Antibody colocalization microarray: a scalable technology for multiplex protein analysis in complex samples

DNA microarrays were rapidly scaled up from 256 to 6.5 million targets, and although antibody microarrays were proposed earlier, sensitive multiplex sandwich assays have only been scaled up to a few tens of targets. Cross-reactivity, arising because detection antibodies are mixed, is a known weakness of multiplex sandwich assays that is mitigated by lengthy optimization. Here, we introduce (1) vulnerability as a metric for assays. The vulnerability of multiplex sandwich assays to cross-reactivity increases quadratically with the number of targets, and together with experimental results, substantiates that scaling up of multiplex sandwich assays is unfeasible. We propose (2) a novel concept for multiplexing without mixing named antibody colocalization microarray (ACM). In ACMs, both capture and detection antibodies are physically colocalized by spotting to the same two-dimensional coordinate. Following spotting of the capture antibodies, the chip is removed from the arrayer, incubated with the sample, placed back onto the arrayer and then spotted with the detection antibodies. ACMs with up to 50 targets were produced, along with a binding curve for each protein. The ACM was validated by comparing it to ELISA and to a small-scale, conventional multiplex sandwich assay (MSA). Using ACMs, proteins in the serum of breast cancer patients and healthy controls were quantified, and six candidate biomarkers identified. Our results indicate that ACMs are sensitive, robust, and scalable.     

Protein microarrays for gene expression and antibody screening.

Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen-antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor-ligand interactions.     

微阵列（microarrays）技术及其应用

微阵列分为cDNA微阵列和寡聚核苷酸微阵列,微阵列上“印”有大量已知部分序列的DNA探针,微阵列技术就是利用分子杂交原理,使同时被比较的标本（用同位素或荧光素标记）与微阵列杂交,通过检测杂交信号强度及数据处理,把他们转化成不同标本中特异基因的丰度,从而全国比较不同标本的基因表达水平的差异,微阵列技术是一种探索基因组功能的有力手段。     

Fabrication of high quality microarrays

Fabrication of DNA microarray demands that between ten (diagnostic microarrays) and many hundred thousands of probes (research or screening microarrays) are efficiently immobilised to a glass or plastic surface using a suitable chemistry. DNA microarray performance is measured by parameters like array geometry, spot density, spot characteristics (morphology, probe density and hybridised density), background, specificity and sensitivity. At least 13 factors affect these parameters and factors affecting fabrication of microarrays are used in this review to compare different fabrication methods (spotted microarrays and in situ synthesis of microarrays) and immobilisation chemistries.     

Analysis of repeatability in spotted cDNA microarrays

We report a strategy for analysis of data quality in cDNA microarrays based on the repeatability of repeatedly spotted clones. We describe how repeatability can be used to control data quality by developing adaptive filtering criteria for microarray data containing clones spotted in multiple spots. We have applied the method on five publicly available cDNA microarray data sets and one previously unpublished data set from our own laboratory. The results demonstrate the feasibility of the approach as a foundation for data filtering, and indicate a high degree of variation in data quality, both across the data sets and between arrays within data sets.     

Functional RNA microarrays for high-throughput screening of antiprotein aptamers

High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.     

Comparative modeling and analysis of microfluidic and conventional DNA microarrays

A theoretical analysis was developed to predict molecular hybridization rates for microarrays where samples flow through microfluidic channels and for conventional microarrays where samples remain stationary during hybridization. The theory was validated by using a multiplexed microfluidic microarray where eight samples were hybridized simultaneously against eight probes using 60-mer DNA strands. Mass transfer coefficients ranged over three orders of magnitude where either kinetic reaction rates or molecular diffusion rates controlled overall hybridization rates. Probes were printed using microfluidic channels and also conventional spotting techniques. Consistent with the theoretical model, the microfluidic microarray demonstrated the ability to print DNA probes in less than 1 min and to detect 10-pM target concentrations with hybridization times in less than 5 min.     

Nanosphere lithography-based platform for developing rapid and high sensitivity microarray systems

A novel gold nanoarray (NA)-based platform was developed for microarray applications. This novel approach is based upon the principle of nanosphere lithography and can be used for one-step antibody immobilization. The developed platform was checked by functionalizing with cysteine followed by capturing biotinylated antibody and detecting it with dye-conjugated steptravidin. An immunoassay was performed with spiked samples containing human fetuin A antigen. The minimum limits of detection (LOD) of human fetuin A for NA-based and conventional microarray platforms were 50 pg/mL and 50 ng/mL, respectively. The developed approach was highly reproducible and unlike conventional microarray approaches the use of a spotting system was omitted because immobilization was controlled and directed on the predefined arrays. This approach could be an ideal alternative for developing microarrays. And, the ease of the strategy also allows the high throughput production of the microarrays.     

