Research Tools : Arrays


Hydrogel Arrays for Screening Cell-Substrate Interactions, Now in Multiwell Format

Building on their previous work, the researchers have now adapted their method to any commercially available, glass or polystyrene-bottom multiwell plate. In the new process, hydrogel is covalently immobilized to the bottom of each well and then selectively polymerized. In this way the spots are completely isolatable, allowing for systemic and independent control of their chemical composition and XYZ physical dimensions.

Once the hydrogel array is formed, each of the spots can be exposed to different soluble factors without risk of diffusion.

Controlling the Formation of Stem Cell Colonies with Tailored SAM Array

Building on their previous work, the researchers have developed a new feature to make SAM arrays an even better tool to control cell aggregation. Specifically, the spots on the array consist of cellular adhesive peptides stuck to the surface by an easy-to-cleave labile bond. The peptides enable layers of cell to form and detach from the array without scraping or other external manipulation.

Any peptide capable of forming such a bond (e.g., a thioester bond) with the SAM surface could be employed.

Hydrogel Arrays for Screening Cell-Substrate Interactions

UW–Madison researchers developed a new method for forming patterned hydrogel arrays featuring any number of test spots possessing different characteristics, such as shape and chemical composition. The arrays can be used to culture a range of cell types and rapidly analyze their behavior (e.g., attachment, spreading, proliferation and differentiation).

The arrays are prepared using a hydrogel precursor solution containing a polymer and crosslinker. The solution is sandwiched between stenciled SAM layers containing hydrophilic (‘water-loving’) and hydrophobic (‘water-hating’) regions, then polymerized and released.

As a result of the process, the array features hydrophilic spots surrounded and isolated by hydrophobic regions, preventing any mixing of contents. The spots can have any desired shape, size and chemical composition.

Controlling Size and Shape of Stem Cell Colonies with SAM Array

UW–Madison researchers have developed a method for generating colonies of stem cells in controlled shapes and sizes.

The method uses self-assembled monolayer (SAM) arrays, which are metal-coated slides patterned with small adherent spots. These tools enable researchers to systematically expose cells to various surface-bound molecules — such as proteins, nucleic acids and polysaccharides — and study how they interact.

The SAM spots can have specified diameters and shapes (e.g., circle, oval or star), and the cells that come in contact with them will adhere accordingly. The cells can be cultured for a sufficient time to form a layer that undergoes a morphogenesis process and then detaches so it can be collected for further analysis.

Assembly of Full-Length Genes from DNA Arrays

UW–Madison researchers have developed a method to produce full-length genes using RNA intermediaries that are produced by run-off transcription from DNA array features. Specifically, an RNA polymerase promoter is appended to the surface-bound oligonucleotides. RNA copies are produced using T7 RNA polymerase and then self-assembled into full RNA transcripts by hybridization and ligation. The RNA transcripts can readily be converted into their corresponding genes using RT-PCR (reverse transcription polymerase chain reaction). These genes then may be employed to express the encoded protein of interest.

Investigating Cell-Surface Interactions via Chemical Array

UW–Madison researchers have developed an improved approach to SAM-based experiments using simple methods to construct arrays of SAMs that can be used to rapidly investigate how selected types and densities of ligands impact cell behaviors such as adhesion, spreading, proliferation, migration and differentiation.

The substrate comprises a slide coated with metal such as gold onto which a polymer stencil is fitted. Through small holes in the stencil, alkanethiol solutions are deposited, forming micrometer-sized spots (up to 120 or more) of SAM. During this deposition process, a range of biological molecules can be tethered to each SAM spot (at varied densities and mixtures), allowing researchers to examine a wide range of possible biological ligands on a single surface. Furthermore, arrays formed using alkanethiolates that prevent biofouling allow researchers to carefully examine the effects of a particular ligand on the behavior of cells cultured on each array spot. Importantly, array fabrication and subsequent examination of cell behavior can be carried out using tools that are standard in biological laboratories such as pipettes and light/fluorescent microscopes.

Using UV Light to Functionalize Metal Oxides for Use in Biosensors, Solar Cells and Other Devices

UW-Madison researchers have developed methods for functionalizing metal oxides, including TiO2, with organic molecules.  The methods use UV light to covalently bond linker precursors to the surface of a metal oxide.  Then other molecules, such as dye molecules or biomolecules like DNA, can be coupled to the linker precursors to further functionalize the metal oxides. 

The functionalized oxides may be used alone or as coatings on various substrates.  They can be incorporated into devices such as biosensors or dye-sensitized solar cells. 

These methods provide functionalized metal oxides with higher densities of organic molecules and greater thermal and chemical stability than functionalized metal oxides prepared using conventional methods.  In addition, these methods do not require high temperatures or an ultra high vacuum, and are simpler and more reproducible than conventional methods.

Synthesis of Low-Cost, High-Density DNA Microarrays

UW-Madison researchers have developed a system and method for synthesizing DNA microarrays using a device that includes a reduction optics assembly and a target assembly. These new components incorporate image reduction and precision stage motion into the synthesis process, increasing the density of the DNA chip to 25 times the density of a traditional microarray while maintaining the cost per feature. As a result, the system offers a significant reduction in the cost of DNA microarrays by increasing the amount of information contained within the microarray while keeping the consumables necessary for the process constant when compared to similar technologies.

Light-Directed DNA Synthesis Using Inverse Capping for Error Reduction

UW-Madison researchers have developed a method of reducing errors in light-directed DNA synthesis by using inverted masks to disable sites where synthesis is not intended to occur. Before light-directed DNA synthesis occurs on a prepared microarray substrate, the substrate is exposed to light via an inverse mask pattern to deprotect inactive regions of the substrate where synthesis is not desired. The deprotected sites are then capped to permanently disable the inactive areas. This inverse capping prevents unwanted DNA synthesis in the inactive areas, resulting in “purer” DNA, even though such areas may be unintentionally exposed to light during DNA synthesis on the active areas of the substrate.

