Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is organic
Reexamination Certificate
2001-07-24
2004-12-21
Ceperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is organic
C436S531000, C436S527000, C436S546000, C435S006120, C530S408000, C530S409000, C530S391100
Reexamination Certificate
active
06833276
ABSTRACT:
BACKGROUND
This invention relates to a polymer brush that features a polymer layer on a substrate surface, the polymer layer being composed of a number of polymer chains each of which include a water-soluble or water-dispersible segment having two termini, one terminus being free and the other being bound to the substrate surface. In one embodiment, the present invention is directed to a sensor wherein probes for biological molecules are attached to these water-soluble or water-dispersible segments. Sensors of this type are particularly useful for analyzing aqueous samples that contain biological materials. The present invention further relates to methods of synthesizing such sensors. In preferred embodiments, the polymers have controlled molecular architectures that allow for tuning the concentration or accessibility of the probes, and thus, for tuning properties (e.g., sensitivity) of the sensor.
Sensors for analyzing biological samples typically have the ability to process samples accurately and rapidly in an aqueous environment. This, in turn, looks to the presence of multiple probes on a single substrate surface capable of selectively interacting with components of the sample. For example, nucleic acid hybridization assays use multiple oligonucleotide probes bound to the substrate surface at pre-selected sites. The oligonucleotide probes, in turn, are available to participate in a hybridization reaction with selected nucleic acid components of the sample. Generally, this interaction of probe and sample relates to the utility of the components of the biological sample, such as the identity, concentration, purity or form of the components being sensed. There are generally many types of probes known, for example, antibodies that may immunoreact with a desired protein in a diagnostic assay, other protein binding assays, and dyes that change color to indicate the concentration of a desired protein, enzyme, small organic molecule, or inorganic molecule such as calcium or lithium.
Attaching probe molecules to surfaces is often difficult because the surfaces lack functional groups that are uniquely reactive in an aqueous system or are readily accessible to the probe molecules as a result of factors such as surface crowding or steric hindrance. This problem becomes particularly acute as the number of functional groups per unit area of surface increases. In addition, once probes are bound to the surface, they must remain accessible to components of the biological sample. Here, too, factors such as steric hindrance may hamper accessibility. Molecular crowding (i.e., density) becomes a critical issue as well, particularly in systems where fluorescence quenching can be an issue.
Controlled free radical polymerization methods with living-type kinetics have been used to covalently bond polymers to the surfaces of substrates and thereby form “polymer brushes.” Husseman et al.,
Macromolecules
1999, 32, 1424-31, for example, describes a variety of polymer brushes prepared using such methods. The resulting brushes, however, were not water-soluble or water-dispersible and thus were not suitable for applications involving aqueous samples such as biological samples. Additionally, Husseman et al. fail to address the importance of controlling the grafting density of, or spacing between, the polymer chains attached to the substrate surface, in order to optimize both the number of probes which may be attached for a given application, as well the efficiency of those probes, once attached, to interact with the target molecules.
Accordingly, a need to continues to exist for a polymer brush, as well as a process for the preparation thereof, having polymer chains of a controlled molecular architecture (i.e., composition, functionality, molecular weight, polydispersity, etc.), as well as spacing or grafting density on the substrate surface, such that the attachment of probe molecules of a given size or type can be optimized. Additionally, a need continues to exist for a sensor (i.e., a polymer brush having probe molecules attached thereto), as well as a process for the preparation thereof, having a controlled structure, such that probe accessibility to the target biological molecules can be optimized.
SUMMARY
Thus it is an object of this invention to provide a platform for selectively interacting with biomolecules, such as sensors for biomolecule recognition and bioseparations, among other applications. In preferred applications, it is an object of the invention to provide a sensor comprising a substrate surface, polymers associated with the surface, and probes attached to the polymers, where each of the polymers have at least a water-soluble or water-dispersible segment having functional groups capable of bonding to the probes. In some contexts, these sensors may be considered to be “polymer brushes.”
It is also an object of this invention to provide biosensors that feature controlled-architecture polymers bound to a surface of a substrate that include water-dispersible or water-soluble segments to which probes for biological molecules are covalently bound via functional groups found on the segments.
Although the invention is more specifically described herein in the context of a biosensor, it is to be understood that the invention is not necessarily to be limited to such applications.
It is another object of this invention to provide polymer modified surfaces or articles.
It is yet another object of this invention to provide a method of making polymer-modified surfaces using free radical polymerization, preferably controlled free radical polymerization having living-type kinetics.
This invention provides the advantage of careful control of the identity, surface grafting density or spacing, and molecular architecture of the functionalized polymer chains. Accordingly, use of this invention provides the ability to prepare a functionalized surface that, in terms relative to the accessibility of surface-bound functional groups to probe molecules, and probe molecules to biological sample components, approximates that of an aqueous solution. At the same time, the probes are tethered to the surface, thereby facilitating analysis following contact with a biological sample.
This invention provides functionalities that bind probes, which are not attached to a flat surface. This means that solution chemistry is more closely simulated, surface effects are reduced, and as a result the sensors of this invention provide increased sensitivity, increased signal, increased signal to noise ratios and increased dynamic range.
In addition, those of skill in the art are able to choose monomers that yield functionalized surface brushes that display a low fluorescent background due to the lack of non-specific binding of the bio-molecule in the sample.
Overall sensitivity of the sensor is also improved, it is believed, due to the spacing of the functional groups that bind the probes from the surface of the substrate. In other words, increased sensitivity has been observed, which is believed to result from the fact that the functional groups that bind the probes are now attached to a polymer chain thereby distancing the probes (and hence the related biochemistry) from the surface of the substrate, reducing, if not completely eliminating, surface effects.
Briefly, therefore, the present invention is directed to a polymer brush for binding a molecule in an aqueous sample in an assay. The polymer brush comprises a substrate having a surface, and a layer on the substrate surface comprising polymer chains. The polymer chains have at least two termini and a water-soluble or water-dispersible intermediate portion between the termini, one terminus being free and the other terminus being bound to the substrate surface. The terminus of each polymer chain that is bound to the substrate surface comprises a residue of a surface bound initiator having the formula
wherein: C is a moiety on the surface of the substrate; L is a linker group capable of bonding to at least one C moiety; q, r and t are independently 0 or 1, provided the sum of
Benoit Didier
Charmot Dominique
Jandeleit Bernd
Klaerner Gerrit
Mansky Paul
Ceperley Mary E.
Senniger Powers
Symyx Technologies Inc.
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