基因芯片技术检测肾综合征出血热病毒核酸的研究

To establish a rapid, sensitive and specific diagnostic assay for Hantavirus with microarray techniques, specific primers and probes were designed according to the conservative and specific DNA sequence of 76-118 strain and R22 strain. The probes were spotted on glass slides to form microarrays.The Cy3-1abled single stranded DNA fragments prepared by dissymmetical PCR were hybridized with the probes on the glass slides. The microarrays were scanned and analyzed with a scanner. The results showed that the DNA microarray could detect the different typed DNA of HTN and SEO with adequate specificity and sensitivity. The developed DNA microarray and techniques might be a very useful method for diagnosis and prevention, and could be widely applied in specific pathogens detection ofinfectious diseases such as hemorrhagic fever with renal syndrome.     

Stripping custom microRNA microarrays and the lessons learned about probe-slide interactions

Microarrays have been used extensively in gene expression profiling and genotyping studies. To reduce the high cost and enhance the consistency of microarray experiments, it is often desirable to strip and reuse microarray slides. Our genome-wide analysis of microRNA expression involves the hybridization of fluorescently labeled nucleic acids to custom-made, spotted DNA microarrays based on GAPSII-coated slides. We describe here a simple and effective method to regenerate such custom microarrays that uses a very low-salt buffer to remove labeled nucleic acids from microarrays. Slides can be stripped and reused multiple times without significantly compromising data quality. Moreover, our analyses of the performance of regenerated slides identifies parameters that influence the attachment of oligonucleotide probes to GAPSII slides, shedding light on the interactions between DNA and the microarray surface and suggesting ways in which to improve the design of oligonucleotide probes.     

百合病毒的DNA芯片检测技术研究

根据已知的黄瓜花叶病毒,百合无症病毒、百合斑驳病毒基因核苷酸序列,设计引物和探针,制备寡核苷酸芯片。用Cy3标记核苷酸引物,不对称RT-PCR扩增产物与芯片上的寡核苷酸探针杂交,荧光扫描仪检测并分析信号。研究制备的基因芯片能够检测侵染百合的3种重要病毒核酸的特异性荧光信号,该项技术具有特异、灵敏、快速的优点。     

Transcriptional signatures of normal human mammary epithelial cells in response to benzo[a]pyrene exposure: a comparison of three microarray platforms

Microarrays are used to study gene expression in a variety of biological systems. A number of different platforms have been developed, but few studies exist that have directly compared the performance of one platform with another. The goal of this study was to determine array variation by analyzing the same RNA samples with three different array platforms. Using gene expression responses to benzo[a]pyrene exposure in normal human mammary epithelial cells (NHMECs), we compared the results of gene expression profiling using three microarray platforms: photolithographic oligonucleotide arrays (Affymetrix), spotted oligonucleotide arrays (Amersham), and spotted cDNA arrays (NCI). While most previous reports comparing microarrays have analyzed pre-existing data from different platforms, this comparison study used the same sample assayed on all three platforms, allowing for analysis of variation from each array platform. In general, poor correlation was found with corresponding measurements from each platform. Each platform yielded different gene expression profiles, suggesting that while microarray analysis is a useful discovery tool, further validation is needed to extrapolate results for broad use of the data. Also, microarray variability needs to be taken into consideration, not only in the data analysis but also in specific probe selection for each array type.     

A general framework for designing and validating oligomer-based DNA microarrays and its application to Clostridium acetobutylicum

While DNA microarray analysis is widely accepted as an essential tool for modern biology, its use still eludes many researchers for several reasons, especially when microarrays are not commercially available. In that case, the design, construction, and use of microarrays for a sequenced organism constitute substantial, time-consuming, and expensive tasks. Recently, it has become possible to construct custom microarrays using industrial manufacturing processes, which offer several advantages, including speed of manufacturing, quality control, no up-front setup costs, and need-based microarray ordering. Here, we describe a strategy for designing and validating DNA microarrays manufactured using a commercial process. The 22K microarrays for the solvent producer Clostridium acetobutylicum ATCC 824 are based on in situ-synthesized 60-mers employing the Agilent technology. The strategy involves designing a large library of possible oligomer probes for each target (i.e., gene or DNA sequence) and experimentally testing and selecting the best probes for each target. The degenerate C. acetobutylicum strain M5 lacking the pSOL1 megaplasmid (with 178 annotated open reading frames [genes]) was used to estimate the level of probe cross-hybridization in the new microarrays and to establish the minimum intensity for a gene to be considered expressed. Results obtained using this microarray design were consistent with previously reported results from spotted cDNA-based microarrays. The proposed strategy is applicable to any sequenced organism.     

