Substrate containing at least one polymer having at least one reactive ion etched surface upon which at least one specific binding agent is immobilized; for assaying or isolating analytes in a sample
Inventors
Rucker, Victor C. • SHEDIAC RENEE, null • Shediac, Renée • Simmons, Blake A. • Havenstrite, Karen L.
Assignees
National Technology and Engineering Solutions of Sandia LLC • Sandia National Laboratories
Publication Number
US-7253008-B2
Publication Date
2007-08-07
Expiration Date
2024-12-28
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Abstract
Disclosed herein are substrates comprising reactive ion etched surfaces and specific binding agents immobilized thereon. The substrates may be used in methods and devices for assaying or isolating analytes in a sample. Also disclosed are methods of making the reactive ion etched surfaces.
Core Innovation
The invention relates to substrates comprising reactive ion etched polymer surfaces, on which specific binding agents are immobilized. These substrates are designed for use in chemical and biological assays, including microarray and nanoarray assays, to assay or isolate analytes in a sample. The reactive ion etched surfaces exhibit altered physical and chemical properties that differ from unetched surfaces, enabling selective immobilization of specific binding agents in desired patterns.
The problem addressed is the challenge in optimizing surface chemistry to enable immobilization of detection antibodies while retaining their bioactivity and minimizing non-specific protein adsorption, which compromises assay sensitivity. Existing glass-based microarrays are limited by non-specific background binding and insufficient sensitivity. Polymers are attractive alternatives due to their desirable bulk physical and chemical properties and cost-effectiveness, but their inherently low surface energy requires modification for applications. There is a need for polymer substrates suitable for patterned microarrays enabling high-throughput assays with increased signal-to-noise ratios and minimal background.
The invention solves this by providing substrates containing at least one polymer having reactive ion etched surfaces, patterned or unetched, with specific binding agents immobilized directly thereon, without requiring blocking agents. The reactive ion etched surfaces demonstrate different affinities for specific binding agents compared to unetched surfaces, depending on the polymer type, allowing for spatially complex arrays of immobilized agents. Methods for fabricating such substrates and using them in assay devices, including for assaying or isolating ligands specifically binding the immobilized agents, are provided.
Claims Coverage
The claims disclose a substrate and related assay devices, methods, and kits with inventive features centered on reactive ion etched polymer surfaces and specific binding agents immobilized directly thereon.
Substrate with reactive ion etched polymethylmethacrylate or cyclo-olefin surfaces having polypeptides bound directly thereon
A substrate comprising at least one reactive ion etched polymethylmethacrylate (PMMA) or cyclo-olefin surface with at least one polypeptide bound directly on that surface, where binding is direct and occurs without any linker, and the polypeptide specifically binds a ligand of interest in a sample.
Presence of unetched polymer surfaces lacking binding and second polymer surfaces with selective binding characteristics
The substrate also contains at least one unetched PMMA or cyclo-olefin surface that fails to bind the polypeptide directly when contacted, and at least one reactive ion etched or unetched surface of a second polymer (e.g., polycarbonate) where the reactive ion etched surface of the second polymer fails to bind the polypeptide directly when contacted.
Patterned arrangements of polymers and surfaces for spatial localization of polypeptides
The reactive ion etched PMMA or cyclo-olefin surfaces and unetched surfaces may be patterned on the substrate, and the first and second polymers may be arranged in a pattern, enabling spatially selective immobilization of polypeptides.
Polypeptides as antigens or antibodies bound without blocking buffers
The polypeptides bound directly on the reactive ion etched PMMA or cyclo-olefin surfaces can be antigens or antibodies, and such binding occurs without the need for non-specific blocking buffers.
Assay device and methods using the substrate to detect or isolate ligands
An assay device comprising the substrate operably attached to a housing; a method of assaying or isolating a ligand in a sample by contacting the substrate with the sample; and a kit comprising the substrate packaged with reagents for assaying or isolating the ligand.
The claims center on substrates with reactive ion etched PMMA or cyclo-olefin surfaces having specific polypeptides immobilized directly thereon, complementary unetched surfaces and second polymer surfaces with selective binding behavior, patterning of these surfaces and polymers, and related assay devices, methods, and kits enabling selective binding and detection of ligands in samples without blocking agents.
Stated Advantages
Minimization of non-specific protein adsorption leading to improved assay sensitivity and enhanced signal-to-noise ratios.
Capability to selectively immobilize specific binding agents on reactive ion etched polymer surfaces without requiring blocking agents or buffers.
Generation of spatially complex, patterned arrays with selective binding characteristics for multiplexed assays.
Use of inexpensive and easily processed polymer substrates with modified surface chemistry to improve robustness in diagnostic protein microarrays and other assays.
Flexibility to use a variety of polymers and patterns to tailor binding affinities and assay designs.
Documented Applications
Use in biological and chemical assays such as microarrays and nanoarrays for qualitative and quantitative determination of analytes including antibodies, antigens, toxins, and nucleic acids.
Medical diagnostics including assays for cancer, abnormal biomolecules, specific nucleic acid sequences, and detection of pathogens and toxins in samples.
Environmental applications for assaying insecticides, pesticides, herbicides, toxins, and bacteria in food supplies or environmental media like water and air.
Detection of chemical and biological warfare agents including bacteria (Bacillus, Mycobacterium, etc.), viruses (Ebola, smallpox, etc.), toxins (ricin, neurotoxins, etc.), and chemical agents (sarin, mustard gas, etc.).
Separation technologies including affinity chromatography, field-flow-fractionation, and thin layer chromatography for isolating or purifying target analytes from mixtures.
Affinity purification applications in biological manufacturing to enrich specific biomolecules such as monoclonal or polyclonal antibodies using immobilized capture reagents on reactive ion etched surfaces.
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