Poly(fluoroalkyl) sugar reagents for surface modification of supports

Poly(fluoroalkyl) sugar reagents are prepared containing a sugar such as a monosaccharide or a disaccharide to which are bonded multiple fluoroalkyl anchor groups capable of attaching to a fluorocarbon surface, and either a reactive group capable of covalent coupling to a biomolecule or a charged group to form an ion-exchanger or a non-ionic group to give a neutral fluorosurfactant. A spacer may be between the reactive group and the sugar. The poly(fluoroalkyl) sugar reagents are strongly adsorbed onto fluorocarbon surfaces to provide supports for such applications as separation and immobilization of biomolecules such as enzymes, carrying out heterogeneous diagnostic assays, and preparation of biosensors.

TECHNICAL FIELD 
This invention relates to a novel group of poly(fluoroalkyl) sugar 
reagents, a method for their use for the modification of the surface of 
solid or liquid supports, and supports used for application in the 
separation of biomolecules, enzyme immobilization, heterogeneous 
diagnostic assays, and biosensors. 
BACKGROUND ART 
Numerous methods have been developed for the immobilization of proteins and 
other biomolecules onto solid or liquid supports. A description of these 
methods can be found in general reviews such as that given by Mosbach, 
1976, Methods in Enzymology, Vol. 44; Weetall, 1975, Immobilized Enzymes, 
Antigens, Antibodies, and Peptides; or Kennedy et al., 1983, Solid Phase 
Biochemistry, Analytical and Synthetic Aspects, Scouten, ed., pp. 253-391. 
The most commonly used methods are adsorption or covalent binding to the 
support. 
Adsorption is the oldest and simplest method for protein immobilization. To 
effect immobilization, a solution of the protein is contacted with a 
support material such as alumina, carbon, an ion-exchange resin, 
cellulose, glass or a ceramic. Although the immobilization procedure may 
be simple, the interactions involved in the adsorption process are complex 
and include charge-charge, van der Waals and hydrophobic interactions, and 
hydrogen bonding. The adsorption method has the advantages of low cost, 
extreme simplicity, mild immobilization conditions and the ability to 
regenerate the support. The main limitation of this method is the 
relatively weak interaction between the protein and the support, which may 
result in desorption of the protein upon changes in pH and ionic strength. 
The often undefined nature of these interactions also can limit their use. 
The most frequently used immobilization technique is the covalent binding 
of the protein to chemically activated solid supports such as glass, 
synthetic polymers, and cross-linked polysaccharides. (Generally, this 
technique results in a protein which is immobilized in a more stable 
fashion than protein immobilized by adsorption.) An example of this method 
is the cyanogen bromide activation of polysaccharide supports, e.g., 
agarose. 
Although these traditional supports have been used in many applications, 
they suffer from some limitations. The polysaccharide supports are 
compressible, which limits their application in column configurations at 
high flow rates. These supports are also susceptible to microbial attack. 
Silica supports are not stable under alkaline conditions. Polymeric 
supports are also not chemically inert, and usually have a specific 
gravity close to 1, which results in long settling times in batch 
operations. Moreover, all of these supports exhibit varying degrees of 
nonspecific binding of unwanted proteins. The use of solid and liquid 
fluorocarbon supports overcome many of these limitations. Fluorocarbons 
are chemically inert and mechanically stable. The high specific gravity of 
fluorocarbon supports results in rapid settling in batch operations. 
However, it is difficult to activate fluorocarbon supports for 
immobilization. 
Fluorocarbon polymers have been used as supports to which biomolecules have 
been attached by adsorption [Fishman U.S. Pat. No. 3,843,443, issued on 
Oct. 22, 1974; WO 8603-840-A filed by Rijskuniv Groningen; Danielson and 
Siergiej, Biotechnol. Bioeng. 23, 1913-1917 (1981); Siergeiej, 
Dissertation Abstracts, Int. B., Volume 44, 153 (1983)]. Because these 
methods rely on simple adsorption of the biomolecule onto the support, the 
attachment is relatively weak. Consequently, some or all of the 
immobilized biomolecule is lost during use. In addition, a significant 
loss of biological activity of the biomolecule results upon adsorption. 
Busby et al. (U.S. Pat. No. 4,317,879, issued Mar. 2, 1982) disclose the 
covalent attachment of the enzyme glucose oxidase to a fluorocarbon 
membrane. The membrane was first etched with a sodium dispersion in 
naphthalene, followed by paraformaldehyde linking of the enzyme. This 
method requires severe chemical conditions to activate the fluorocarbon 
surface for covalent binding to the enzyme. 
