Juan J. Diaz-Mochon

Microarray platforms for enzymatic and cell-based assays

This tutorial review introduces the uninitiated to the world of microarrays (or so-called chips) and covers a number of basic concepts such as substrates and surfaces, printing and analysis. It then moves on to look at some newer applications of microarray technology, which include enzyme analysis (notably kinases and proteases) as well as the growing enchantment with so-called cell-based microarrays that offer a unique approach to high-throughput cellular analysis. Finally, it looks forwards and highlights future possible trends and directions in the microarray arena.

Inkjet fabrication of hydrogel microarrays using in situ nanolitre-scale polymerisation

Polymer hydrogel microarrays were fabricated by inkjet printing of monomers and initiator, allowing up to 1800 individual polymer features to be printed on a single glass slide.

Decoding a PNA Encoded Peptide Library by PCR: The Discovery of New Cell Surface Receptor Ligands

The ability to screen and identify new ligands for cell surface receptors has been a long-standing goal as it might allow targeting of pharmaceutically relevant receptors, such as integrins or G protein coupled receptors. Here, we present a method to amplify hits from a library of PNA-tagged peptides. To this end, human cells, overexpressing either integrins or the CCR6 receptor, were treated with a 10,000 member PNA-encoded peptide library. Extraction of the PNA tags from the surface of the cells was followed by a PNA-tag to DNA translation and amplification enabling decoding of the tags via microarray hybridization. This approach to ligand discovery facilitates screening for differences in surface-receptor ligands and/or receptor expression between different cell types, and opens up a practical approach to PNA-tag amplification.

Parallel and multiplexed bead-based assays and encoding strategies.

Advances in high throughput screening (HTS), together with the rapid progress in combinatorial chemistry, genomic and proteomic sciences have dramatically stimulated the development of a variety tools to enable the drug discovery process to become more efficient. Major future challenges in HTS include obtaining high density and good quality data based on assays that are rapid, reliable, inexpensive, sensitive, simple and miniaturised. This paper reviews the development and role of bead-based assays for HTS including DNA and single nucleotide polymorphism (SNP) assays, particularly from a multiplex perspective and evaluating the recent advances in bead-based arrays. The encoding strategies that are commonly used in bead-based assays are highlighted, while the importance of magnetic beads in genomic and proteomic purifications is discussed. In conclusion, bead-based assays offer a powerful promising approach for many aspects of drug discovery.

Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast

In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors.

Combining nebulization-mediated transfection and polymer microarrays for the rapid determination of optimal transfection substrates.

In this manuscript, we report how transfection efficiencies vary as a function of the substrate upon which cells adhere using a polymer microarray platform to allow rapid analysis of a large number of substrates. During these studies, traditional transfection protocols were nonsatisfactory, and thus we developed an approach in which an ultrasonic nebulizer was used to dispense lipoplexes onto cell-based microarrays in the absence of liquid. Under these conditions, droplets were directly deposited onto the cells thereby enhancing transfection. This approach was successfully applied to the transfection of various cell lines immobilized on a library of polyacrylates and permitted the identification of highly efficient transfection/polymer combinations, while showing that specific polymer-cell interactions may promote the efficacy of chemical transfection.

Novel Biopolymers to Enhance Endothelialisation of Intra-vascular Devices

Rapid endothelisation is of critical importance in the prevention of adverse remodelling after device implantation. Currently, there is a need for alternative strategies to promote re-endothelialisation for intravascular stents and vascular grafts. Using polymer microarray technology 345 polymers are comprehensively assessed and a matrix is identified that specifically supports both progenitor and mature endothelial cell activity in vitro and in vivo while minimising platelet attachment.

Novel Biochip Platform for Nucleic Acid Analysis

This manuscript describes the use of a novel biochip platform for the rapid analysis/identification of nucleic acids, including DNA and microRNAs, with very high specificity. This approach combines a unique dynamic chemistry approach for nucleic acid testing and analysis developed by DestiNA Genomics with the STMicroelectronics In-Check platform, which comprises two microfluidic optimized and independent PCR reaction chambers, and a sequential microarray area for nucleic acid capture and identification by fluorescence. With its compact bench-top “footprint” requiring only a single technician to operate, the biochip system promises to transform and expand routine clinical diagnostic testing and screening for genetic diseases, cancers, drug toxicology and heart disease, as well as employment in the emerging companion diagnostics market.

The use of solid supports to generate nucleic acid carriers.

Nucleic acids are the foundation stone of all cellular processes. Consequently, the use of DNA or RNA to treat genetic and acquired disorders (so called gene therapy) offers enormous potential benefits. The restitution of defective genes or the suppression of malignant genes could target a range of diseases, including cancers, inherited diseases (cystic fibrosis, muscular dystrophy, etc.), and viral infections. However, this strategy has a major barrier: the size and charge of nucleic acids largely restricts their transit into eukaryotic cells. Potential strategies to solve this problem include the use of a variety of natural and synthetic nucleic acid carriers. Driven by the aim and ambition of translating this promising therapeutic approach into the clinic, researchers have been actively developing advanced delivery systems for nucleic acids for more than 20 years. A decade ago we began our investigations of solid-phase techniques to construct families of novel nucleic acid carriers for transfection. We envisaged that the solid-phase synthesis of polycationic dendrimers and derivatized polyamimes would offer distinct advantages over solution phase techniques. Notably in solid phase synthesis we could take advantage of mass action and streamlined purification procedures, while simplifying the handling of compounds with high polarities and plurality of functional groups. Parallel synthesis methods would also allow rapid access to libraries of compounds with improved purities and yields over comparable solution methodologies and facilitate the development of structure activity relationships. We also twisted the concept of the solid-phase support on its head: we devised miniaturized solid supports that provided an innovative cell delivery vehicle in their own right, carrying covalently conjugated cargos (biomolecules) into cells. In this Account, we summarize the main outcomes of this series of chemically related projects.

Encoded peptide libraries and the discovery of new cell binding ligands

Cells over-expressing integrins or CCR6 were incubated on a DNA microarray, pre-hybridized with a 10,000 member PNA-encoded peptide library allowing novel cell specific ligands for integrins and CCR6 to be identified.