Immobilization of biologically active protein on a support with a 7-18 carbon spacer and a bifunctional phospholipid

Enzymes and certain other bioactive substances are immobilized on solid substrates which have sufficient functional groups such as hydroxyl or carboxyl. The bioactive substances are linked to the substrates through spacer compounds having a long open alkyl chain with 7-18 carbon atoms and also through phospholipid intermediates. The spacer compound is chemically linked to the substrate. The phospholipid is covalently linked to the spacer compound. Immobilized bioactive substances of the invention exhibit a marked increase in activity and stability. In a preferred embodiment, immobilized enzymes having a high degree of resistance to thermal inactivation are prepared.

This invention relates to the preparation of immobilized enzymes with a 
high degree of resistance to thermal inactivation through covalent 
attachment to phospholipid layers, which are already covalently linked to 
solid supports through long chain spacer molecules. Also included in the 
invention are processes for the synthesis of appropriately functionalized 
phospholipids suitable for the dual role of binding to bioactive 
substances as well as to modified solid supports. 
BACKGROUND OF THE INVENTION 
Enzymes find extensive applicability in diverse areas such as food 
processing, enantioselective organic synthesis, production of 
pharmaceuticals, clinical diagnosis/treatment, extracorporeal affinity 
chromatography, waste management, environmental analysis/pollution control 
and biosensors. As industrial catalysts, they offer a number of advantages 
over conventional chemical catalysts due to their high catalytic activity, 
substrate specificity, the mild conditions involved in their use, minimal 
by-product formation and no environmental pollution risk. However the two 
main disadvantages relating to their utility are their instability and the 
economic factor. The practical use of enzymes often requires elevated 
temperatures to increase productivity, prevent microbial contamination, 
improve the solubility of substrates and reduce the viscosity of the 
reaction medium. On the other hand, the stability of enzymes is affected 
by conditions such as heat, contact with chemicals and organic solvents, 
all of which cause denaturation. Amongst these, heat is by far the most 
important factor for the loss of the biological activity of enzymes and 
some correlation exists between thermal stability and other kinds of 
stabilization such as resistance to proteolysis. Thermal inactivation of 
enzymes is initiated by the partial reversible unfolding of their native 
structure which is followed by irreversible configurational/conformational 
changes. Processes such as aggregation, formation of "scrambled 
structures", cleavage of disulfide bridges, peptide bond hydrolysis, 
racemization of amino acid residues, deamidation, dissociation of 
prosthetic groups, isopeptide bond formation and oxidation of thiol/indole 
groups have been implicated during heat mediated denaturing of enzymes. 
Enhancement of the thermal stability could alleviate most, if not all, of 
the problems associated with the use of native enzymes for various 
applications. Thermostabilization strategies followed during the past 
three decades consist of (i) addition of substances, (ii) chemical 
modification, (iii) cross-linking, (iv) use of anhydrous solvents 
(non-aqueous media), (v) protein engineering and (vi) immobilization. Of 
these, the immobilization technique is the most extensively used one for 
imparting thermal stability to enzymes. Enzymes immobilized on suitable 
substrates possess considerable advantages over those used in the soluble 
phase. They often show marked increase in stability and may be used in 
bioreactors for continuous processing, thereby cutting down on the costs 
in comparison with reactors utilizing these biocatalysts in solution. For 
example, using immobilized aminoacylase, the cost of amino acid production 
is reduced by 40% as against the soluble enzyme. In addition, immobilized 
biocatalysts are easily removable from reaction mixtures and have enhanced 
shelf life. 
By definition, an immobilized enzyme is a protein physically localized in a 
certain region of space or converted from a water-soluble mobile state to 
a water-insoluble immobile condition. Protocols used for immobilizing 
enzymes can be categorized according to whether the protein becomes 
immobile by chemical binding or by physical retention. These consist of 
(i) binding of enzyme molecules to carriers through covalent bonds, (ii) 
by adsorptive interactions (physisorption), (iii) entrapment into gels, 
beads or fibres, (iv) cross-linking or co-crosslinking with bifunctional 
reagents and (v) encapsulation in microcapsules or membranes. Of these, 
the adsorptive procedures have become more or less obsolete due to the 
fact that the surfaces produced are too unstable to withstand mechanical 
stresses and chemical treatments involved in industrial processes. 