Hato et al., (U.S. Pat. No. 4,619,897, issued Oct. 23, 1986) disclose the 
immobilization of enzymes onto a fluorine resin membrane which is made 
hydrophilic on one side by penetration of a perfluoroalkyl surface active 
agent to a prescribed depth. The asymmetrically functional membrane 
obtained is then treated with an enzyme and a cross-linking agent such as 
glutaraldehyde to effect immobilization. In this approach, the 
fluorocarbon surface is not activated for covalent attachment of the 
enzyme. Rather, the enzyme is crosslinked within the pores of the wetted 
membrane. This approach is limited to porous fluorocarbon membranes. 
The use of perfluorocarbon polymer-based supports for enzyme immobilization 
and affinity chromatography is described in U.S. Pat. No. 4,885,250 issued 
Dec. 5, 1989. In this method the biomolecule is first modified by reaction 
with a perfluoroalkylating agent such as perfluorooctylpropylisocyanate 
described in copending application Ser. No. 134,028, now U.S. Pat. No. 
4,954,444. Then, the modified protein is adsorbed onto the fluorocarbon 
support to effect immobilization. This procedure works well for the 
immobilization of many biomolecules, particularly immunoglobulins. 
However, substantial loss of biological activity results for some proteins 
because of the need to use organic solvents (16% v/v) in the 
perfluoroalkylation reaction, the hydrophobic nature of the fluorocarbon 
support, and the need for multipoint modification of protein to obtain 
secure immobilization. Multipoint modification of the biomolecule is 
required because of the mono(fluoroalkyl) reagents used. In addition, the 
mono(fluoroalkyl) reagents desorb from the support in the presence of high 
levels of organic solvents, e.g., about 50% or greater. 
A revised method for immobilization onto fluorocarbon surfaces or 
non-fluorocarbons which contain a fluorocarbon interlayer attached to the 
surface of the core has been described in copending application Ser. No. 
413,867, now U.S. Pat. No. 5,079,155. In this revised method, a 
perfluoroalkylating reagent containing a mono(fluoroalkyl) anchor group, a 
hydrophilic spacer arm, and a reactive group for covalent coupling to the 
protein is adsorbed onto the surface of the support. An aqueous solution 
of the protein is added to effect immobilization. This approach overcomes 
many of the limitations of the original method. Higher retention of 
activity, particularly for enzymes, is obtained because totally aqueous 
solutions of the enzyme are used, and the surface of the support is more 
hydrophilic. This approach also provides a preactivated support which is 
more convenient to use. However, the support with the adsorbed reagent is 
not stable because the reagent is secured by only one fluoroalkyl anchor 
group. In addition, multipoint attachment of the biomolecule is required 
for secure immobilization. 
In copending application Ser. No. 413,867 a method for forming solid 
supports for size exclusion and ion-exchange separations is described. The 
support is prepared by forming a fluorocarbon interlayer by attaching a 
fluorocarbon to the surface of a solid carrier, followed by treatment with 
a fluorosurfactant. The use of a neutral fluorosurfactant results in a 
size exclusion support, while a charged fluorosurfactant provides an 
ion-exchange support. The stability of these supports is limited because 
of the mono(fluoroalkyl) anchor group which adsorbs to the fluorocarbon 
surface. 
Giaver, (U.S. Pat. No. 4,619,904, issued Oct. 28, 1986) describes the use 
of fluorocarbon emulsions in agglutination immunoassays. The emulsions 
were formed by adding a fluorinated polar molecule such as 
pentafluorobenzoyl chloride to a fluorocarbon liquid. The resulting 
emulsion was contacted with an aqueous solution of the protein. Again, 
mono(fluoroalkyl) anchor groups were used to immobilize the protein. 
Lowe et al. in copending application Ser. No. 428,154, describe the 
attachment of biomolecules to fluorocarbon surfaces by means of a polymer 
such as poly(vinyl alcohol), which has been chemically modified to contain 
a significant number of perfluoroalkyl groups. Although this approach 
provides multiple fluoroalkyl anchor groups for secure attachment to the 
fluorocarbon surface, the number of anchor groups is difficult to control 
and reproduce. 
