Fluorous-based carbohydrate microarrays

The success of microarrays, such as DNA chips, for biosample screening with minimal sample usage has led to a variety of technologies for assays on glass slides. Unfortunately, for small molecules, such as carbohydrates, these methods usually rely on covalent bond formation, which requires unique functional handles and multiple chemical steps. A new simpler concept in microarray formation is based on noncovalent fluorous-based interactions. A fluorous tail is designed not only to aid in saccharide purification but also to allow direct formation of carbohydrate microarrays on fluorous-derivatized glass slides for biological screening with lectins, such as concanavalin A. The noncovalent interactions in the fluorous-based array are even strong enough to withstand the detergents used in assays with the Erythrina crystagalli lectin. Additionally, the utility of benzyl carbonate protecting groups on fucose building blocks for the formation of alpha-linkages is demonstrated.     

Fabrication of DNA microarrays on nanoengineered polymeric ultrathin film prepared by self-assembly of polyelectrolyte multilayers

Microarray-based technology is in need of flexible and cost-effective chemistry for fabrication of oligonucleotide microarrays. We have developed a novel method for the fabrication of oligonucleotide microarrays with unmodified oligonucleotide probes on nanoengineered three-dimensional thin films that are deposited on glass slides by consecutive layer-to-layer adsorption of polyelectrolytes. Unmodified oligonucleotide probes were spotted and immobilized on these multilayered polyelectrolyte thin films (PET) by electrostatic adsorption and entrapment on the porous structure of the PET film. The PET provides higher probe binding capacity and thus higher hybridization signal than that of the traditional two-dimensional aminosilane and poly-L-lysine coated slides. Immobilized probe densities of 3.4 x 10(12)/cm2 were observed for microarray spots on PET with unmodified 50-mer oligonucleotide probes, which is comparable to the immobilized probe densities of alkyamine-modified 50-mer probes end-tethered on an aldehyde-functionalized slide. The study of hybridization efficiency showed that 90% of immobilized probes on PET film are accessible to target DNA to form duplex format in hybridization. The DNA microarray fabricated on PET film has wider dynamic range (about 3 orders of magnitude) and lower detection limit (0.5 nM) than the conventional amino- and aldehyde-functionalized slides. Oligonucleotide microarrays fabricated on these PET-coated slides also had consistent spot morphology. In addition, discrimination of single nucleotide polymorphism of 16S rRNA genes was achieved with the PET-based oligonucleotide microarrays. The PET microarrays constructed by our self-assembly process is cost-effective, versatile, and well suited for immobilizing many types of biological active molecules so that a wide variety of microarray formats can be developed.     

Carbohydrate chips for studying high-throughput carbohydrate-protein interactions

Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.     

Surface behavior and molecular recognition in DNA microarrays from N,N-dimethylacrylamide terpolymers with activated esters as linking groups

A series of terpolymers made of DMA, NAS and MAPS were synthesized by free radical copolymerization and used as functional coatings for the fabrication of glass slide DNA microarrays. The surface properties of coated glass slides were investigated through contact angle measurements, ellipsometry and atomic force microscopy. The terpolymer molecular weight showed a moderate effect on surface tension (gamma(s) = 56-62 mN x m(-1)), but no clear effect on polymeric layer thickness (5-8 nm) and roughness. Hybridization experiments with amine-functionalized oligonucleotides gave the best fluorescence intensity results for microarrays coated with intermediate-molecular-weight terpolymers. Finally, an accelerated ageing test of the microarray in a humidity chamber showed a nice relationship between decay curves of contact angle against water and fluorescence intensity.     

Preparation of carbohydrate arrays by using Diels-Alder reactions with inverse electron demand

Carbohydrate microarrays are an emerging tool for the high-throughput screening of carbohydrate-protein interactions that represent the basis of many biologically and medicinally relevant processes. The crucial step in the preparation of carbohydrate arrays is the attachment of carbohydrate probes to the surface. We examined the Diels-Alder reaction with inverse-electron-demand (DARinv) as an irreversible, chemoselective ligation reaction for that purpose. After having shown the efficiency of the DARinv in solution, we prepared a series of carbohydrate-dienophile conjugates that were printed onto tetrazine-modified glass slides. Binding experiments with fluorescently labeled lectins proved successful and homogeneous immobilization was achieved by the DARinv. For immobilization of nonfunctionalized reducing oligosaccharides we developed a bifunctional chemoselective linker that enabled the attachment of a dienophile tag to the oligosaccharides through oxime ligation. The conjugates obtained were successfully immobilized on glass slides. The presented strategies for the immobilization of both synthetic carbohydrate derivatives and unprotected reducing oligosaccharides facilitate the preparation of high-quality carbohydrate microarrays by means of the chemoselective DARinv. This concept can be readily adapted for the preparation of other biomolecule arrays.     