Protein-Acrylamide Copolymer Hydrogels for Measuring Protein Concentration and Activity

UW-Madison researchers have developed a method of specifically attaching proteins to glass surfaces via copolymerization with a polyacrylamide hydrogel. They also have developed techniques for using the arrays formed using this process to detect proteins and measure their concentrations, binding affinities and kinetics.

Their method uses a surface functionalized with an acrylic acid- or acrylamide-based hydrogel. Proteins are labeled with an acrylic moiety and then attached to the functionalized surface through copolymerization with the hydrogel. The attached protein is then available for use in a variety of assays.

Image Locking System for DNA Microarray Synthesis

UW–Madison researchers have developed an image locking system for DNA microarray synthesis that will stabilize or “lock” the image with respect to an image capture device such as a camera or microscope. The system includes the use of detection or reference marks. When a shift in image position is detected, a correction signal is sent to one of two mirrors, moving the image to correct for the change in image position.

Plasma-Enhanced Functionalization of Inorganic Oxide Surfaces

UW-Madison researchers have developed an efficient, two-step technique for covalently attaching epoxide functionalities to glass, silicon, quartz, and other inorganic surfaces. First, an oxide surface is exposed to a cold plasma to create hydroxyl functionalities on the surface. Next, these hydroxyl groups are reacted with epoxy group-containing molecules in the absence of plasma to form surface-bound spacer chains. Biomolecules can then be immobilized on the resulting functionalized surface by reacting the biomolecules with the spacer chains.

Plasma-Enhanced Functionalization of Carbon-Containing Substrates

UW-Madison researchers have developed an efficient, dry plasma technique for functionalizing surfaces that holds several advantages over traditional wet chemical approaches. The technique involves two steps. First, a carbon containing substrate is exposed to an inert plasma (e.g., argon or hydrogen) that generates reactive active sites, such as free radicals or ions, on the surface of the substrate. Next, the surface is exposed to volatile compounds in the absence of plasma. The compounds react with the active sites to produce surface-bound spacer chain molecules containing one or more functional groups. These functional groups, in turn, can react with molecules of DNA or protein.

Methods and Devices for Precisely Dispensing Microvolumes of Fluids

UW-Madison researchers have developed a microplotter device that can deposit spots or lines on the order of 5 micrometers in size for several applications, including biological microarrays and polymer-based circuits. The device consists of a nozzle for depositing fluid, which is connected to a positioning system that is controlled by customized software on a desktop computer. The nozzle, composed of a micropipette fastened to a piece of piezoelectric, deposits small features through ultrasonics.

Fusion Protein Arrays on Metal Substrates for Surface Plasmon Resonance Imaging

UW-Madison researchers have developed surface plasmon resonance-capable arrays in which molecules, such as proteins and nucleic acids, or whole cells, are adhered to a metal substrate. Proteins are immobilized on a chemically modified gold surface in an array format by exploiting an interaction between the enzyme glutathione S-transferase (GST) and the tri-peptide glutathione (GSH). First, a GSH array is created on a chemically modified gold surface. Next, GSH-GST binding is used to immobilize GST fusion proteins onto the gold surface. The GST portion of the fusion protein acts as an anchor to the surface, while the remainder of the protein is accessible for interaction studies with peptides, nucleotides, small molecules or other proteins in solution.

Modified Carbon, Silicon & Germanium Surfaces for Biomolecular Arrays

UW-Madison researchers have developed a method for creating modified carbon, silicon and germanium surfaces. The method starts with an alkene molecule that has been modified to contain a protected, reactive moiety, preferably at a terminal carbon. The modified and protected alkene is reacted with an unoxidized carbon, silicon or germanium substrate to yield an array of alkane molecules covalently bonded to the substrate. Preferably, this reaction is photo-initiated by contacting the alkene with the substrate and exposing it to a suitable wavelength of UV light. The points of attachment can be photopatterned by controlling where the light falls upon the substrate/alkene reactants. Once the alkane molecules are deprotected, biomolecules such as nucleic acids or proteins can be attached directly to the reactive moiety on the alkane to yield a surface of biomolecules immobilized on the substrate. Alternatively, a crosslinker can be interposed between the biomolecule and alkane.

Process for Intercalation of Spacer Molecules between Substrates and Active Biomolecules

UW–Madison researchers have developed a method of functionalizing the surface of a wide variety of substrates so that spacer molecules are attached. The substrates are exposed to a cold plasma ignited in dichlorosilane, silicon tetrachloride or hexachlorodisilane gas to implant silicon-chlorine functionalities in the substrate surface. The plasma implanted surface functionalities then are utilized to initiate second stage gas phase derivatization reactions to form linker molecules attached to the substrate. Active biomolecules such as enzymes are bound to the exposed linker molecules to bind the bioactive molecules to the substrate while allowing freedom of movement and conformation of the bound molecule comparable to that of the free molecule.

Flow Cell Synthesis of DNA Probes

UW-Madison researchers have developed an improved flow cell for use in the synthesis of arrays of DNA probe sequences and polypeptides. The flow cell precisely aligns a substrate with respect to an image former, while distributing a reagent-containing fluid through the active volume and over the active exposed surface of the flow cell.

The flow cell includes a base with a central window opening and a registration surface against which the substrate may be mounted. A press block is engaged against a gasket mounted on the active surface of the substrate to fully enclose an active volume between the press block, the peripheral walls of the gasket’s central opening and the active surface of the substrate. Channels in the press block extend to the extension openings in the gasket, allowing the flow of reagents into and out of the active volume.