Immobilization through cross-linking has met with limited success because 
of the large amounts of enzyme required, the uncontrollable nature of the 
reaction which may lead to inactivation and the unsuitable mechanical 
properties of the resulting surfaces. The main disadvantages of the 
microencapsulation technique are that the molecular weight of the 
substrate has to be very low to allow diffusion across the membranous 
barrier and the capsules are very prone to enzyme leakage as they are 
relatively fragile. Furthermore, the polysaccharide-based polymeric 
materials used for entrapping enzymes into gels or beads suffer from the 
fact that strict sterile operating conditions must be maintained to 
prevent the growth of bacteria and fungii. With acrylamide monomers used 
for entrapment purposes, the conditions of photopolymerization may 
generate localized temperatures up to 60.degree. C. causing denaturing of 
the enzyme. With other polymeric systems, problems of enzyme loading, 
viability and stability have to be overcome for industrial applications. 
Several reviews have appeared in the scientific and patent literature on 
the available choices of substrates and the protocols for covalently 
binding enzymes on them. The substrates in vogue range from inorganic 
materials such as porous glass, ceramics, silica and metal/metal oxides to 
organic materials such as the natural polymers cellulose, chitin and 
agarose and synthetic products like acrylates, polyamides, derivatized 
polystyrene and redox systems like polypyrrole. Biomolecules like the 
avidin-biotin system or bovine serum albumin are also being utilized. 
However, because of the problems of microbial growth on organic supports, 
and the consequent loss of activity, collapse of the structure and product 
contamination, there has been an increasing interest in the use of 
inorganic support materials, especially silica, controlled pore glass and 
ceramics. 
The two factors to be considered in the selection of a method for the 
covalent linkage of an enzyme to a support are: the type of functional 
groups on the protein through which binding to the support is to be 
accomplished (and consequently the type of chemical reactions to the 
employed) and the physical/chemical characteristics of the support 
material with appropriate reactive functionalities grafted onto their 
surface. The functional groups on the enzymes which are available for 
covalent bonding are (1) amino (eta-amino groups of lysine and arginine 
and the N-terminal amino moieties of the polypeptide chains), (2) carboxyl 
groups of aspartic and glutamic acid and the C-terminal moieties, (3) 
phenol rings of tyrosine, (4) sulfhydryl groups of cysteine, (5) hydroxyls 
of serine, threonine and tyrosine, (6) the imidazole groups of histidine 
and (7) the indole groups of tryptophan. In practice, most of the covalent 
coupling reactions involve the amino, carboxy and mercapto moieties on the 
amino acids in the protein structure. The solid supports, in turn, must 
carry functional groups such as carboxyl, amino, formyl, epoxy, halo 
(chloro or bromo) and hydroxyl. A majority of solid supports either carry 
hydroxyls on their surfaces or can be easily modified by chemical or 
electrochemical means to introduce such hydroxylic groups. 
Chemical reactions most commonly used for the interaction of the 
functionalities in the enzyme with those on the support materials consist 
of (1) the nucleophilic displacement of the surface hydroxyls on the 
supports activated with a sulphonyl chloride, 2-fluoro pyridinium tosylate 
or cyanuric chloride by the amino group on the protein, (2) nucleophilic 
addition of the protein amino group to a surface hydroxyl on the support 
which is activated with cyanogen bromide or carbonyldiimidazole or a 
chloroformate; or an analogous nucleophilic addition of the protein amino 
group to a carboxyl on the support surface which is activated as its 
N-hydroxysuccinimide ester, azide or with a diimide, (3) electrophilic 
addition of a diazonium functionality formed from an aromatic amino moiety 
on the support to the tyrosine residues on the enzyme, (4) electrophilic 
addition of the mercapto group on the cysteine moiety of the enzyme to a 
maleimide function introduced onto the surface of the support, and (5) 
cross-linking a surface amino group on the support to an amino group on 
the enzyme with a bifunctional reagent such as glutaraldehyde. 
