Developing ideal surface coatings for protein attachment

[Home | Technology ]

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. Protein microarrays or protein chips have been subjects of considerable excitement in the last few years. Although the field is still in its infancy, there have been an exceptionally large number of review articles and commentaries. On the other hand, successful experimental demonstrations of protein microarray technology are few and far in between. What is the reason for such a unique situation? Answer to this question can be found at the exceptional excitement in potential applications of protein microarray technology, as well as the exceptional difficulty in developing such a technology.
A well accepted argument is that the cutting edge of biology is increasingly found at the level of proteome simply because answers to most diseases lie in the proteins, not in the DNAs. 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. The interest and expectation in protein microarray technology also stems to a great extent from the hugely successful DNA microarray technology, as illustrated by the widespread use of cDNA and oligonucleotide arrays in research; the former is exemplified by the GeneChip platform of Affymetrix, Inc.
A wide range of applications have been envisioned and/or demonstrated for protein microarrays. One can divide these into three general categories: expression profiling, interaction profiling, and functional identification. The first application is most obvious. The concentration profile of proteins depends on age, physical/chemical environment, and more importantly, disease state. This profile cannot be obtained from mRNA levels alone because of the varying efficacies of translation and post-translational modification. Thus, knowing protein levels is the most direct way to phenotype cells and to diagnose disease state, stage, and response to treatment. This task is possible and has already been explored with antibody arrays. The second application is critical to drug discovery. Given the large number of proteins and the fact that their activities are often intimately related to mutual interactions, it is a daunting task to identify and understand the vast possibility of protein-protein interactions. One may envision the preparation of protein microarrays with the whole or subset of human proteome and use them for the large scale categorizing of protein-protein interactions, including the identification of specific domain-domain interactions. These microarrays can also be used in drug discovery since many drugs function by disrupting protein-protein interactions. The last application is most difficult but important for fundamental understanding. The functions of only a small population of proteins are known at the present time and the main goal of proteomics is to associate each protein with particular functions. A protein microarray may be used to screen for corresponding ligands. The reciprocal process is to use a small molecular array to screen for binding with proteins. In many ways, functional profiling overlaps with interaction profiling.
Despite all the potential and expectations, it is perhaps naïve to assume that the success story of DNA microarrays in genomics can be simply duplicated for protein microarrays. The availability of the PCR technique has made producing DNAs molecules a routine task. Synthesis of oligonucleotides is also automated and can be carried out on solid surfaces. However, techniques equivalent to PCR do not exist for proteins. Proteins are produced in small quantities either recombinantly in cells or in cell-free translation systems, neither of which is as simple as PCR. Producing and purifying antibodies from biological samples of animal models based on the natural immune systems is also a difficult and labor intensive process. Besides the production issue, proteins are much more difficult to handle than DNAs are. DNAs are relatively simple polyanions and can be chemically modified and easily immobilized on solid surfaces based on electrostatic interactions or covalent bonding through functional groups on either terminus. Protein molecules are much more complex. They possess delicate three-dimensional (3D) structures and varying chemical and physical properties (e.g., hydrophobic, hydrophilic, and ionic domains). Because the activity or function of a protein molecule is critically dependent on its 3D structure which is very sensitive to local physical and chemical environment, keeping an immobilized protein molecule in native state, with its 3D structure intact, and with its active domains accessible, is a major challenge. The tremendous variability in the nature of proteins and consequently in the requirement of their detection and identification also makes the development of protein chips a particularly challenging task. There are four major barriers in protein microarray development: 1) Background; 2) Protein native state and orientation; 3) Protein detection and identification; 4) Speed of protein or antibody production and purification.
MSI is developing technologies to overcome first two challenges which are manifested in the stringent demand on surface chemistry. There are two inherent difficulties associated with protein immobilization: i) Background. Proteins tend to adsorb nonspecifically to most solid surfaces. This is because a protein molecule has various hydrophobic or charged domains that can bind strongly with hydrophobic or oppositely charged surfaces. The hydrophobic interaction is particularly prevalent and is the dominant reason for fouling of a surface. The excessive interaction between a protein molecule and a solid surface often results in the disruption of its 3D structure and eventually denaturation, i.e., the complete loss of activity; ii) Conformation and orientation. A protein interacts with other molecules through specific functional domains. However, chemical forces responsible for adsorption on a solid surface are oblivious of the presence of any functional domain. If we let nature take its course, chances are we will not have protein molecules with the desired orientation on a solid surface. We must engineer specific chemical functionality to differentiate the domain responsible for immobilization from those of chemical/biological activity. Ideally, we would like the surface chemistry for protein microarrays to meet the following criteria:

The surface is inherently inert and resists non-specific adsorption;
The surface contains functional groups for the facile immobilization of protein molecules of interest;
Bonding between a protein molecule and a solid surface is strong enough to retain the molecule on the surface, but also sufficiently non intrusive to have minimal effect on the delicate 3D structure;
The linking chemistry allows the control of protein orientation and the local chemical environment favors the immobilized protein molecules to retain their native conformation;
The immobilization chemistry is highly specific and does not require pre-purification of protein samples.