A generic small-molecule microarray immobilization strategy

Small-molecule microarrays are often limited by the requirement for each compound undergoing immobilization to contain a common functional group or by the need to prepare glass slides containing photo-reactive groups. Herein, we present a generic strategy that allows any compound library to be immobilized. This was achieved by printing a fluorous-tagged photo-reactive 3-aryl-3-trifluoromethyldiazirine, which undergoes non-selective insertion into compounds following UV-activation, onto fluorous-functionalized glass slides. The arrays could be reused following aqueous stripping and re-assessment of the compounds with the same protein or another target of interest.     

Preparation of Carbohydrate Arrays by Using Diels–Alder Reactions with Inverse Electron Demand

Carbohydrate microarrays are an emerging tool for the high‐throughput screening of carbohydrate–protein interactions that represent the basis of many biologically and medicinally relevant processes. The crucial step in the preparation of carbohydrate arrays is the attachment of carbohydrate probes to the surface. We examined the Diels–Alder reaction with inverse‐electron‐demand (DARinv) as an irreversible, chemoselective ligation reaction for that purpose. After having shown the efficiency of the DARinv in solution, we prepared a series of carbohydrate–dienophile conjugates that were printed onto tetrazine‐modified glass slides. Binding experiments with fluorescently labeled lectins proved successful and homogeneous immobilization was achieved by the DARinv. For immobilization of nonfunctionalized reducing oligosaccharides we developed a bifunctional chemoselective linker that enabled the attachment of a dienophile tag to the oligosaccharides through oxime ligation. The conjugates obtained were successfully immobilized on glass slides. The presented strategies for the immobilization of both synthetic carbohydrate derivatives and unprotected reducing oligosaccharides facilitate the preparation of high‐quality carbohydrate microarrays by means of the chemoselective DARinv. This concept can be readily adapted for the preparation of other biomolecule arrays.     

Dark-to-bright optical responses of liquid crystals supported on solid surfaces decorated with proteins

Protein assays are critical analytical tools performed in various biochemical laboratories to quantify the concentration of proteins. In this study, we report the optical responses of a thin layer of liquid crystals supported on glass slides decorated with proteins and the utility of this phenomenon as a new "all-or-nothing" type of protein assay. It was found that the orientations of liquid crystals are very sensitive to the concentration of protein solution applied to the surface. When the protein concentration exceeds a critical value (IgG 5.0 microg/mL, BSA 6.0 microg/mL, FTIC-anti-biotin 0.40 microg/mL, and FITC-anti-IgG 0.37 microg/mL), the thin layer of liquid crystals gives a very sharp dark-to-bright optical response within a small concentration range. This characteristic is not observed in any traditional protein assays, which are based on the adsorption of UV or visible light. The optical response is also very precise and reproducible. It is not affected by the thickness of the liquid crystal cell or the amount of organosilanes coated on the glass slides. The liquid crystal-based protein assay may be very useful for screening purposes, especially when a simple positive or negative answer is desired.     

Glass surfaces grafted with high-density poly(ethylene glycol) as substrates for DNA oligonucleotide microarrays

Surfaces carrying a dense layer of poly(ethylene glycol) (PEG) were prepared, characterized, and tested as substrates for DNA oligonucleotide microarrays. PEG bis(amine) with a molecular weight of 2000 was grafted onto silanized glass slides bearing aldehyde groups. After grafting, the terminal amino groups of the PEG layer were derivatized with the heterobifunctional cross-linker succinimidyl 4-[p-maleimidophenyl]butyrate to permit the immobilization of thiol-modified DNA oligonucleotides. The stepwise chemical modification was validated with X-ray photoelectron spectroscopy. Goniometry indicated that the PEG grafting procedure reduced surface inhomogeneities present after the silanization step, while atomic force microscopy and ellipsometry confirmed that the PEG layer was dense and monomolecular. Hybridization assays using DNA oligonucleotides and fluorescence imaging showed that PEG grafting improved the yield in hybridization 4-fold compared to non-PEGylated maleimide-derivatized surfaces. In addition, the PEG layer reduced the nonspecific adsorption of DNA by a factor of up to 13, demonstrating that surfaces with a dense PEG layer represent suitable substrates for DNA oligonucleotide microarrays.     