The thermal stability of enzymes covalently attached to support materials 
is significantly enhanced in comparison with the native enzyme. For 
example, Hayashi et al. (J. Appl. Polym. Sci. 1992, 44, 143) have observed 
that papain immobilized on polymethyl L-glutamate exhibited an activity up 
to three times higher than the native enzyme when maintained at 70.degree. 
C. in buffer solution for one hour. The free papain loses 90% of its 
initial activity at 75.degree. C. within 45 minutes. Raghunath and 
coworkers (Biotechnol. Bioeng. 1984, 26, 104) have demonstrated that 
urease immobilized on collagen-poly(glycidyl methacrylate) graft copolymer 
support was thermally stable up to 70.degree. C. and 40 days when stored 
at 4.degree. C. in a buffer solution. Davidenko et al. (Chem. Abstr. 1985, 
102, 127894) have reported that urease adsorbed on carbon fibres is stable 
up to 65.degree. C. and retained 90% of its activity when stored for a 
month at 4.degree.-5.degree. C. Thermal stabilization up to 70.degree. C. 
in buffer solutions was also reported for chymotripsin by multi-point 
covalent attachment to aldehyde-agarose gels (Guisen et al. Biotechnol. 
Bioeng. 1991, 38, 1144) and for glucoamylase on periodate oxidized dextran 
(Lenders and Chricton, Biotechnol. Bioeng. 1988, 31,267). Asakura et al. 
(Polym.-Plast. Technol. Eng. 1989, 28, 453) immobilized alkaline 
phosphatase on Bombyx mori silk fibroin by cyanogen bromide and diazo 
coupling methods and have shown that while the free enzyme was totally 
deactivated at 65.degree. C., the enzyme coupled by the diazonium 
procedure retained 30% of its activity, in comparison with 10% for the 
cyanogen bromide-modified product. Yabushita and coworkers (Chem. Pharm. 
Bull. 1988, 36, 954) have shown that urokinase immobilized on an 
ethylene-vinyl acetate copolymer matrix retained more than 50% of its 
initial activity when kept for 8 hours at 45.degree. C., while the soluble 
enzyme lost almost all of its activity in 3 hours. 
Margolin and coworkers (Eur. J. Biochem. 1985, 146, 625) effected a 
comparative evaluation of the stability and activity of enzymes 
immobilized on water-soluble and water-insoluble supports. Employing poly 
(N-ethyl-4-vinyl pyridinium bromide) (a polycationic support) and poly 
(methylacrylic acid) (a polyanionic support) for immobilizing a series of 
enzymes, these authors showed that pronounced thermal stabilization of 
penicillin amidase and urease could be achieved only if these enzymes are 
on the precipitated supports (in the insoluble form) and covalently 
attached to the polyelectrolyte nucleus. Thus, the thermal stability of 
polyelectrolyte complex-bound penicillin amidase increased seven-fold at 
pH 5.7, 60.degree. C. and three hundred-fold at pH 3.1, 25.degree. C., 
compared to the native enzyme. For urease, the thermal stabilization 
increases twenty-fold at pH 5, 70.degree. C. 
The role of phospholipids as protective agents for maintaining the activity 
of antibodies, enzymes and receptors is well-documented. There is 
considerable evidence concerning the requirement of a lipid environment 
for sustaining the activity of enzymes. For example, it has been shown 
that a lipid-modified glucose oxidase enzyme electrode offers greater 
selectivity and stability for the analysis of glucose. Phospholipids may 
act as modulators of enzymatic reactions in addition to their role as 
obligatory cofactors for some membrane enzymes. Thus, it was shown 
(Niedzwiecka et al., Acta Biochim. Biophys. Hung., 1990, 25,47), that the 
purified lymphocyte 5'-nucleotidase reconstituted into lipid bilayer 
demonstrates remarkable stability on storage at 4.degree. C. The liposome 
incorporated enzyme from chicken gizzard is five times more stable at 
56.degree. C. than the enzyme in the detergent solution, indicating that 
the phospholipids play a role in preventing the denaturing process. 