To satisfy all above requirements, we have developed proprietary surface coating technology consisting of a high-density poly-ethylene-glycol (PEG) brush. It is intrinsically inert and offers exceptionally low background. This starting surface offers an ideal environment for biomolecular adsorption. The surface of the PEG brush is further functionalized to meet the particular need of biomolecular immobilization. The immobilized biomolecule (e.g., protein) has little interaction with the surface besides the linker, thus maintaining optimal activity in the solution environment for its interaction with target. This strategy has been successfully demonstrated for a number of applications by our customers, such as protein micrarrays, single molecule spectroscopy, biosensors, microfluidics, etc.

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 SBIR grants from NIH and NSF for product developments

Recent Publications

Y. Deng, X.-Y. Zhu, T. Kienlen, A. Guo “Transport at the air/water interface is the reason for rings in protein microarrays” J. Am. Chem. Soc. 128 (2006) 2768-2769. download a pdf file.

Guo, X.-Y. Zhu “The critical role of surface chemistry in protein microarrays” in Functional Protein Microarrays: Pathways to Discovery, edt. Paul Predki (CRC press, 2006). download a pdf file.

T.-W. Cha, A. Guo, and X.-Y. Zhu “Formation of supported phospholipid bilayers on molecular surfaces: role of surface charge density and electrostatic interaction” Biophys. J. 90 (2006) 1270-1274. download a pdf file. .

T.-W. Cha, A. Guo, X.-Y. Zhu “Enzymatic activity on a chip: the critical role of protein orientation” Proteomics, 5 (2005) 416-419. download a pdf file.

T. Cha, A. Guo, Y. Jun, D.-Q. Pei, X.-Y. Zhu, “Immobilization of oriented protein molecules on poly (ethylene glycol) coated Si(111)”, Proteomics, 4 (2004) 1965-1976. download a pdf file.

Low background aldehyde slides for protein microarray makers

MSI's Coating Technology for Microarrays

[Home | Technology ]

