Protein microarrays on hybrid polymeric thin films prepared by self-assembly of polyelectrolytes for multiple-protein immunoassays

We report here the development and characterization of protein microarrays fabricated on nanoengineered 3-D polyelectrolyte thin films (PET) deposited on glass slide by consecutive adsorption of polyelectrolytes via self-assembly technique. Antibodies or antigens were immobilized in the PET-coated glass slides by electrostatic adsorption and entrapment of porous structure of the 3-D polymer film and thus establishing a platform for parallel analysis. Both antigen and antibody microarrays were fabricated on the PET-coated slides, and direct and indirect immunoassays on protein microarrays for multiple-analyte detection were demonstrated. Microarrays produced on these PET-coated slides have consistent spot morphology and provide performance features needed for proteomic analysis. The protein microarrays on the PET films provide LOD as low as 6 pg/mL and dynamic ranges up to three orders of magnitude, which are wider than the protein microarrays fabricated on aldehyde and poly-L-lysine functionalized slides. The PET films constructed by self-assembly technique in aqueous solution is green chemistry based, cost-effective method to generate 3-D thin film coatings on glass surface, and the coated slide is well suited for immobilizing many types of biological molecules so that a wide variety of microarray formats can be developed on this type of slide.     

A cancer protein microarray platform using antibody fragments and its clinical applications

Antibody microarrays have shown great potential for measurement of either a spectrum of target proteins in proteomics or disease-associated antigens in molecular diagnostics. Despite its importance, the applications of antibody microarrays are still limited by a variety of fundamental problems. Among them, cross-reactivity significantly limits the multiplexing ability in parallel sandwich immunoassays. As a result, it is very important to design new capture probes in order to incorporate a universal label into the assay configuration. In this report, an antibody fragments (F(ab')2) microarray platform for serum tumor markers was developed. Each antigen was detected at different concentrations to assemble its calibration curve, and combinations of different markers were tested to examine the specificity of simultaneous detection based on the F(ab')2 microarrays. Diagnostics of serum samples with this cancer antibody microarray platform and immunoradiometric assays (IRMA) were also performed. Wide range calibration curves (0-1280 U mL(-1)) were obtained for each tumor marker. Comparative studies demonstrated that such F(ab')2 microarrays exhibited both moderately improved sensitivity and better specificity than full-sized monoclonal antibody microarrays. It is also demonstrated that this microarray platform is quantitative, highly specific and reasonably sensitive. More importantly, clinical applications of our F(ab')2 microarray platform for upwards of 100 patient serum samples clearly show its potential in cancer diagnostics.     

Novel 3-dimensional dendrimer platform for glycolipid microarray

Glycolipids are important biological molecules that modulate cellular recognitions and pathogen adhesions. In this paper, we report a sensitive glycolipid microarray for non-covalently immobilizing glycolipids on a microarray substrate and we perform a set of immunoassays to explore glycolipid-protein interactions. This substrate utilizes a three-dimensional hydrazide-functionalized dendrimer monolayer attached onto a microscopic glass surface, which possesses the characteristics to adsorb glycoliplids non-covalently and facilitates multivalent attributes on the substrate surface. In the proof-of-concept experiments, gangliosides such as GM1, FucGM1, GM3, GD1b, GT1b, and GQ1b, and a lipoarabinomannan were tested on the substrate and interrogated with toxins and antibodies. The resulting glycolipid microarrays exhibited hypersensitivity and specificity for detection of glycolipid-protein interactions. In particular, a robust and specific binding of a pentameric cholera toxin B subunit to the GM1 glycolipid spotted on the array has demonstrated its superiority in sensitivity and specificity. In addition, this glycolipid microarray substrate was used to detect lipoarabinomannan in buffer within a limit-of-detection of 125 ng/mL. Furthermore, Mycobacterium tuberculosis (Mtb) Lipoarabinomannan was tested in human urine specimens on this platform, which can effectively identify urine samples either infected or not infected with Mtb. The results of this work suggest the possibility of using this glycolipid microarray platform to fabricate glycoconjugate microarrays, which includes free glycans and glycolipids and potential application in detection of pathogen and toxin.     

