Automated analytical microarrays: a critical review

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple analytes in a single experiment. The specific affinity reaction of nucleic acids (hybridization) and antibodies towards antigens is the most common bioanalytical method for generating multiplexed quantitative results. Nucleic acid-based analysis is restricted to the detection of cells and viruses. Antibodies are more universal biomolecular receptors that selectively bind small molecules such as pesticides, small toxins, and pharmaceuticals and to biopolymers (e.g. toxins, allergens) and complex biological structures like bacterial cells and viruses. By producing an appropriate antibody, the corresponding antigenic analyte can be detected on a multiplexed immunoanalytical microarray. Food and water analysis along with clinical diagnostics constitute potential application fields for multiplexed analysis. Diverse fluorescence, chemiluminescence, electrochemical, and label-free microarray readout systems have been developed in the last decade. Some of them are constructed as flow-through microarrays by combination with a fluidic system. Microarrays have the potential to become widely accepted as a system for analytical applications, provided that robust and validated results on fully automated platforms are successfully generated. This review gives an overview of the current research on microarrays with the focus on automated systems and quantitative multiplexed applications. Figure MCR 3: A fully automated chemiluminescence microarray reader for analytical microarrays     

Protein microarrays and quantum dot probes for early cancer detection

We describe here a novel approach for detection of cancer markers using quantum dot protein microarrays. Both relatively new technologies; quantum dots and protein microarrays, offer very unique features that together allow detection of cancer markers in biological specimens (serum, plasma, body fluids) at pg/ml concentration. Quantum dots offer remarkable photostability and brightness. They do not exhibit photobleaching common to organic fluorophores. Moreover, the high emission amplitude for QDs results in a marked improvement in the signal to noise ratio of the final image. Protein microarrays allow highly parallel quantitation of specific proteins in a rapid, low-cost and low sample volume format. Furthermore the multiplexed assay enables detection of many proteins at once in one sample, making it a powerful tool for biomarker analysis and early cancer diagnostics.

In a series of multiplexing experiments we investigated ability of the platform to detect six different cytokines in protein solution. We were able to detect TNF-, IL-8, IL-6, MIP-1β, IL-13 and IL-1β down to picomolar concentration, demonstrating high sensitivity of the investigated detection system.

We have also constructed and investigated two different models of quantum dot probes. One by conjugation of nanocrystals to antibody specific to the selected marker—IL-10, and the second by use of streptavidin coated quantum dots and biotinylated detector antibody. Comparison of those two models showed better performance of streptavidin QD–biotinylated detector antibody model. Data quantitated using custom designed computer program (CDAS) show that proposed methodology allows monitoring of changes in biomarker concentration in physiological range.     

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.     

157-nm Laser ablation of polymeric layers for fabrication of biomolecule microarrays

A new methodology for protein microarray fabrication is proposed based on the ablation of polymer film using laser at 157 nm (F₂). The polymer has been selected among others with the criterion of negligible protein adsorption. Improved results have been obtained by pretreatment of the polymer surface with an inert protein. The use of 157-nm laser radiation allowed very good depth control during the polymeric layer ablation process. In addition the importance of laser ablation at 157 nm is based on the fact that irradiated surfaces indicate limited chemical change due to the fact that laser ablation at 157 nm is only photochemical, thus avoiding excessive surface heating and damage. Results of protein microarray fabrication are presented to illustrate the viability of the proposed method.     

Fluorous-based peptide microarrays for protease screening

As ever more protease sequences are uncovered through genome sequencing projects, efficient parallel methods to discover the potential substrates of these proteases becomes crucial. Herein we describe the first use of fluorous-based microarrays to probe peptide sequences and begin to define the scope and limitations of fluorous microarray technologies for the screening of proteases. Comparison of a series of serine proteases showed that their ability to cleave peptide substrates in solution was maintained upon immobilization of these substrates onto fluorous-coated glass slides. The fluorous surface did not serve to significantly inactivate the enzymes. However, addition of hydrophilic components to the peptide sequences could induce lower rates of substrate cleavage with enzymes such as chymotrypsin with affinities to hydrophobic moieties. This work represents the first step to creating robust protease screening platforms using noncovalent microarray interface that can easily incorporate a range of compounds on the same slide.     

