in DNA microarrays. The experimental setup presented in this article enables the on-chip microarray to be hybridized and monitored at several different stringency conditions during a single assay. The performance of the chip and the setup is demonstrated by on-line measurements of a hybridization of a DNA target solution to a microarray. A presented numerical model indicates that the hybridization process in microfluidic hybridization assays is diffusion limited, due to the low values of the diffusion coefficients D of the DNA and RNA molecules involved.     

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.     

Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to function optimally under one condition only. Microarrays are often burdened with a significant degree of cross-hybridization, because of a poor combination of assay conditions and probe choice. As reviewed here, a number of promising microfluidics-based technologies can provide automatic processing of arrays under different assay conditions. These new array processors provide researchers and assay developers with novel possibilities to construct highly specific DNA arrays even towards regions of DNA greatly varying in G?+?C content. These array processors are also a powerful development tool for building arrays, because they combine high sample throughput with investigation of optimal assay conditions. The array processors can increase specificity in all DNA microarray assays, e.g. for gene expression, and microRNA and mutation analysis. Increased specificity of the array will also benefit microarray-based loci selection prior to high-throughput sequencing.     

How to Make a DNA Chip

Microarrays are one of the hottest areas in biological research today. Microarrays have been mostly applied to nucleic acid analysis, specifically to the assessment of which genes are being expressed and at what level. Early microarrays were prepared by using photolithographic methods, which were more commonly used for integrated circuit (“computer chip”) production. Hence the colloquial term “DNA chip” came into being. The completion of the sequencing of the human genome and that of many other organisms makes the determination of gene function an important next step in understanding the role of DNA in the processes of life. DNA microarrays are an excellent tool to address this question because their numerous probe sites enable the analysis of many genes simultaneously. With good experience in this initial use, many further applications of microarrays are being developed, including genotyping in research and genetic diagnosis in medicine. DNA microarrays have made abundantly clear the power of vast parallelism in biological analysis, which is raising interest in other types of microarrays (small‐molecule, protein). Many applications for DNA microarrays have been developed and clearly many more will emerge through the creativity of the scientists who use them. In early studies, users produced their own microarrays. The apparent power of microarrays has demanded improvements in production methods, and technologies from physical sciences and engineering are now being applied to DNA chips. Many branches of chemistry can contribute to improved methods: from synthetic chemistry (to attach or prepare DNA), to the physical chemistry of surfaces, to analytical chemistry (to assess surface reactions).     

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．     

不相交主成分分析(PCA)和遗传算法(GA)用于差异表达基因的识别

建立了一种基于不相交主成分分析(Disjoint PCA)和遗传算法(GA)的特征变量选择方法, 并用于从基因表达谱(Gene expression profiles)数据中识别差异表达的基因. 在该方法中, 用不相交主成分分析评估基因组在区分两类不同样品时的区分能力; 用GA寻找区分能力最强的基因组; 所识别基因的偶然相关性用统计方法评估. 由于该方法考虑了基因间的协同作用更接近于基因的生物过程, 从而使所识别的基因具有更好的差异表达能力. 将该方法应用于肝细胞癌(HCC)样品的基因芯片数据分析, 结果表明, 所识别的基因具有较强的区分能力, 优于常用的基因芯片显著性分析(Significance analysis of microarrays, SAM)方法.     

Enhancing the fluorescence intensity of DNA microarrays by using cationic surfactants

DNA microarrays have been used as powerful tools in genomics studies and single nucleotide polymorphisms analysis. However, the fluorescence detection used in most conventional DNA microarrays is still limited by its sensitivity. The aim of this study is to use a cationic surfactant, cetyl trimethylammonium bromide (CTAB), to enhance the fluorescence intensity of 6-carboxy-fluorescene (FAM)-labeled DNA probes immobilized on a DNA microarray. We show that in the presence of CTAB the immobilized FAM-labeled DNA probes is 11-fold brighter than that without exposure to CTAB. Similarly, when we hybridize FAM-labeled DNA targets to a DNA microarray and treat the surface with CTAB solution, the fluorescence intensity shows a 26-fold increase for perfect-match DNA targets. More importantly, the contrast between perfect-match and 1-mismatch DNA is also increased from 1.3-fold to 15-fold. This method offers a simple and efficient technique to enhance the detection limit of DNA microarrays.     

Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays

DNA microarray technology has become an important research tool for biotechnology and microbiology. It is now possible to characterize genetic diversity and gene expression in a genomewide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Grampositive Corynebacterium glutamicum. Both bacteria are widely used for biotechnological amino acid production. In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C. glutamicum and E. coli. We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C. glutamicum and E. coli.     