Rosenberg, Jones and Vadgama (Biochim. Biophys. Acta 1992, 1115, 157) 
encapsulated glucose oxidase in liposomes and found that electrodes coated 
with a nitro-cellulose membrane carrying these liposome-enzyme 
formulations exhibited extended linear range of response. The enzyme 
activity was found to be partially dictated by the liposomal bilayer 
permeability, and therefore, the enzyme affinity for its substrate could 
be regulated by using liposomes prepared from different lipids such as 
dimyristoyl, dipalmitoyl and distearoyl-phosphatidylcholine. It has also 
been shown by Kotowski and Tien (Bioelectrochem. Bioenerg. 1988, 19, 277) 
that glucose oxidase could be covalently immobilized on a 
polypyrrole-supported bilayer lipid membrane surface and the 
enzyme-substrate reaction could be followed by cyclic voltammetry. The 
phospholipid functions as an electric switch during this analysis, besides 
supplying the natural biomembrane-type environment to the enzyme. 
Besides thermal inactivation, the extent of activity exhibited by an 
immobilized enzyme is also dependent upon aspects such as the chemical 
procedure used to effect immobilization, the spacer chain length and the 
pH of the buffering medium in which the enzyme-substrate reactions are 
carried out. For example, Comfort et al. (Biotechnol. Bioeng. 1988, 32, 
554) evaluated the immobilization yields of heparinase and bilurubin 
oxidase on agarose and acrylic beads activated by four different reagents, 
viz. cyanogen bromide, carbonyldiimidazole, oxirane and tresyl chloride, 
respectively. They found that while heparinase was bound in 90% yield 
(with 50% active enzyme) by the cyanogen bromide method, bilurubin oxidase 
was preferentially linked. (50-55% maximum yield, with 25-30% active 
enzyme) by the tresyl chloride and oxirane displacement. However, in both 
cases, nearly 40-50% of the immobilized enzymes were leached out when 
allowed to stand in buffer for a short time. Przybyt and Sugier (Anal. 
Chim. Acta 1990, 239, 269) investigated the activity of urease immobilized 
on oxidized tungsten electrodes by electrochemistry. The covalent binding 
protocol followed by these authors consisted of initially silanizing the 
metal oxide surface with gamma-aminopropyltriethoxysilane and then 
cross-linking the enzyme with either cyanuric chloride or hexamethylene 
diisocyanate or glutaraldehyde. They found that the lifetime of the enzyme 
electrodes with the cyanuric chloride linker was only one day. In 
comparison, the lifetimes of electrodes prepared by employing 
glutaraldehyde and the diisocyanate cross-linkers were 29 and 22 days, 
respectively. The life-time of the enzyme electrode, obtained by the 
direct cross-linking of the metal oxide surface with the enzyme through 
hexamethylene diisocyanate (without prior silanization) was 19 days. 
Furthermore, these authors noted profound effects on the electrode 
response due to factors such a the nature of the buffer, its concentration 
and ionic strength. 
The importance of the spacer chain length towards the retention of the 
activity of an immobilized enzyme on a given surface has been demonstrated 
by several groups of workers. For instance, Kennedy and Cabral (in Methods 
in Enzymology, Vol. 135, pp. 117-130, Academic Press, San Diego, 1987) 
examined the linking of glucoamylase to control pore glass activated with 
titanium tetrachloride. The substrates were initially treated with ammonia 
(no carbon spacer), 1,2-diaminoethane (a two-carbon spacer) and 
hexamethylene diamine (a 6-carbon spacer) and then cross-linked with the 
enzyme through glutaraldehyde. The six carbon spacer-carrying substrate 
exhibited an activity retention of 12% relative to the activity of the 
soluble enzyme, while the figures were 1.5% and 3.2% for the no carbon and 
two carbon spacer, respectively. Jayakumari and Pillai (J. Appl. Polym. 
Sci. 1991, 42, 583) observed that the direct coupling of papain to 
carboxylated polystyrene yielded only 5% active enzyme, while binding of 
the same enzyme to the same support through glutaric anhydride 
cross-linker produced 30% of active enzyme. However, the maximum activity 
retention (54%) was obtained when papain was linked to hydroxymethyl 
polystyrene through polyethylene glycol (PEG 600) cross-linker. These 
authors also demonstrated that increasing cross-link densities decreased 
the total immobilization yields as well as the amount of active enzyme. 
Furthermore, rigid supports lowered total/active enzyme yields in 
comparison with flexible supports. Schuhmann et al. (J. Amer. Chem. Soc. 