Cowpea mosaic virus nanoscaffold as signal enhancement for DNA microarrays

Previous studies have shown that a functionalized viral nanoparticle can be used as a fluorescent signal-generating element and enhance detection sensitivity for immunoassays and low density microarrays. In this study, we further tested this ability in commercial DNA microarrays, including Affymetrix high density resequencing microarray. Optimum conditions for NeutrAvidin and dye coupling to a double-cysteine mutant of cowpea mosaic virus (CPMV) were found to be comparable to the commonly used streptavidin-phycoerythrin (SAPE) for high density resequencing microarray. A 3-fold signal enhancement in comparison to Cy5-dCTP controls was obtained when using nanoparticles on control scorecard expression microarrays. Hybridization results from commercially available 8000 rat expression arrays indicate an increment of 14% on the detected features when the virus complex was used as the staining reagent in comparison to Cy5-dCTP controls. The current work shows the utility of the CPMV-dye nanoparticles as a detection reagent in well-established detection platforms.     

From combinatorial chemistry to chemical microarray

Combinatorial chemistry was first applied to the generation of peptide arrays in 1984. Since then, the field of combinatorial chemistry has evolved rapidly into a new discipline. There is a great need for the development of methods to examine the proteome functionally at a global level. Using many of the techniques and instruments developed for DNA microarrays, chemical microarray methods have advanced significantly in the past three years. High-density chemical microarrays can now be synthesized in situ on glass slides or be printed through covalent linkage or non-specific adsorption to the surface of the solid-support with fully automatic arrayers. Microfabrication methods enable one to generate arrays of microsensors at the end of optical fibers or arrays of microwells on a flat surface. In conjunction with the one-bead one-compound combinatorial library method, chemical microarrays have proven to be very useful in lead identification and optimization. High-throughput protein expression systems, robust high-density protein, peptide and small-molecule microarray systems, and automatic mass spectrometers are critical tools for the field of functional proteomics.     

Development of miniaturized immunoassay: Influence of surface chemistry and comparison with enzyme-linked immunosorbent assay and Western blot

Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. To implement functional protein microarrays, appropriate surface chemistry must be designed so that both the protein structure and the biological activity can be retained. In the current study, two surface chemistries for protein microarrays and immunofluorescent assays were developed. Glass slides were functionalized with N-hydroxysuccinimide (NHS) ester via a monofunctional silane or maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays was then evaluated for the detection of anti-histone autoantibodies present in the sera of patients suffering from a systemic autoimmune disease, namely systemic lupus erythematosus (SLE), and the results were compared with those of the classical enzyme-linked immunosorbent assay (ELISA) and Western blot. The detection limit of our MAMVE copolymer microarrays was 50-fold lower than that of the classical ELISA. Furthermore, 100-fold less volume of biological samples was required with these miniaturized immunoassays.     

The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters

Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching (PM) complementary sequence and other spots with one or two mismatches (MM) : in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays.     

A model of base-call resolution on broad-spectrum pathogen detection resequencing DNA microarrays

Oligonucleotide microarrays offer the potential to efficiently test for multiple organisms, an excellent feature for surveillance applications. Among these, resequencing microarrays are of particular interest, as they possess additional unique capabilities to track pathogens’ genetic variations and perform detailed discrimination of closely related organisms. However, this potential can only be realized if the costs of developing the detection microarray are kept at a manageable level. Selection and verification of the probes are key factors affecting microarray design costs that can be reduced through the development and use of in silico modeling. Models created for other types of microarrays do not meet all the required criteria for this type of microarray. We describe here in silico methods for designing resequencing microarrays targeted for multiple organism detection. The model development presented here has focused on accurate base-call prediction in regions that are applicable to resequencing microarrays designed for multiple organism detection, a variation from other uses of a predictive model in which perfect prediction of all hybridization events is necessary. The model will assist in simplifying the design of resequencing microarrays and in reduction of the time and costs required for their development for new applications.     

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.     