Active bead-linked immunoassay on protein microarrays

Protein microarrays are becoming a powerful tool in proteome, biochemical, and clinical studies. In addition to the quality of arrayed immobilized probe molecules, sensitivity of the microarray-based assay is highly dependent on the detection technique. Here we suggest four simple techniques for rapid detection of analytes bound to protein microarrays. The techniques employ functionalized magnetic and non-magnetic beads moved to, from, or along the array surface by external forces. In contrast to other labeling techniques actively controlled physical labels: (i) make detection extremely fast to allow microarray reading in seconds; (ii) provide a low background due to active removal of weakly bound beads; and (iii) provide a highly sensitive detection, since one antigen-antibody bond is capable of holding bead immobilized on the array surface. In combination with the electrophoretically assisted active immunoassay we described recently such active reading allows to reduce total indirect immunoassay time to 7-10 min while having sensitivity in the femtomolar concentration range. High speed, sensitivity, and specificity make active bead-linked detection an ideal choice in rapid high-throughput screening and in emergency diagnostics.     

DNA microarray: a high throughput approach for methylation detection

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.     

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.     

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.     

Microarray technology as a universal tool for high-throughput analysis of biological systems

Over the last years microarray technology has become one of the principal platform technologies for the high-throughput analysis of biological systems. Starting with the construction of first DNA microarrays in the 1990s, microarray technology has flourished in the last years and many different new formats have been developed. Peptide and protein microarrays are now applied for the elucidation of interaction partners, modification sites and enzyme substrates. Antibody microarrays are envisaged to be of high importance for the high-throughput determination of protein abundances in translational profiling approaches. First cell microarrays have been constructed to transform microarray technology from an in vitro technology to an in vivo functional analysis tool. All of these approaches share a common prerequisite: the solid support on which they are generated. The demands on this solid support are thereby as manifold as the applications themselves. This review is aimed to display the recent developments in surface chemistry and derivatization, and to summarize the latest developments in the different application areas of microarray technology.     

Discriminatory power, typability, and accuracy of single nucleotide extension microarrays

Despite great conceptual promise, the use of microarrays in typing approaches has not yet gained wide acceptance. The establishment of proper criteria for determining discriminatory power as well as typability and the accuracy of microarray data remains to be solved. Purely experimental estimations of these parameters would far exceed what is experimentally practical. We therefore used simulations in combination with experimental results in parameter estimations. Our assay was based on 26 single nucleotide polymorphisms (SNPs) identified in the Campylobacterjejuni Multi Locus Sequence Typing (MLST) database (http:///pubmist.org/campylobacter/). The SNPs were detected using a single nucleotide extension (SNE) typing microarray. Unknown isolates were assigned to the known sequence type(s) by calculating weighted sum of matches minus a weighted sum of mismatches between predicted and candidate genotype. The weights were set according to the Bayesian posterior probability of the SNP classification. These studies showed that any typing or profiling method based on binary data requires an accuracy of <2-3% error for each datapoint (in our case SNPs) to classify the isolates to the correct allelic profile in 90% of the cases. The classification error for our experimental data was 3.2% (after removing 5 high error SNPs). We therefore conclude that SNE microarrays are promising for future high-throughput typing of bacteria.     

Fluorescence, XPS, and TOF-SIMS surface chemical state image analysis of DNA microarrays

Performance improvements in DNA-modified surfaces required for microarray and biosensor applications rely on improved capabilities to accurately characterize the chemistry and structure of immobilized DNA molecules on micropatterned surfaces. Recent innovations in imaging X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) now permit more detailed studies of micropatterned surfaces. We have exploited the complementary information provided by imaging XPS and imaging TOF-SIMS to detail the chemical composition, spatial distribution, and hybridization efficiency of amine-terminated single-stranded DNA (ssDNA) bound to commercial polyacrylamide-based, amine-reactive microarray slides, immobilized in both macrospot and microarray diagnostic formats. Combinations of XPS imaging and small spot analysis were used to identify micropatterned DNA spots within printed DNA arrays on slide surfaces and quantify DNA elements within individual microarray spots for determination of probe immobilization and hybridization efficiencies. This represents the first report of imaging XPS of DNA immobilization and hybridization efficiencies for arrays fabricated on commercial microarray slides. Imaging TOF-SIMS provided distinct analytical data on the lateral distribution of DNA within single array microspots before and after target hybridization. Principal component analysis (PCA) applied to TOF-SIMS imaging datasets demonstrated that the combination of these two techniques provides information not readily observable in TOF-SIMS images alone, particularly in identifying species associated with array spot nonuniformities (e.g., "halo" or "donut" effects often observed in fluorescence images). Chemically specific spot images were compared to conventional fluorescence scanned images in microarrays to provide new information on spot-to-spot DNA variations that affect current diagnostic reliability, assay variance, and sensitivity.     

Chemiluminescence microarrays in analytical chemistry: a critical review

Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review. Figure

Achievements in the development of CL microarray analysis platforms