A Systematic Investigation of Proteoforms with N-Terminal Glycine and Their Dynamics Reveals Its Impacts on Protein Stability

The N-termini of proteins can regulate their degradation, and the same protein with different N-termini may have distinct dynamics. Recently, it was found that N-terminal glycine can serve as a degron recognized by two E3 ligases, but N-terminal glycine was also reported to stabilize proteins. Here we developed a chemoenzymatic method for selective enrichment of proteoforms with N-terminal glycine and integrated dual protease cleavage to further improve the enrichment specificity. Over 2000 unique peptides with protein N-terminal glycine were analyzed from >1000 proteins, and most of them are previously unknown, indicating the effectiveness of the current method to capture low-abundance proteoforms with N-terminal glycine. The degradation rates of proteoforms with N-terminal glycine were quantified along with those of proteins from the whole proteome. Bioinformatic analyses reveal that proteoforms with N-terminal glycine with the fastest and slowest degradation rates have different functions and localizations. Membrane proteins with N-terminal glycine and proteins with N-terminal glycine from the N-terminal methionine excision degrade more rapidly. Furthermore, the secondary structures, adjacent amino acid residues, and protease specificities for N-terminal glycine are also vital for protein degradation. The results advance our understanding of the effects of N-terminal glycine on protein properties and functions.     

A miniature cytometry platform for capture and characterization of T-lymphocytes from human blood

Given the clinical and diagnostic importance of blood analysis, there is considerable interest in developing novel miniature devices for rapid characterization of blood constituents. The present paper describes development of a miniature cytometry platform aimed at analysis of T-lymphocytes from peripheral human blood. Microarrays of T-cell-specific antibodies (Abs), including anti-CD3, -CD4, -CD8 and mouse IgG (negative control) were robotically printed onto glass slides coated with a non-fouling poly(ethylene glycol) (PEG) hydrogel. The glass substrates containing Ab arrays were incubated with 100 μL of red blood cell (RBC)-depleted whole human blood for 15 min and then exposed to a controlled shear of ∼2 dyn cm⁻² for additional 10 min. This process led to the removal of non-specific leukocytes and “development” of patterns of T-cells captured on the Ab spots. The immunofluorescent staining of the surface-bound cells revealed the presence of purified CD4⁺ and CD8⁺ T-cells (purity >94%) on their respective Ab spots. Importantly, the proportions of CD4⁺ and CD8⁺ T-cells captured on the Ab spots correlated closely (R² − 0.9) with flow cytometry analysis of T-cell subsets in blood. Overall, this cytometry platform allowed to rapidly (under 30 min) capture pure T-cell subsets from minimally processed human blood. Significantly, our device provided quantitative information about subset abundance solely based on the location of cells within the microarray. This cytometry platform is envisioned as a miniature immunology tool for determination of T-cell phenotype and will have immediate applications in HIV diagnostics and research.     

Increasing robustness and sensitivity of protein microarrays through microagitation and automation

Assay systems that employ protein microarrays for the analysis of complex samples are powerful tools to generate a high amount of data from a limiting amount of sample. Due to miniaturization, these systems are susceptible to fluctuations during signal generation and the use of uniform conditions for sample incubation and during the assay procedure is required to get reproducible results. Diffusion limits may prevent constant conditions on all parts of the array and can lead to the decease of the sensitivity of the array. Therefore, we set-up an automated assay system integrating a novel microagitation device using surface acoustic wave (SAW) technology. Multiplexed assays for the detection of autoantibodies from human serum and sandwich immunoassay for the detection of matrix metalloproteases (MMPs) were used to evaluate the system. Diffusion-rate limited solid phase reactions were enhanced by microagitation using the SAW technology resulting in up to three-fold higher signals.     

High performance protein microarrays based on glycidyl methacrylate-modified polyethylene terephthalate plastic substrate

There is a great challenge to immobilize high density of probe molecules for high performance protein microarrays, and this is achieved in this work by using polyethylene terephthalate (PET) plastic substrate onto which glycidyl methacrylate (GMA) photopolymer is grafted under mild conditions to introduce high density of epoxy groups for covalent immobilization of proteins. The poly(GMA)-grafted PET (PGMA-PET) surface was characterized with atomic force microscope (AFM) and attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy. For high density of protein immobilization and good quality of microspots, experiments were conducted to optimize the printing buffer, and an optimal buffer was found out to be PBS with 10% glycerol + 0.003% triton X-100. According to the studies of loading capacity and immobilization kinetics, the optimal protein probe concentration and incubation time for the efficient immobilization are 200 μg mL⁻¹ and 8 h, respectively. The performance of the PGMA-PET-based protein microarrays is evaluated with sandwich immunoassay using rat IgG and anti-rat IgG as model proteins, demonstrating a limit of detection (LOD) of 10 pg mL⁻¹ and a dynamic range of five orders of magnitude which are better than or very comparable with the reported or commercially available immunoassays, while providing a high-throughput approach. The work renders a simple and economic method to manufacture high performance protein microarrays and is expected to have great potentials in broad applications related to clinic diagnosis, drug discovery and proteomic research.     

