Processing microtitre plates for covalent immobilization chemistries
Inventors
North, Stella H. • Lock, Evgeniya H. • Walton, Scott G. • Taitt, Chris Rowe
Assignees
Publication Number
US-9962676-B2
Publication Date
2018-05-08
Expiration Date
2030-11-17
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Abstract
Disclosed herein is a method of: treating an organic polymer with an electron beam-generated plasma; exposing the treated polymer to air or an oxygen- and hydrogen-containing gas, generating hydroxyl groups on the surface of the polymer; reacting the surface with an organosilane compound having a chloro, fluoro, or alkoxy group and a functional or reactive group that is less reactive with the surface than the chloro, fluoro, or alkoxy group; and covalently immobilizing a biomolecule to the functional or reactive group or a reaction product thereof.
Core Innovation
The invention disclosed is a method for processing organic polymer microtitre plates by treating them with an electron beam-generated plasma followed by exposure to air or an oxygen- and hydrogen-containing gas to generate hydroxyl groups on the polymer surface. Subsequently, the surface is reacted with an organosilane compound that has a chloro, fluoro, or alkoxy group and a functional or reactive group less reactive with the surface. This functionalized surface enables covalent immobilization of biomolecules to the organosilane's reactive group or its reaction products.
The problem addressed by the invention relates to the limitations of standard polystyrene microtitre plates used in assays, which suffer from poor chemical resistance, uncontrolled surface chemistry, protein denaturation, desorption, and loss of biomolecule activity due to non-covalent adsorption. Existing covalent attachment methods rely on coatings or use single attachment chemistries limiting biomolecule immobilization versatility and often involve wet chemical treatments that cause non-specific attachment and surface damage. There is a need for an advanced surface engineering approach that independently controls chemical functionality and surface morphology at the nanoscale to enable versatile and stable covalent biomolecule immobilization on polymer microtitre plates.
The method employs electron beam-generated plasma to selectively modify only the top few nanometers of the polymer surface, generating reactive hydroxyl groups without significantly altering bulk properties or surface roughness. This plasma has low ion energies and low photon production, thereby reducing undesirable etching or subsurface changes. The plasma-treated surface is then covalently modified with organosilane compounds bearing various functional or reactive terminal groups, enabling a broad array of crosslinking chemistries. Finally, biomolecules can be covalently immobilized using different crosslinkers or direct attachment chemistries in discrete wells, allowing on a single plate multiple immobilization chemistries tailored to distinct biomolecules.
Claims Coverage
The claims include two independent claims that cover the core method of treating a polymer microtitre plate with an electron beam-generated plasma and the conditions for generating hydroxyl groups on the surface.
Electron beam-generated plasma treatment of organic polymer microtitre plates
A method comprising treating an organic polymer microtitre plate with an electron beam-generated plasma and exposing the treated plate to air or an oxygen- and hydrogen-containing gas, whereby hydroxyl groups are generated on the plate surface.
Specific plasma generation conditions for polymer surface modification
The plasma is generated in argon with an argon gas pressure of 90 mTorr ±10%, the treatment time is 2 minutes ±10%, the electron beam is pulsed at 50 Hz ±10%, and the electron beam pulse width is 2 ms ±10%.
The independent claims cover a method for generating hydroxyl groups on the surface of an organic polymer microtitre plate by electron beam-generated plasma treatment under specific conditions, enabling subsequent covalent biomolecule immobilization.
Stated Advantages
Plasma treatment produces a uniform, functionalized surface amenable to a broad range of immobilization chemistries.
The method allows multiple covalent immobilization chemistries on a single microtitre plate, enabling bespoke attachment of diverse biomolecules in different wells.
Electron beam-generated plasma uses low ion energies and low photon production, minimizing surface etching or degradation and preserving surface morphology.
The dry plasma treatment reduces hazardous chemical wastes and offers a tunable alternative to wet chemical surface modification methods.
The method achieves a reproducible, thin monolayer of functional organosilane on the polymer surface with well-oriented reactive groups for enhanced biomolecule activity.
Plasma-treated plates show superior biomolecule immobilization efficiency and retention compared to untreated plates and surpass certain commercial preactivated plates, offering a cost-effective alternative.
Documented Applications
Enzyme-linked immunosorbent assays (ELISAs) utilizing polystyrene microtitre plates with improved covalent biomolecule attachment.
High-throughput multiplexed biomolecule interaction screening using microtitre plates capable of multiple covalent immobilization chemistries.
Immobilization of a variety of biomolecules including peptides, antimicrobial peptides, carbohydrates, lipids, antibodies, and proteins for biological assays.
Applications in clinical diagnostics, biotechnology, vaccine and therapeutics development, and basic biomedical research requiring robust biomolecule immobilization.
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