1991, 113, 1394) showed that the electrical communication between the 
redox centres of glucose oxidase and vitreous carbon electrodes is more 
effective when a long chain diamine was used to cross-link the aldehyde 
functionalities of ferrocene and those of glucose oxidase obtained by the 
oxidation with periodate. Reduction of electron-transfer distances between 
the redox centre of the enzyme and the peripherally bound ferrocene relay 
and between the relay and the electrode due to penetration of the relay to 
a sufficient depth by the enzyme was postulated to be responsible for 
their observations. Kobayashi et al. (J. Colloid Interface Sci. 1991, 141, 
505) have reacted microfine magnetic particles of magnetite with APTES and 
then cross-linked the surface with a protease; thermolysin, with 
glutaraldehyde. They also utilized 
omega-aminohexylaminopropyltrimethoxysilane, 
4-aminobutylaminopropyltrimethoxysilane and 
2-aminoethyl-aminopropyltrimethoxysilane and showed that maximum enzymatic 
activity was exhibited by the hexyl-silane (50% higher than with APTES). 
The report of Williamson et al. (Anal. Letters 1989, 22, 803), however, 
contradicts the above findings on the spacer length, when an antibody, 
rather than an enzyme, is immobilized to a support. These authors 
covalently attached anti-T.sub.2 mycotoxin monoclonal antibodies on quartz 
fibres by three techniques. The first two consist of the activation of the 
surface hydroxyls of quartz with p-toluene sulphonyl chloride or 
p-nitrophenylchloroformate, followed by the direct attachment of the 
antibody. The third method involves initial silanization of quartz with 
APTES followed by cross-linking of the antibody with glutaraldehyde. 
Almost the same amount of activity was found to be exhibited by the 
antibody on all of the above three surfaces. However, the thermal 
stability of the antibody on the APTES-modified surface at 50.degree. C. 
was considerably better than the antibody surfaces prepared with the other 
two reagents. Significantly, treatment of the sulphonyl chloride or 
chloroformate activated quartz with hexamethylene diamine, prior to the 
immobilization of the antibody with glutaraldehyde, did not improve the 
activity of the bound antibody, in spite of the six-carbon spacer. 
The above brief summary of the thermal and a thermal factors responsible 
for the deactivation of enzymes indicates that even immobilized enzymes 
are not stable above 60.degree.-70.degree. C. In a number of instances, 
nearly 50% of the immobilized enzyme is leached out by washing with a 
buffer or detergent. Use of cross-linkers during the immobilization of the 
enzymes also has a detrimental effect on the retention of the activity by 
the immobilized biomolecules. Recent advances in the isolation of 
thermostable enzymes utilize thermophilic bacteria and considerable 
thermal stability has been claimed for the enzymes made by this route. 
However, a recent report by Brosnan and coworkers (Eur. J. Biochem. 1992, 
203, 225) demonstrates that alpha-amylase isolated from Bacillus 
stearothermophilus is irreversibly deactivated at 90.degree. C. in 1.9 
minutes at pH 5.0. 
Although a large number of publications in documented literature have 
clearly indicated that phospholipids exert a stabilizing effect on the 
activity of enzymes, enzyme preparations so far known have only utilized 
encapsulations in phospholipid liposomes. In two earlier patents (U.S. 
Pat. No. 4,824,529 {1989] and U.S. Pat. No. 4,637,861 [1987]), as well as 
in a recent publication (Anal. Chim. Acta 1989, 225, 369), we have 
demonstrated that phospholipids can be covalently attached to different 
kinds of supports. As analogues of natural biomembranes, these 
phospholipids are expected to impart greater stability than hitherto known 
to enzymes, if the two bio-entities could be covalently linked. It is 
envisaged that the combination of a suitable spacer chain and 
immobilization to a support through a phospholipid would enable the 
formation of thermally very stable enzyme systems with extended 
operational and storage stabilities in the solid state (without any 
buffers), for a variety of applications. 
It is therefore an object of the present invention to provide new compounds 
suitable as spacers as well as linkers for the covalent immobilization of 
enzymes and other biologically active substances either directly or 
through an intermediate compound, onto a substrate. 