Protein arrays on high-surface-area plasma-nanotextured poly(dimethylsiloxane)-coated glass slides

Treatment of poly(dimethylsiloxane) (PDMS) surfaces with SF(6) plasma results in the creation of high-surface-area nanotextured surfaces that considerably favour protein adsorption with respect to untreated ones. In order to employ such nanotextured surfaces as substrates for microarrays to be created and analysed using standard instrumentation, we fabricated thin PDMS films on top of standard low-cost microscope glass slides. The properties of both untreated and plasma-treated PDMS-coated slides towards spotting of protein solutions were evaluated in terms of spot signal intensity and homogeneity as well as of spot shape and size. It was found that the plasma-treated PDMS-coated glass slides provided highly homogeneous spots (mean intra-spot variation 7.6%) with spot signal intensity 6-times higher than that obtained using the untreated ones. In addition, comparison with commercially available polystyrene and aminosilanized-glass microarray slides showed that the proposed slides provided 3-times higher spot signal intensity and 2-times lower intra-spot signal variation. In addition, the implementation of long-aged-after-plasma-treatment nanotextured PDMS-coated glass slides provided spots whose shape and size matched those of the spotting tip. As a consequence, denser arrays of variable spot shape can be created using SF(6) plasma-treated PDMS-coated slides instead of standard microarray slides opening new potentials for bioanalytical applications.     

Corrigendum to “Deposition of chemically reactive and repellent sites on biosensor chips for reduced non-specific binding” [Colloids Surf. B 79 (2010) 270–275]

Treatment of poly(dimethylsiloxane) (PDMS) surfaces with SF₆ plasma results in the creation of high-surface-area nanotextured surfaces that considerably favour protein adsorption with respect to untreated ones. In order to employ such nanotextured surfaces as substrates for microarrays to be created and analysed using standard instrumentation, we fabricated thin PDMS films on top of standard low-cost microscope glass slides. The properties of both untreated and plasma-treated PDMS-coated slides towards spotting of protein solutions were evaluated in terms of spot signal intensity and homogeneity as well as of spot shape and size. It was found that the plasma-treated PDMS-coated glass slides provided highly homogeneous spots (mean intra-spot variation 7.6%) with spot signal intensity 6-times higher than that obtained using the untreated ones. In addition, comparison with commercially available polystyrene and aminosilanized-glass microarray slides showed that the proposed slides provided 3-times higher spot signal intensity and 2-times lower intra-spot signal variation. In addition, the implementation of long-aged-after-plasma-treatment nanotextured PDMS-coated glass slides provided spots whose shape and size matched those of the spotting tip. As a consequence, denser arrays of variable spot shape can be created using SF₆ plasma-treated PDMS-coated slides instead of standard microarray slides opening new potentials for bioanalytical applications.     

Allergen microarrays on high-sensitivity silicon slides

We have recently introduced a silicon substrate for high-sensitivity microarrays, coated with a functional polymer named copoly(DMA-NAS-MAPS). The silicon dioxide thickness has been optimized to produce a fluorescence intensification due to the optical constructive interference between the incident and reflected lights of the fluorescent radiation. The polymeric coating efficiently suppresses aspecific interaction, making the low background a distinctive feature of these slides. Here, we used the new silicon microarray substrate for allergy diagnosis, in the detection of specific IgE in serum samples of subjects with sensitizations to inhalant allergens. We compared the performance of silicon versus glass substrates. Reproducibility data were measured. Moreover, receiver-operating characteristic (ROC) curves were plotted to discriminate between the allergy and no allergy status in 30 well-characterized serum samples. We found that reproducibility of the microarray on glass supports was not different from available data on allergen arrays, whereas the reproducibility on the silicon substrate was consistently better than on glass. Moreover, silicon significantly enhanced the performance of the allergen microarray as compared to glass in accurately identifying allergic patients spanning a wide range of specific IgE titers to the considered allergens.     

Synthesis and characterization of nitric oxide-releasing sol-gel microarrays

Diazeniumdiolate-modified sol-gel microarrays capable of releasing low levels of nitric oxide are reported as a viable means for improving the blood compatibility of a surface without fully modifying the underlying substrate. Several parameters are characterized including: (1) NO surface flux as a function of sol-gel composition and microarray geometry; (2) microstructure dimensions and spacing for optimal blood compatibility; and (3) the effect of sol-gel surface modification on analyte accessibility to platinum electrodes. The sol-gel microarrays release biologically relevant levels of NO under physiological conditions for >24 h. In vitro platelet adhesion assays indicate that a NO surface flux of 2.2 pmol cm(-2) s(-1) effectively reduces platelet adhesion to glass substrates modified with sol-gel microstructures separated by 50 microm. The blood compatibility observed for these micropatterned surfaces is comparable to NO-releasing sol-gel films. When the separation between NO-releasing microstructures is reduced to 10 microm, the NO surface flux required to reduce platelet adhesion is lowered to 0.4 pmol cm(-2) s(-1). Finally, the oxygen response of platinum electrodes modified with NO-releasing sol-gel microarrays indicates that selective modification via micropatterning enhances analyte accessibility to the sensor surface.     