		MSI's Coating Technology for Microarrays [Home \| Technology ] 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. Protein microarrays or protein chips have been subjects of considerable excitement in the last few years. Although the field is still in its infancy, there have been an exceptionally large number of review articles and commentaries. On the other hand, successful experimental demonstrations of protein microarray technology are few and far in between. What is the reason for such a unique situation? Answer to this question can be found at the exceptional excitement in potential applications of protein microarray technology, as well as the exceptional difficulty in developing such a technology. A well accepted argument is that the cutting edge of biology is increasingly found at the level of proteome simply because answers to most diseases lie in the proteins, not in the DNAs. 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. The interest and expectation in protein microarray technology also stems to a great extent from the hugely successful DNA microarray technology, as illustrated by the widespread use of cDNA and oligonucleotide arrays in research; the former is exemplified by the GeneChip platform of Affymetrix, Inc. A wide range of applications have been envisioned and/or demonstrated for protein microarrays. One can divide these into three general categories: expression profiling, interaction profiling, and functional identification. The first application is most obvious. The concentration profile of proteins depends on age, physical/chemical environment, and more importantly, disease state. This profile cannot be obtained from mRNA levels alone because of the varying efficacies of translation and post-translational modification. Thus, knowing protein levels is the most direct way to phenotype cells and to diagnose disease state, stage, and response to treatment. This task is possible and has already been explored with antibody arrays. The second application is critical to drug discovery. Given the large number of proteins and the fact that their activities are often intimately related to mutual interactions, it is a daunting task to identify and understand the vast possibility of protein-protein interactions. One may envision the preparation of protein microarrays with the whole or subset of human proteome and use them for the large scale categorizing of protein-protein interactions, including the identification of specific domain-domain interactions. These microarrays can also be used in drug discovery since many drugs function by disrupting protein-protein interactions. The last application is most difficult but important for fundamental understanding. The functions of only a small population of proteins are known at the present time and the main goal of proteomics is to associate each protein with particular functions. A protein microarray may be used to screen for corresponding ligands. The reciprocal process is to use a small molecular array to screen for binding with proteins. In many ways, functional profiling overlaps with interaction profiling. Despite all the potential and expectations, it is perhaps naïve to assume that the success story of DNA microarrays in genomics can be simply duplicated for protein microarrays. The availability of the PCR technique has made producing DNAs molecules a routine task. Synthesis of oligonucleotides is also automated and can be carried out on solid surfaces. However, techniques equivalent to PCR do not exist for proteins. Proteins are produced in small quantities either recombinantly in cells or in cell-free translation systems, neither of which is as simple as PCR. Producing and purifying antibodies from biological samples of animal models based on the natural immune systems is also a difficult and labor intensive process. Besides the production issue, proteins are much more difficult to handle than DNAs are. DNAs are relatively simple polyanions and can be chemically modified and easily immobilized on solid surfaces based on electrostatic interactions or covalent bonding through functional groups on either terminus. Protein molecules are much more complex. They possess delicate three-dimensional (3D) structures and varying chemical and physical properties (e.g., hydrophobic, hydrophilic, and ionic domains). Because the activity or function of a protein molecule is critically dependent on its 3D structure which is very sensitive to local physical and chemical environment, keeping an immobilized protein molecule in native state, with its 3D structure intact, and with its active domains accessible, is a major challenge. The tremendous variability in the nature of proteins and consequently in the requirement of their detection and identification also makes the development of protein chips a particularly challenging task. There are four major barriers in protein microarray development: 1) Background; 2) Protein native state and orientation; 3) Protein detection and identification; 4) Speed of protein or antibody production and purification. MSI is developing technologies to overcome first two challenges which are manifested in the stringent demand on surface chemistry. There are two inherent difficulties associated with protein immobilization: i) Background. Proteins tend to adsorb nonspecifically to most solid surfaces. This is because a protein molecule has various hydrophobic or charged domains that can bind strongly with hydrophobic or oppositely charged surfaces. The hydrophobic interaction is particularly prevalent and is the dominant reason for fouling of a surface. The excessive interaction between a protein molecule and a solid surface often results in the disruption of its 3D structure and eventually denaturation, i.e., the complete loss of activity; ii) Conformation and orientation. A protein interacts with other molecules through specific functional domains. However, chemical forces responsible for adsorption on a solid surface are oblivious of the presence of any functional domain. If we let nature take its course, chances are we will not have protein molecules with the desired orientation on a solid surface. We must engineer specific chemical functionality to differentiate the domain responsible for immobilization from those of chemical/biological activity. Ideally, we would like the surface chemistry for protein microarrays to meet the following criteria: The surface is inherently inert and resists non-specific adsorption; The surface contains functional groups for the facile immobilization of protein molecules of interest; Bonding between a protein molecule and a solid surface is strong enough to retain the molecule on the surface, but also sufficiently non intrusive to have minimal effect on the delicate 3D structure; The linking chemistry allows the control of protein orientation and the local chemical environment favors the immobilized protein molecules to retain their native conformation; The immobilization chemistry is highly specific and does not require pre-purification of protein samples. To satisfy all above requirements, we have developed proprietary surface coating technology consisting of a high-density poly-ethylene-glycol (PEG) brush. It is intrinsically inert and offers exceptionally low background. This starting surface offers an ideal environment for biomolecular adsorption. The surface of the PEG brush is further functionalized to meet the particular need of biomolecular immobilization. The immobilized biomolecule (e.g., protein) has little interaction with the surface besides the linker, thus maintaining optimal activity in the solution environment for its interaction with target. This strategy has been successfully demonstrated for a number of applications by our customers, such as protein micrarrays, single molecule spectroscopy, biosensors, microfluidics, etc. 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 SBIR grants from NIH and NSF for product developments Recent Publications Y. Deng, X.-Y. Zhu, T. Kienlen, A. Guo “Transport at the air/water interface is the reason for rings in protein microarrays” J. Am. Chem. Soc. 128 (2006) 2768-2769. download a pdf file. Guo, X.-Y. Zhu “The critical role of surface chemistry in protein microarrays” in Functional Protein Microarrays: Pathways to Discovery, edt. Paul Predki (CRC press, 2006). download a pdf file. T.-W. Cha, A. Guo, and X.-Y. Zhu “Formation of supported phospholipid bilayers on molecular surfaces: role of surface charge density and electrostatic interaction” Biophys. J. 90 (2006) 1270-1274. download a pdf file. . T.-W. Cha, A. Guo, X.-Y. Zhu “Enzymatic activity on a chip: the critical role of protein orientation” Proteomics, 5 (2005) 416-419. download a pdf file. T. Cha, A. Guo, Y. Jun, D.-Q. Pei, X.-Y. Zhu, “Immobilization of oriented protein molecules on poly (ethylene glycol) coated Si(111)”, Proteomics, 4 (2004) 1965-1976. download a pdf file.

	MicroSurfaces, Inc. 4001 Stinson Blvd., Suite 430 Minneapolis, MN 55421 Tel: 612-789-0104; Fax: 612-788-4543; e-mail: Copy Right © 2007 Athena Guo. All rights reserved.