Immunoassays based on electrochemical detection using microelectrode arrays

We show that CombiMatrix's VLSI arrays of individually addressable electrodes, using conventional CMOS integrated circuitry, can be used in detecting various analytes via immunoassay protocols. These microarrays provide over 1000 electrodes per square centimeter. The chips are coated with a porous material on which specific affinity tags are synthesized proximate to selected electrode sites. CombiMatrix microarrays are used to develop spatially multiplexed assay formats for biological entities over a wide range of sizes, from small molecules to cells. Antibodies are tagged with coded affinity labels and then allowed to self-assemble on the appropriate electrode assay sites. Each analyte-specific antibody is chaperoned to individual, predetermined locations by the self-assembly process. The resulting chip can perform numerous different analyte-specific immunoassays, simultaneously. We present new detection technologies based upon the use of the active individually addressable microelectrodes on the chip: redox enzyme amplified electrochemical detection. The results for human alpha1 acid glycoprotein, ricin, M13 phage, Bacillus globigii spores, and fluorescein indicate that this method is one of the most sensitive available, with limits of detection in the attomole range. The detection range is 4-5 logs of analyte concentration, with an assay volume of 50 microl or less. The system provides for a host of multiplexed immunoassays because of the large number of electrodes available. We show how the assays can be optimized for maximum performance on the CombiMatrix microarray platform.     

蛋白微阵列研究进展

蛋白微阵列是随着基因微阵列技术发展起来的,用于基因微阵列的制备方法、信号的检测及分析系统,也可用于蛋白微阵列。各种蛋白微阵列基质的发展,提高了蛋白的固定效率。放射性同位数、化学发光、激光共聚焦荧光扫描等技术都已用于微阵列的检测。重组蛋白技术的发展,提高了蛋白微阵列检测的通量和灵敏度。蛋白微阵列具有通量高、使用样品少、重复性好、可定量的特点,使其在生物医药科学研究中得到了广泛应用。本综述了蛋白微阵列的制备及其在免疫检测、医学诊断及蛋白组研究中的应用。     

Novel Microarrays for Simultaneous Serodiagnosis of Multiple Antiviral Antibodies

We developed an automated diagnostic system for the detection of virus-specific immunoglobulin Gs (IgGs) that was based on a microarray platform. We compared efficacies of our automated system with conventional enzyme immunoassays (EIAs). Viruses were immobilized to microarrays using a radical cross-linking reaction that was induced by photo-irradiation. A new photoreactive polymer containing perfluorophenyl azide (PFPA) and poly(ethylene glycol) methacrylate was prepared and coated on plates. Inactivated measles, rubella, mumps, Varicella-Zoster and recombinant Epstein-Barr viruse antigen were added to coated plates, and irradiated with ultraviolet light to facilitate immobilization. Virus-specific IgGs in healthy human sera were assayed using these prepared microarrays and the results obtained compared with those from conventional EIAs. We observed high correlation (0.79–0.96) in the results between the automated microarray technique and EIAs. The microarray-based assay was more rapid, involved less reagents and sample, and was easier to conduct compared with conventional EIA techniques. The automated microarray system was further improved by introducing reagent storage reservoirs inside the chamber, thereby conserving the use of expensive reagents and antibodies. We considered the microarray format to be suitable for rapid and multiple serological diagnoses of viral diseases that could be developed further for clinical applications.     

DNA microarray for discrimination between pathogenic 0157:H7 EDL933 and non-pathogenic Escherichia coli strains

The primary technique currently used to detect biological agents is based on immunoassays. Although sensitive and specific, currently employed immunoassays generally rely on the detection of a single epitope, and therefore often cannot discriminate subtle strain-specific differences. Since DNA microarrays can hybridize hundreds to thousands of genomic targets simultaneously and do not rely on phenotypic expression of these genetic features for identification purposes, they have enormous potential to provide inexpensive, flexible and specific strain-specific detection and identification of pathogens. In this study, pathogenic Escherichia coli O157:H7-specific genes, non-pathogenic K12-specific genes, common E. coli genes, and negative control genes were polymerase chain reaction-amplified and spotted onto the surface of treated glass slides. After labeled bacterial cDNA samples were hybridized with probes on the microarray, specific fluorescence patterns were obtained, enabling identification of pathogenic E. coli O157:H7 and non-pathogenic E. coli K12. To test the utility of this microarray device to detect genetically engineered bacteria, E. coli BL21 (a B strain derivative with antibiotic resistance gene, ampR) and E. coli JM107 (a K12 strain derivative lacking the gene ompT) were also employed. The array successfully confirmed the strain genotypes and demonstrated that antibiotic resistance can also be detected. The ability to assess multiple data points makes this array method more efficient and accurate than a typical immunoassay, which detects a single protein product.     

