Liquid crystalline properties of type I collagen: Perspectives in tissue morphogenesis

Collagen molecules form the major part of tissues like bone, cornea or tendon where they organize into ordered fibrillar networks. The acid-soluble protein spontaneously assembles in liquid crystalline phases, characterized in polarized light microscopy and X-ray diffraction. Collagen fibrillogenesis obtained in condensed media establishes a link between the fibrillar networks described in vivo and the mesomorphic states obtained in vitro. Cell–matrix interactions on these biomimetic materials are currently analysed with perspectives in tissue engineering. In a morphogenetic context, we propose the hypothesis of a liquid crystalline order, between soluble precursor molecules, preceding fibrillogenesis.     

Functional GPCR microarrays

This paper describes G-protein-coupled receptor (GPCR) microarrays on porous glass substrates and functional assays based on the binding of a europium-labeled GTP analogue. The porous glass slides were made by casting a glass frit on impermeable glass slides and then coating with gamma-aminopropyl silane (GAPS). The emitted fluorescence was captured on an imager with a time-gated intensified CCD detector. Microarrays of the neurotensin receptor 1, the cholinergic receptor muscarinic 2, the opioid receptor mu, and the cannabinoid receptor 1 were fabricated by pin printing. The selective agonism of each of the receptors was observed. The screening of potential antagonists was demonstrated using a cocktail of agonists. The amount of activation observed was sufficient to permit determinations of EC50 and IC50. Such microarrays could potentially streamline drug discovery by helping integrate primary screening with selectivity and safety screening without compromising the essential functional information obtainable from cellular assays.     

Membrane protein microarrays

This paper describes the fabrication of microarrays consisting of G protein-coupled receptors (GPCRs) on surfaces coated with gamma-aminopropylsilane (GAPS). Microspots of model membranes on GAPS-coated surfaces were observed to have several desired properties-high mechanical stability, long range lateral fluidity, and a thickness corresponding to a lipid bilayer in the bulk of the microspot. GPCR arrays were obtained by printing membrane preparations containing GPCRs using a quill-pin printer. To demonstrate specific binding of ligands, arrays presenting neurotensin (NTR1), adrenergic (beta1), and dopamine (D1) receptors were treated with fluorescently labeled neurotensin (BT-NT). Fluorescence images revealed binding only to microspots corresponding to the neurotensin receptor; this specificity was further demonstrated by the inhibition of binding in the presence of excess unlabeled neurotensin. The ability of GPCR arrays to enable selectivity studies between the different subtypes of a receptor was examined by printing arrays consisting of three subtypes of the adrenergic receptor: beta1, beta2, and alpha2A. When treated with fluorescently labeled CGP 12177, a cognate antagonist analogue specific to beta-adrenergic receptors, binding was only observed to microspots of the beta1 and beta2 receptors. Furthermore, binding of labeled CGP 12177 was inhibited when the arrays were incubated with solutions also containing ICI 118551, and in a manner consistent with the higher affinity of ICI 118551 for the beta2 receptor relative to that for the beta1 receptor. The ability to estimate binding affinities of compounds using GPCR arrays was examined using a competitive binding assay with BT-NT and unlabeled neurotensin on NTR1 arrays. The estimated IC(50) value (2 nM) for neurotensin is in agreement with the literature; this agreement suggests that the receptor -G protein complex is preserved in the microspot. This first ever demonstration of direct pin-printing of membrane proteins and ligand-binding assays thereof fills a significant void in protein microchip technology--the lack of practical microarray-based methods for membrane proteins.     

Rewritable DNA microarrays

Thiol-terminated single-stranded deoxyribonucleic acids (ssDNA) can be immobilized onto pulsed plasma deposited poly(allylmercaptan) surfaces via disulfide bridge chemistry and are found to readily undergo nucleic acid hybridization. Unlike other methods for oligonucleotide attachment to solid surfaces, this approach is shown to be independent of substrate material or geometry, and amenable to highly efficient rewriting.     

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.     

MALDI imaging mass spectrometry reveals multiple clinically relevant masses in colorectal cancer using large‐scale tissue microarrays

For identification of clinically relevant masses to predict status, grade, relapse and prognosis of colorectal cancer, we applied Matrix‐assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) to a tissue micro array containing formalin‐fixed and paraffin‐embedded tissue samples from 349 patients. Analysis of our MALDI‐IMS data revealed 27 different m/z signals associated with epithelial structures. Comparison of these signals showed significant association with status, grade and Ki‐67 labeling index. Fifteen out of 27 IMS signals revealed a significant association with survival. For seven signals (m/z 654, 776, 788, 904, 944, 975 and 1013) the absence and for eight signals (m/z 643, 678, 836, 886, 898, 1095, 1459 and 1477) the presence were associated with decreased life expectancy, including five masses (m/z 788, 836, 904, 944 and 1013) that provided prognostic information independently from the established prognosticators pT and pN. Combination of these five masses resulted in a three‐step classifier that provided prognostic information superior to univariate analysis. In addition, a total of 19 masses were associated with tumor stage, grade, metastasis and cell proliferation. Our data demonstrate the suitability of combining IMS and large‐scale tissue micro arrays to simultaneously identify and validate clinically useful molecular marker. Copyright © 2017 John Wiley & Sons, Ltd.     