It is a further object of the present invention to provide new 
phospholipids suitable for covalent binding to the substrate through a 
spacer compound and to the bioactive molecule. 
It is another object of the present invention to provide preparations 
comprising immobilized biologically active substances, e.g. enzymes, bound 
to the substrate through the spacer compounds and optionally also through 
the phospholipids. 
It is still another object of the present invention to provide methods for 
the preparation of the spacers and phospholipids utilized in the present 
invention. 
SUMMARY OF THE INVENTION 
According to the invention, it is proposed to link enzymes (or other 
biologically active molecules) to selected substrates through certain 
spacer compounds, for example alkoxysilanes and preferably also through 
phospholipid intermediates which are bound to the silanized substrate and 
to the biologically active substance. 
Preferably, the substrate is a solid material having sufficient functional 
groups selected from hydroxyl, carboxylic, amino, mercapto and aldehyde 
groups to enable the spacer compound (alkoxysilane or a diamine or a 
dicarboxylic acid) to be attached to the substrate. 
The substrate may be an inorganic material such as a metal, semiconductor 
(silica or quartz) or ceramic (e.g. alumina); an organic polymer (either a 
naturally occurring material such as cellulose or chitin or agarose, or a 
synthetic product, like modified teflon) and a biomolecule, e.g. protein 
or whole cell, provided that the above-defined functional groups are 
present or can be incorporated onto the surface of this substrate. On 
metallic substrates, hydroxyl groups can be incorporated by oxidation and 
subsequent hydration. 
The biologically active substances, referred to herein also as bioactive 
substances, suitable for the purpose of the present invention, are 
enzymes, antibodies, antigens and other proteins, i.e. compounds with 
polypeptide structure. Certain other molecules such as DNA or hormones 
(with polypeptide structure) are also suitable. 
The enzyme or another bioactive substance is covalently linked to the 
phospholipid as opposed to encapsulation in liposomes proposed in the 
prior art. 
Accordingly, this invention relates, in one aspect, to new preparations 
comprising, in general terms, a solid substrate and a biologically active 
substance linked covalently to the substrate through a spacer compound 
having 7-18 carbon atoms in its alkyl chain. 
The spacer compound may be an alkoxysilane, a dicarboxylic acid or a 
diamine. 
In another aspect, the invention relates to new preparations comprising a 
substrate, a phospholipid covalently linked to the substrate through a 
spacer compound having 7-18 carbon atoms in its alkyl chain, and a 
bioactive substance covalently linked to the phospholipid. 
In yet another aspect, the present invention proposes a new method of 
making the above-defined structures, the method comprising: 
(a) providing a selected solid substrate having sufficient functional 
groups selected from hydroxyl, carboxyl, amino, mercapto and aldehyde on 
its surface, 
(b) binding an alkoxysilane (or a long chain dicarboxylic acid or a long 
chain diamine) to the functional groups of the substrate, and, 
(c) binding the biologically active substance to said alkoxysilane (or 
other spacer). 
Alternatively, the method comprises the following steps: 
(a) providing a selected solid substrate having the required functional 
groups selected from carboxyl, hydroxyl, amino, mercapto or aldehyde on 
its surface, 
(b) binding a spacer compound to the functional groups of the substrate, 
(c) binding a phospholipid to the spacer molecule, and 
(d) binding a biologically active substance to the phospholipid. 
In a preferred embodiment of the invention, the bioactive substance is an 
enzyme. Urease was selected for laboratory tests, but the invention is not 
limited thereto. 
Where a phospholipid is a part of the immobilized structure of the 
invention, a number of spacer compounds may be utilized for bonding the 
phospholipid to the support. Alkoxysilanes with an open chain having from 
7 to 18 carbons atoms, aliphatic dicarboxylic acids and diamines with 
similar alkyl chains can be used as the spacer compounds.

DETAILED DESCRIPTION OF THE INVENTION 
Experimental 
Silicon wafers were used as substrates. The wafers (10 cm in diameter, 
p-doped, natural oxide grown) were purchased from Avrel Colo., Santa 
Clara, Calif. Silica gel Davisil (trademark), Grade 645, 60-100 mesh, 150 
.ANG. (angstrom), 99+% purity) was obtained from Aldrich. 