Making a new technology work: the standardization and regulation of microarrays

The translation of laboratory innovations into clinical tools is dependent upon the development of regulatory arrangements designed to ensure that the new technology will be used reliably and consistently. A case study of a key post-genomic technology, gene chips or microarrays, exemplifies this claim. The number of microarray publications and patents has increased exponentially during the last decade and diagnostic microarray tests already are making their way into the clinic. Yet starting in the mid-1990s, scientific journals were overrun with criticism concerning the ambiguities involved in interpreting most of the assumptions of a microarray experiment. Questions concerning platform comparability and statistical calculations were and continue to be raised, in spite of the emergence by 2001 of an initial set of standards concerning several components of a microarray experiment. This article probes the history and ongoing efforts aimed at turning microarray experimentation into a viable, meaningful, and consensual technology by focusing on two related elements:     

Salt Concentration Effects on Equilibrium Melting Curves from DNA Microarrays

DNA microarrays find applications in an increasing number of domains where more quantitative results are required. DNA being a charged polymer, the repulsive interactions between the surface of the microarray and the targets in solution are increasing upon hybridization. Such electrostatic penalty is generally reduced by increasing the salt concentration. In this article, we present equilibrium-melting curves obtained from dedicated physicochemical experiments on DNA microarrays in order to get a better understanding of the electrostatic penalty incurred during the hybridization reaction at the surface. Various salt concentrations have been considered and deviations from the commonly used Langmuir adsorption model are experimentally quantified for the first time in agreement with theoretical predictions.     

Evaluation of surface chemistries for antibody microarrays

Antibody microarrays are an emerging technology that promises to be a powerful tool for the detection of disease biomarkers. The current technology for protein microarrays has been derived primarily from DNA microarrays and is not fully characterized for use with proteins. For example, there are a myriad of surface chemistries that are commercially available for antibody microarrays, but there are no rigorous studies that compare these different surfaces. Therefore, we have used a sandwich enzyme-linked immunosorbent assay (ELISA) microarray platform to analyze 17 different commercially available slide types. Full standard curves were generated for 23 different assays. We found that this approach provides a rigorous and quantitative system for comparing the different slide types based on spot size and morphology, slide noise, spot background, lower limit of detection, and reproducibility. These studies demonstrate that the properties of the slide surface affect the activity of immobilized antibodies and the quality of data produced. Although many slide types produce useful data, glass slides coated with aldehyde silane, poly-l-lysine, or aminosilane (with or without activation with a crosslinker) consistently produce superior results in the sandwich ELISA microarray analyses we performed.     

Electrostatic readout of DNA microarrays with charged microspheres

DNA microarrays are used for gene-expression profiling, single-nucleotide polymorphism detection and disease diagnosis. A persistent challenge in this area is the lack of microarray screening technology suitable for integration into routine clinical care. Here, we describe a method for sensitive and label-free electrostatic readout of DNA or RNA hybridization on microarrays. The electrostatic properties of the microarray are measured from the position and motion of charged microspheres randomly dispersed over the surface. We demonstrate nondestructive electrostatic imaging with 10-mum lateral resolution over centimeter-length scales, which is four-orders of magnitude larger than that achievable with conventional scanning electrostatic force microscopy. Changes in surface charge density as a result of specific hybridization can be detected and quantified with 50-pM sensitivity, single base-pair mismatch selectivity and in the presence of complex background. Because the naked eye is sufficient to read out hybridization, this approach may facilitate broad application of multiplexed assays.