Catching proteases in action with microarrays

Proteases regulate many essential functions in biology, yet their precise roles are only beginning to be unraveled. In this issue, two related papers describe a novel method to dissect specific protease activities from complex mixtures.     

PNA-encoded protease substrate microarrays

Our current understanding of the role and regulation of protease activity in normal and pathogenic processes is limited by our ability to measure and deconvolute their enzymatic activity. To address this limitation, an approach was developed that utilizes rhodamine-based fluorogenic substrates encoded with PNA tags. The PNA tags address each of the substrates to a predefined location on an oligonucleotide microarray through hybridization, thus allowing the deconvolution of multiple signals from a solution. A library of 192 protease substrates was prepared by split and mix combinatorial synthesis. The methodology and validation of this approach for profiling proteolytic activity from single proteases and from those in crude cell lysates as well as clinical blood samples is described.     

Small-molecule microarrays as tools in ligand discovery

Small molecules that bind and modulate specific protein targets are increasingly used as tools to decipher protein function in a cellular context. Identifying specific small-molecule probes for each protein in the proteome will require miniaturized assays that permit screening of large collections of compounds against large numbers of proteins in a highly parallel fashion. Simple and general binding assays involving small-molecule microarrays can be used to identify probes for nearly any protein in the proteome. The assay may be used to identify ligands for proteins in the absence of knowledge about structure or function. In this tutorial review, we introduce small-molecule microarrays (SMMs) as tools for ligand discovery; discuss methods for manufacturing SMMs, including both non-covalent and covalent attachment strategies; and provide examples of ligand discovery involving SMMs.     

Expression profiling in cancer using cDNA microarrays

Currently there are over 1,000,000 human expressed sequence tag (EST) sequences available on the public database, representing perhaps 50-90% of all human genes. The cDNA microarray technique is a recently developed tool that exploits this wealth of information for the analysis of gene expression. In this method, DNA probes representing cDNA clones are arrayed onto a glass slide and interrogated with fluorescently labeled cDNA targets. The power of the technology is the ability to perform a genome-wide expression profile of thousands of genes in one experiment. In our review we describe the principles of the microarray technology as applied to cancer research, summarize the literature on its use so far, and speculate on the future application of this powerful technique.     

Polymer microarrays for cellular adhesion

Microarray screening of polymer libraries for cellular adhesion was developed utilising a thin film of agarose to allow unsurpassed localisation of cell binding onto the array substrate and the discovery of cell specific polymers.     

Protein microarrays for diagnostic assays

Protein microarray technology has enormous potential for in vitro diagnostics (IVD). Miniaturized parallelized immunoassays are perfectly suited to generating a maximum of diagnostically relevant information from minute amounts of sample whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first products are already on the market. This article reviews the current state of protein microarrays and discusses developments and future demands relating to protein arrays in their role as multiplexed immunoassays in the field of diagnostics.

Surface characterization of carbohydrate microarrays

Carbohydrate microarrays are essential tools to determine the biological function of glycans. Here, we analyze a glycan array by time-of-flight secondary ion mass spectrometry (ToF-SIMS) to gain a better understanding of the physicochemical properties of the individual spots and to improve carbohydrate microarray quality. The carbohydrate microarray is prepared by piezo printing of thiol-terminated sugars onto a maleimide functionalized glass slide. The hyperspectral ToF-SIMS imaging data are analyzed by multivariate curve resolution (MCR) to discern secondary ions from regions of the array containing saccharide, linker, salts from the printing buffer, and the background linker chemistry. Analysis of secondary ions from the linker common to all of the sugar molecules employed reveals a relatively uniform distribution of the sugars within the spots formed from solutions with saccharide concentration of 0.4 mM and less, whereas a doughnut shape is often formed at higher-concentration solutions. A detailed analysis of individual spots reveals that in the larger spots the phosphate buffered saline (PBS) salts are heterogeneously distributed, apparently resulting in saccharide concentrated at the rim of the spots. A model of spot formation from the evaporating sessile drop is proposed to explain these observations. Saccharide spot diameters increase with saccharide concentration due to a reduction in surface tension of the saccharide solution compared to PBS. The multivariate analytical partial least squares (PLS) technique identifies ions from the sugars that in the complex ToF-SIMS spectra correlate with the binding of galectin proteins.     

Fluorous tagged small molecule microarrays

The affinity fluorous interaction between fluorous tagged small molecules and a fluoroalkyl modified glass surface was shown to facilitate the detection of protein-ligand binding interactions in the fabrication and screening of small molecule microarrays.     

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.     

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.