Adhesive microarrays for multipurpose diagnostic tools

We are reporting here a new technology for the straightforward production of integrated microarrays. The approach is based on the use of adhesive supports enabling (i) the immobilization of biomolecules as microarrays (up to 2500 spots per cm(2)) and (ii) the easy assembly of these microarrays with complex 3D structures such as 96-well bottomless microplates or polymer and glass microfluidic networks. The analytical performances of the system were demonstrated for sandwich protein detection (C-reactive protein) and hybridization assays, both in classical 96-well microplate format and microfluidic environment.     

Analysis of Enzyme Structure and Activity by Protein Engineering

Structure-activity relationships of enzymes can now be analyzed for the first time by the systematic alteration of protein structure. Recent developments in the chemical synthesis of DNA fragments and recombinant DNA technology enable the facile modification of proteins by highly specific mutagenesis of their genes. Kinetic analysis of the mutant enzymes combined with high-resolution structural data from protein X-ray crystallography allow direct measurements on the relationships between structure and function. In particular, the strength and nature of enzyme-substrate interactions and their detailed roles in catalysis and specificity can now be studied. We have developed such analysis of enzyme structure-function by site-directed mutagenesis of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus, concentrating so far on the subtle role of hydrogen bonding in both substrate specificity and catalysis. We find that the energetics of tyrosine and ATP binding must be analyzed in terms of an exchange reaction with solvent water. Based on this idea and structural data, we have engineered an enzyme of improved enzyme-substrate affinity, and there thus appear to be real prospects of engineering proteins of new specificities, activities, and structural properties. We are also using protein engineering to gather direct information on the nature of enzyme catalysis. For example, we find the catalysis of formation of Tyr-AMP from Tyr and ATP is due largely to electrostatic and hydrogen bonding interactions that are stronger in the transition state than in the ground state—a “strain” mechanism rather than acid-base or covalent catalysis.     

蛋白质芯片及其分析应用新进展

蛋白质芯片是一种快速、高效、高通量的蛋白质组研究新技术。目前,它已成为人们研究的热点之一。本文就近年来蛋白质芯片及其分析应用新进展做一简要的评述。     

Optimization of on-chip elongation for fabricating double-stranded DNA microarrays

The sequence-specific recognitions between DNA and proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays: (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extension was about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiements showed that the elongation temperature of 50 °C and the Mg²⁺ concentration of 2.5 mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.     

Preparation and use of microarrays containing synthetic heparin oligosaccharides for the rapid analysis of heparin-protein interactions

Heparin is a highly sulfated, linear polymer that participates in a plethora of biological processes by interaction with many proteins. The chemical complexity and heterogeneity of this polysaccharide can explain the fact that, despite its widespread medical use as an anticoagulant drug, the structure-function relationship of defined heparin sequences is still poorly understood. Here, we present the chemical synthesis of a library containing heparin oligosaccharides ranging from di- to hexamers of different sequences and sulfation patterns. An amine-terminated linker was placed at the reducing end of the synthetic structures to allow for immobilization onto N-hydroxysuccinimide activated glass slides and creation of heparin microarrays. Key features of this modular synthesis, such as the influence of the amine linker on the glycosidation efficiency, the use of 2-azidoglucose as glycosylating agents for oligosaccharide assembly, and the compatibility of the protecting group strategy with the sulfation-deprotection steps, are discussed. Heparin microarrays containing this oligosaccharide library were constructed using a robotic printer and employed to characterize the carbohydrate binding affinities of three heparin-binding growth factors. FGF-1, FGF-2 and FGF-4 that are implicated in angiogenesis, cell growth and differentiation were studied. These heparin chips aided in the discovery of novel, sulfated sequences that bind FGF, and in the determination of the structural requirements needed for recognition by using picomoles of protein on a single slide. The results presented here highlight the potential of combining oligosaccharide synthesis and carbohydrate microarray technology to establish a structure-activity relationship in biological processes.