Developing ideal surface coatings for protein microarrays

Low background aldehyde slides for protein microarray makers

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Advanced Surface Coating Technologies for Microarrays & Microfluidics

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MSI’s Coating Technology for Microarrays

 

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The conventional approach to drug discovery and development is a time-consuming, labor intensive, and hit-or-miss process. Microarrays promise to revolutionize disease diagnosis and drug discovery. With great advances in genomics, such as the completion of human genome sequencing, the next grand challenge becomes apparent: understanding biological functions of proteins encoded by genes. Proteins are the primary structural, functional and signaling elements in the human body, thus, a comprehensive analysis of proteins is required to obtain a complete picture of normal and disease processes in the body. Using the microarray technology, thousands of proteins or antibodies could be studied in parallel to establish their biochemical properties and biological activities. Such a high throughput analysis of protein function is essential to the pharmaceutical industry and human health because most drugs we use today are either proteins or alter the functions of proteins. Specific examples may include protein microarrays for mechanistic studies of drug action, monitoring antibodies contained in serum such as in the diagnostics of auto-immune diseases, and recombinant antibody library screening, etc.

Despite its importance, the protein microarray market is just starting, i.e., at the same point what the DNA array market was five years ago. The development of protein array technology is hindered by the complexity of protein molecules. There are millions of different proteins (much greater than the number of genes) in a human body, most of them have yet to be identified and characterized. The tremendous variability in the nature of proteins and consequently in the requirement of their detection and identification makes the development of protein chips a particularly challenging task. Additionally, protein molecules must be immobilized on a matrix in a way that preserve their native structures and are accessible to their targets. This is not easy! Unlike DNA, a protein molecule has complex three dimensional structure. The immobilization chemistry must be compatible with preserving protein molecules in native states. This requires good control of local molecular environments for the immobilized protein molecule. There are four major barriers in protein microarray development: 1) Background. Proteins tend to adsorb nonspecifically to solid substrates, leading to background problems (less sensitivity and low signal to noise ratio). 2) Protein native state and orientation. Because proteins have complex structures and activities, the immobilization chemistry has to be such that it preserves a protein in native state and with optimal orientation for protein ­ target interaction; 3) Protein detection and identification. Because different proteins take up fluorescent tags to different extents, labeling all proteins in a sample with a tag, as with mRNAs detected by conventional DNA microarrays, is not a viable option. Other methods, such as FLAME, SPR, etc., are currently under development; 4) Speed of protein or antibody production and purification. The conventional method of producing proteins and antibodies is too labor intensive and time consuming. Several companies are exploring ways of making thousands of antibodies or proteins available for arraying.

MSI is developing technologies to overcome the first two barriers in protein microarray development. We have developed novel surface technologies for the immobilization of proteins in a microarray that possess the following attributes: (1) the surface chemistry assures negligible background; (2) the orientation of proteins immobilized on the surface is uniform and controllable; (3) the immobilized proteins are in their native states and easily accessible by proteins or other molecular targets in the solution;. (4) the linking chemistry is highly selective and facile; Essentially, our proprietary coating technologies allow us to covalently attach a monolayer of molecules on a solid surface to create functional glass slides. Depending on the application, the functional groups on a slide can be -CHO, epoxy, -NH2, -SH etc. For example, figure 1 compares the exceptionally low background of MSI’s coated slide with products from a major competitor. We arrayed various concentrations of membrane-type matrix metalloproteinase (MT-MMP5, panel B) and antibody raised against MT-MMP5 (panel A), respectively ( for other low background functional slides, see products for detail). Figure 1 clearly indicates that functional glass slides made with MSI’s coating technology yielded much lower background and produced sharper images. In addition, spot diffusion is minimal with MSI’s slides.

We have also developed glass or silicon surfaces coated with high density brushes of poly-ethylene-glycol (PEG). The PEG brush is intrinsically inert towards the adsorption of proteins, peptides, cells, and other biomolecules, thus providing a zero background starting surface in a variety of biomedical experiments. Regions of PEG molecules may be selectively removed by a variety of lithographic technical to prepare micro- or nano-patterned surfaces. On the other hand, standard bioconjugation chemistry may be used to covalently link biomolecules to -OH groups on the otherwise zero background PEG brush. In addition, we have developed surface immobilization technology to preserve protein native state and to provide optimal orientation for protein-target interaction. Figure 2 shows green fluorescent protein (GFP) immobilized on a chip. The protein only fluoresces when it is in its native state. More importantly, GFP was immobilized from a crude preparation expressed by bacteria cells without pre-purification. This result demonstrates the exceptionally high selectivity of MSI’s immobilization chemistry.

Based on a report from BioInsights of Redwood, Calif., the current protein chip market is $45 millions, and will reach $500 millions by 2006. Major players in the protein chip market include, among others, Biacore, Ciphergen, Zyomyx and Phylos. Most of these companies are developing prefabricated protein chips, each in a special format and requires the use of special fluidic devices and scanners. This kind of prefabricated, special protein chips account for ~1/3 of the market. Customers who need to make specific microarrays on site using standard arrayers and scanners already in place for DNA chip research require blank slides in standard formats. MicroSurfaces, Inc. targets this larger sector of the market and supplies customers with functional glass slides and associated surface technology for on-site protein microarray fabrication. MSI’s glass slide is more advantageous over current glass slides on the market. This advantage is reflected in its exceptionally low background, high uniformity, and high chemical reactivity. MSI has also received an SBIR grant from NIH for further development.


low backgroundFigure 1. Fluorescence microscopy images of protein arrays printed on MSI’s low background aldehyde slides (right in each panel) and on commercially available slides from a leading competitor (left in each panel). Various concentrations of MT-MMP5 (Panel B) or antibody raised against MT-MMP5 (Panel A) were arrayed onto aldehyde glass slides with a BioRobotic arrayer. Protein or primary antibody spots were detected using Cy3-conjugated secondary antibody.

 
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Oriented protein arrayFigure 2. Green fluorescence protein on MSI’s functional slide.
GFP was expressed in bacteria cells and an aliquote of crude preparation was arrayed on MSI’s coated slide. A slide without proper functionality was used as a control to show the specificity of the immobilization chemistry.