Analysis of Density‐Dependent Binding of Glycans by Lectins Using Carbohydrate Microarrays

To investigate the density‐dependent binding of glycans by lectins using carbohydrate microarrays, a number of C‐terminal hydrazide‐conjugated neoglycopeptides with various valences and different spatial arrangements of the sugar ligands were prepared on a solid support. The synthetic strategy includes (1) assembly of alkyne‐linked peptides possessing C‐terminal hydrazide on a solid support, (2) coupling of azide‐linked, unprotected sugars to the alkyne‐linked peptides on the solid support utilizing click chemistry, and (3) release of the neoglycopeptides from the solid support. By using this synthetic methodology, sixty five neoglycopeptides with a valency ranging from 1 to 4 and different spatial arrangements of the carbohydrate ligands were generated. Carbohydrate microarrays were constructed by immobilizing the prepared neoglycopeptides on epoxide‐derivatized glass slides and were used to analyze the density‐dependent binding of glycans by lectins. The results of binding property determinations show that lectin binding is highly dependent on the surface glycan density.     

Construction of an antibody microarray based on agarose-coated slides

The antibody microarray, a high-throughput multiplex immunoassay method, has become a significant tool for quantitative proteomics studies. We describe here the strategies for optimizing the condition of antibody microarray building based on agarose-coated slides. In this study, modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 (MCP-1), then dilutions of the cytokine were applied to the arrays, and the protein was detected with biotin-labeled antibody coupled with Cy3-conjugated streptavidin. Thus a protein profiling microarray based on sandwich immunoassay has been established. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, shelf life of the postprinting slides, blocking buffers, and reproducibility of the system. A calibration curve with a correlation coefficient of 0.9995 was established which suggested that the matrix can retain arrayed proteins in near-quantitative fashion. The results revealed high signal uniformity and reproducibility with regard to intra-array (1.3%) and the interarray (8.7%) variation at the capture antibody concentration of 125 microg/mL. Besides, the printed arrays could be stored for at least two months without any apparent change of the performance parameters.     

A miniaturized glucocorticoid receptor translocation assay using enzymatic fragment complementation evaluated with qHTS

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z' of 0.55. qHTS yielded EC(50) values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 microM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3sigma or 6sigma threshold, respectively. This corresponds to a confirmation rate of approximately 30% or approximately 50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.     

Hydrogel droplet microarrays with trapped antibody-functionalized beads for multiplexed protein analysis

Antibody microarrays are a powerful tool for rapid, multiplexed profiling of proteins. 3D microarray substrates have been developed to improve binding capacity, assay sensitivity, and mass transport, however, they often rely on photopolymers which are difficult to manufacture and have a small pore size that limits mass transport and demands long incubation time. Here, we present a novel 3D antibody microarray format based on the entrapment of antibody-coated microbeads within alginate droplets that were spotted onto a glass slide using an inkjet. Owing to the low concentration of alginate used, the gels were highly porous to proteins, and together with the 3D architecture helped enhance mass transport during the assays. The spotting parameters were optimized for the attachment of the alginate to the substrate. Beads with 0.2 μm, 0.5 μm and 1 μm diameter were tested and 1 μm beads were selected based on their superior retention within the hydrogel. The beads were found to be distributed within the entire volume of the gel droplet using confocal microscopy. The assay time and the concentration of beads in the gels were investigated for maximal binding signal using one-step immunoassays. As a proof of concept, six proteins including cytokines (TNFα, IL-8 and MIP/CCL4), breast cancer biomarkers (CEA and HER2) and one cancer-related protein (ENG) were profiled in multiplex using sandwich assays down to pg mL(-1) concentrations with 1 h incubation without agitation in both buffer solutions and 10% serum. These results illustrate the potential of beads-in-gel microarrays for highly sensitive and multiplexed protein analysis.     

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.     

Rhodamine B doped silica nanoparticle labels for protein microarray detection

A core-shell Rhodamine B-doped SiO2 nanoparticle was synthesized and its fluorescent intensity was found to be 1000 times higher than that of individual Rhodamine B molecule. The doped nanoparticles were further conjugated with streptavidin and the resulting nanoparticles were used in the detection of reverse-phase protein microarrays, in which human IgG of various concentrations was first immobilized on aldehyde-modified glass slides and then biotinlyated goat anti human IgG as well as the labeled nanopart...