Printing of protein microarrays via a capillary-free fluid jetting mechanism

Current proteomics experiments rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the requirements needed for successful spotting of proteins to perform high-throughput, array-based proteomic profiling. Biological laser printing (BioLP) is a spotting technology that does not rely on solid pins, quill pins, or capillary-based fluidics. The non-contact mechanism of BioLP utilizes a focused laser pulse to transfer protein solutions, thereby eliminating the potential for orifice clogging, air bubbles, and unnecessary volume loss potentially encountered in commercially available spotting technologies. The speed and spot-to-spot reproducibility of BioLP is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30 microm and approximately 500 fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. Arraying a solution of BSA with subsequent immunodetection demonstrates the reproducible spotting of protein in an array format with CVs of <3%. Printing of the enzyme alkaline phosphatase followed by a positive reaction with a colorimetric substrate demonstrates that functional protein can be spotted using this laser-based printer.     

A General Framework for Designing and Validating Oligomer-Based DNA Microarrays and Its Application to Clostridium acetobutylicum

While DNA microarray analysis is widely accepted as an essential tool for modern biology, its use still eludes many researchers for several reasons, especially when microarrays are not commercially available. In that case, the design, construction, and use of microarrays for a sequenced organism constitute substantial, time-consuming, and expensive tasks. Recently, it has become possible to construct custom microarrays using industrial manufacturing processes, which offer several advantages, including speed of manufacturing, quality control, no up-front setup costs, and need-based microarray ordering. Here, we describe a strategy for designing and validating DNA microarrays manufactured using a commercial process. The 22K microarrays for the solvent producer Clostridium acetobutylicum ATCC 824 are based on in situ-synthesized 60-mers employing the Agilent technology. The strategy involves designing a large library of possible oligomer probes for each target (i.e., gene or DNA sequence) and experimentally testing and selecting the best probes for each target. The degenerate C. acetobutylicum strain M5 lacking the pSOL1 megaplasmid (with 178 annotated open reading frames [genes]) was used to estimate the level of probe cross-hybridization in the new microarrays and to establish the minimum intensity for a gene to be considered expressed. Results obtained using this microarray design were consistent with previously reported results from spotted cDNA-based microarrays. The proposed strategy is applicable to any sequenced organism.     

Gene expression profiling: methodological challenges,results, and prospects for addiction research

This review describes the current methods used to profile gene expression. These methods include microarrays, spotted arrays, serial analysis of gene expression (SAGE), and massive parallel signature sequencing (MPSS). Methodological and statistical problems in interpreting microarray and spotted array experiments are also discussed. Methods and formats such as minimum information about microarray experiments (MIAME) needed to share gene expression data are described. The last part of the review provides an overview of the application of gene-expression profiling technology to substance abuse research and discusses future directions.     

Protein microarrays: Reduced autofluorescence and improved LOD

In protein microarray performance, the choice of an appropriate surface is a crucial factor. Three‐dimensional substrates like nitrocellulose are known to have higher binding capacities than planar surfaces. Furthermore, they can enable the immobilization of proteins in a functional manner. One disadvantage of today's nitrocellulose‐based microarrays is the high background fluorescence, which can interfere with the detection of low‐abundance proteins. We have developed an innovative black nitrocellulose membrane‐based protein microarray that exhibits low autofluorescence in combination with increased sensitivity and improved LOD (limit of detection). The applicability of the novel material was demonstrated with main focus on reversed‐phase microarray experiments. In comparison to various commercially available microarrays, a higher sensitivity in regard to the spotted protein was achieved. In contrast to other porous nitrocellulose‐based microarrays, the black nitrocellulose provides a significant lower autofluorescence and background intensity.     

A comparison of cDNA, oligonucleotide, and Affymetrix GeneChip gene expression microarray platforms.

We have conducted a study to compare the variability in measured gene expression levels associated with three types of microarray platforms. Total RNA samples were obtained from liver tissue of four male mice, two each from inbred strains A/J and C57BL/6J. The same four samples were assayed on Affymetrix Mouse Genome Expression Set 430 GeneChips (MOE430A and MOE430B), spotted cDNA microarrays, and spotted oligonucleotide microarrays using eight arrays of each type. Variances associated with measurement error were observed to be comparable across all microarray platforms. The MOE430A GeneChips and cDNA arrays had higher precision across technical replicates than the MOE430B GeneChips and oligonucleotide arrays. The Affymetrix platform showed the greatest range in the magnitude of expression levels followed by the oligonucleotide arrays. We observed good concordance in both estimated expression level and statistical significance of common genes between the Affymetrix MOE430A GeneChip and the oligonucleotide arrays. Despite their apparently high precision, cDNA arrays showed poor concordance with other platforms.