De Miguel et al., Chromatographia, Vol. 24, 849-853, 1987, describe the 
strong retention of phenyl-D-glucopyranoside, modified with multiple 
fluorocarbon chains, on fluorocarbon bonded phases under reversed phase 
conditions. The authors speculate that such strong retention may allow 
dynamic anchoring of biomolecules. No examples were provided. The 
compounds described cannot be used for immobilization because they contain 
no reactive group to couple to the biomolecule. The major difference 
between the phenyl-D-glucopyranosides of De Miguel et al., and the present 
invention is that their compounds do not contain a spacer arm and reactive 
group for covalent binding to the biomolecule. 
One object of this invention is to provide a superior composition for 
modifying fluorocarbon surfaces that permit good retention of 
biomolecules. 
Another object of this invention is to provide a reagent for facilitating 
immobilization of biomolecules on a support with retention of biological 
activity. 
Yet another object of this invention is to provide a method for forming a 
solid support using the superior reagents described herein. 
Still another object of this invention is to provide a process for 
immobilizing a biomolecule onto a fluorocarbon surface. 
SUMMARY OF THE INVENTION 
This invention relates to poly(fluoroalkyl) sugar reagents for modification 
of the surfaces of supports used in immobilizing biomolecules. 
The poly(fluoroalkyl) sugar reagents claimed in this invention are 
comprised of a sugar template to which are bonded multiple fluoroalkyl 
anchor groups and an optional spacer, attached to a reactive group to 
covalently bind to biomolecules, a charged group to form an ion-exchanger, 
or a non-ionic group to give a neutral fluorosurfactant. 
These reagents are adsorbed onto fluorocarbon surfaces to provide supports 
for such applications as the separation of biomolecules, enzyme 
immobilization, heterogeneous diagnostic assays, and biosensors. 
The reagents and supports described herein offer advantages over those of 
the prior art because the poly(fluoroalkyl) sugar reagents are adsorbed on 
fluorocarbon support surfaces more strongly than mono(fluoroalkyl) 
reagents. Examples of novel compositions claimed herein are as follows: 
(a) a composition of the structure: 
##STR1## 
wherein: R is selected from 
##STR2## 
m=1-5; n=3-20; 
x=1-10; and 
R' is selected from the following structures: 
##STR3## 
(b) a composition of the structure: 
##STR4## 
wherein: R is selected from CO(CH.sub.2).sub.m (CF.sub.2).sub.n CF.sub.3 
; 
x=1-10; 
m=1-5; 
n=3-20; and 
R' is 
##STR5## 
(c) a composition of the structure: 
##STR6## 
wherein: R is CO(CH.sub.2).sub.m (CF.sub.2).sub.n CF.sub.3 ; 
x=2-10; and 
R' is selected from 
##STR7## 
(d) a composition of the structure: 
##STR8## 
wherein: R is CO(CH.sub.2).sub.m (CF.sub.2).sub.n CF.sub.3 ; 
m=1-5; 
n=3-20; 
R' is CH.sub.2 OCH.sub.2 Ph, H, phenyl, CH.sub.3 or C.sub.2 H.sub.5 ; and 
x=1-20. 
The invention also concerns: 
(1) a support containing an attached binder for a biomolecule consisting 
essentially of: 
(a) a fluorocarbon surface; and 
(b) a polyfluoro sugar composition comprised of a sugar template to which 
are attached multiple fluoroalkyl anchor groups securely attached to the 
surface of said fluorocarbon interlayer, said sugar template further 
consisting of a spacer containing a reactive group capable of coupling to 
a biomolecule. 
(2) a process for immobilizing a biomolecule on a fluorocarbon surface 
comprising the steps of: 
(a) activating the fluorocarbon surface by contacting the fluorocarbon 
surface with a reactive poly(fluoroalkyl) sugar reagent containing a sugar 
template to which are attached a plurality of fluoroalkyl anchor groups, 
an optional spacer and a reactive group by causing the fluorocarbon 
surface to adsorb the reagent; then 
(b) adding a solution of a biomolecule to the activated fluorocarbon 
surface to attach to the sugar reagent to immobilize the biomolecule on 
the fluorocarbon surface. 
(3) a process for immobilizing a biomolecule on a fluorocarbon surface 
comprising the step of: 
(a) attaching a poly(fluoroalkyl) sugar reagent to a biomolecule to form a 
conjugate; and then 
(b) adsorbing the resulting conjugate on a fluorocarbon surface.