Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1995-05-08
2002-03-26
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S030000, C435S041000, C435S180000, C436S518000, C436S531000, C436S532000
Reexamination Certificate
active
06361936
ABSTRACT:
TECHNICAL FIELD
Novel highly functionalized solid substrates are provided in conjunction with methods for their preparation and conjugation of the functionalities to a wide variety of compositions. The subject articles find extensive use in diagnosis, therapy, processing, as well as other applications, where binding of a particular moiety to a solid surface is desired.
BACKGROUND
Solid surfaces play a significant role in the ability to separate a component from a mixture of components, to localize one or more moieties to a specific area, to investigate characteristics of various molecules, molecular aggregations, cells or tissue, or in other situations where having a moiety in a defined region, optionally in relation to a particular medium, is of interest. Thus, solid substrates find application in diagnostics, for haptens, antigens and nucleic acids, affinity chromatography, apheresis, cytophoresis as well as other applications. There are many characteristics of interest with the solid substrate, such as degree of non-specific binding, clarity, ease of fabrication, availability, etc. The characteristics are of varying importance, depending upon the particular application. For purposes of the subject invention essential characteristics include the ease with which functionalities may be introduced, the convenience of such functionalties for linking other groups, the interaction between such functionalities and the moieties attached to such functionalities, the density of functionalization available for linkage, reproducibility of the functionalization and the methods employed for linkage. There is also an interest in being able to easily view the contents of a container, such as a bottle or flask.
In many situations, it is highly desirable to have a high density of a particular moiety on the surface. By having a high density a greater number of interactions can be achieved in a predetermined surface area. This allows for smaller volumes to be used, shorter contact times, greater intensity of signal, as well as other advantages.
There is, therefore, a continuing interest in being able to develop new methods and compositions which allow for improvements of solid substrates. Such improvements can have affects on the sensitivity and efficiency of diagnostic assays, the efficiency of separation of components in a complex mixture, the ability to pack particular moieties into a small surface area, as well as other improvements.
Relevant Literature
Olah et al.,
J. Am. Chem. Soc
. (1962) 84:3687 reports nitration in tetramethylenesulfone. Functionalization of polystyrene is described in U.S. Pat. Nos. 3,956,219; 3,886,486; 3,974,110; 3,995,094; and 4,226,958. Linking through a Schiff's base to a solid support is described in U.S. Pat. Nos. 4,419,444 and 4,217,338.
SUMMARY OF THE INVENTION
Novel functionalized solid substrates comprising addition polymerized aromatic monomers are provided. The novel substrates are polynitrilo substituted substrates produced by nitration of the aromatic polymer surface by an electrophilic agent, particularly a nitrating agent, in a solvent substantially incapable of dissolving the polymer, containing a small amount of a polar coordinating agent. A high proportion of the aromatic groups are substituted with nitro groups. The nitro groups may then be reduced to nitrilo groups, particularly amino groups or other reduced nitrogen groups which may serve as a site for linking to a wide variety of molecules. The resulting solid substrates find particular use for separations or high density packing of a moiety of interest.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Novel functionalized aromatic substrates are provided, where substantially the entire surface of a solid aromatic substrate is functionalized. Highly functionalized surfaces are achieved employing electrophilic agents capable of aromatic electrophilic substitution, particularly a nitrating agent, and a solvent which is relatively non-polar and in which the polymer is substantially immiscible and not swelled. Included in the reaction medium is a small amount of a polar substance, particularly a hydroxylic compound, which can coordinate with the electrophilic agent.
The-solid substrate will be a homo- or co-polymer, usually non-cross-linked or having a low level of cross-links (<2.0% polyene monomer) wherein at least 50 mol %, more usually at least about 80 mol % will be aromatic addition polymerizable hydrocarbon monomers, particularly having aryl groups of from 1 to 3 rings and having from 0 to 1 alkyl substituents, from 1 to 3 carbon atoms, usually at other than the para-position. For the most part, the substrate will be polystyrene which is produced in accordance with conventional ways and appears in a variety of forms, such as vials, microtiter plates, Petri dishes, plates, containers, fabricated articles, and the like. Instead of containers, the solid substrate may be particles, hollow fibers, strands, chopped strands, membranes, or the like. The polymer may be molded, extruded, machined or the like, usually resulting in a clear relatively smooth surface.
For the most part, electrophilic agents are employed, particularly nitrating agents, where the nitronium ion is coordinated with an anion which allows for significant ionization in the solvent medium, particularly a complex anion, more particularly a polyhalide anion. Illustrative polyhalide anions include tetrafluoroborate, antimony hexafluoride, phosphorous hexafluoride, etc.
The solvent will be one in which the polymer is neither soluble nor swelled, particularly tetramethylenesulfone (TMS). Included in the solvent will be a polar coordinating compound, which is able to coordinate with a nitronium ion. Conveniently, water may be used, but other oxy compounds may also find use, such as methanol. The polar compound should be capable of displacing both the anion and the solvent from coordination with the nitronium ion. The polar component will preferably be present in not greater than about equimolar amount to the nitronium ion concentration, usually present in at least about 0.05, more usually in at least about 0.25, and generally at least about 0.5 times the molar concentration of nitronium ion.
The nitronium ion will generally be at a concentration of at least about 0.05 M, usually at least about 0.1 M, and usually not greater than about 0.8 M. The particular concentration is primarily one of convenience, so long as there is a sufficient amount of reactant to substantially completely functionalize the polymeric surface.
The temperature of the reaction will vary with the nature of the reagent, generally being at least about 4° C., and not more than about 60° C., usually not more than about 40° C., preferably ranging from about 30° C. to 40° C. The time for the reaction will vary with the temperature, so that longer times will be related to lower temperatures. Generally, the reaction will take at least 1 hr, and not more than about 48 hrs, generally running from about 2 to 24 hrs.
The reaction medium is contacted with the polymeric surface to provide an amount of reactant stoichiometric with the available aromatic groups at the surface, usually at least about 1.5 excess, where greater molar excess may be employed as convenient. In some instances it may be desirable to prepare a number of reaction mixtures using differing amounts of nitronium ion to optimize the level of functionalization of the surface.
After completion of the reaction, the reaction mixture may be discarded and the surface washed to remove any residual materials. Various solvents may be used for cleaning the surface, such as methanol, ethanol, dimethylsulfoxide (DMSO), water, nitromethane or the like. The particular material employed for cleaning is not critical to this invention and is primarily one of convenience, so long as the polymer is insoluble and is not swelled by the solvent.
The resulting polymeric surface is found by electron spectroscopy for chemical analysis (ESCA) to have a ratio of nitrogen to aryl of at least 0.1, usually at least about 0.15, more usually
Aventis Pharmaceuticals Products Inc.
Forman B J
Fredman Jeffrey
Morrison & Foerster / LLP
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