All solvents were reagent grade samples further purified by drying with an 
appropriate drying agent and distilled prior to use. The following 
products: glutaric anhydride, 10-undecylenic acid, 11-aminoundecanoic 
acid, chloroplatinic acid, t-butyldimethylsilylchloride, triethoxysilane, 
dimethylaminopyridine, N-hydroxysuccinimide, carbonyldiimidazole, 
dicyclohexylcarbodiimide, di-t-butylcarbonate, sebacoyl chloride, 
aminopropyltriethoxysilane and trifluoroacetic anhydride were purchased 
from Aldrich. 
Glycerophosphoryl choline cadmium chloride complex, 
ethyldimethylaminopropyl carbodiimide, Urease (U2000) were purchased from 
Sigma. 
Lyso 1-palmitoyl phosphatidylethanolamine was supplied by Avanti, Urease 
(URE3) was supplied by Biozyme, Urease substrate solution by J. D. 
Biologicals and aminopropyldimethylethoxysilane by Petrarch. 
Synthesis of Omega-Functional Alkyl Triethoxysilanes 
1. Preparation of 11-triethoxysilylundecanoic acid methyl ester: 
a) 10-Undecylenic acid (20 mmoles) was dissolved in methanol (25 ml) and 
concentrated hydrochloric acid (0.5 ml) added. The mixture was reflexed 
for five hours, the excess methanol distilled off and the residue treated 
with cold sodium bicarbonate solution (5% aq., 200 ml). The crude methyl 
ester was extracted with ether, the ether layer washed with water, dried 
with magnesium sulphate and concentrated on a rotary evaporator to yield a 
colorless liquid (yield almost quantitative). Distillation of this product 
under vacuum gave the pure ester, b.p. 104.degree.-5.degree. C./0.1 mm. 
b) Hydrosilylation--The pure methyl ester (2 g) was treated with 
triethoxysilane (3 ml) under nitrogen with the addition of chloroplatinic 
acid (10 mg). After stirring at room temperature for 12 hours, the mixture 
was refluxed for 2 hours. The excess silane reagent was removed under 
vacuum and the residue extracted with pentane under nitrogen. The extract 
was filtered and the filtrate concentrated under vacuum to yield the 
desired product as a colorless liquid Yield 3.5 g). FABMS: MH.sup.+, m/z 
363, [MH-EtOH].sup.+, m/z 318 (100%); FTIR: .sup..nu. C=O 1731 cm.sup.-1, 
.sup..nu. Si-O 1102, 1081 cm.sup.-1. 
2. Preparation of 11-triethoxysilyl undecylenic acid t-butyldimethysilyl 
ester: 
a) 10-Undecylenic acid (20 mmoles) was dissolved in DMF (20 ml) to which 
t-butyldimethylsilyl chloride (4.5 g) and imidazole (100 mg) were added. 
The mixture was stirred at room temperature under nitrogen for 24 hours. 
Excess solvent and silane reagent were removed under vacuum and the 
residue extracted with pentane. The pentane extract was dried and 
concentrated on a rotary evaporator to yield the crude ester, which was 
purified by distillation under vacuum, b.p. 110.degree.-112.degree. C./0.1 
mm, yield quantitative. FTIR: .sup..nu. C=O 1716 cm.sup.-1. 
b) Hydrosilylation--This reaction was carried out under the same conditions 
as described under 1b. The product was characterized by FABMS: MH.sup.+, 
m/z 449 (10%), [MH-EtOH].sup.+, m/z 403 (100%); FTIR: .sup..nu. C=O 1716 
cm.sup.-1, .sup..nu. Si-O 1102 and 1088 cm.sup.-1. 
Generation of the Omega-Carboxylic Silylated Surfaces 1 and 2 
1. Formation of the carboxylic surface 1 (see FIG. 1) 
a) Silanization of silica surfaces by 3-dimethylethoxysilyl 1 propanamine 
was carried out by treating the cleaned surfaces with a solution of 
gamma-aminopropyldimethylethoxysilane 3 (2% in toluene, 20 ml) and 
refluxing for six hours under nitrogen. The substrates were then removed 
and washed with chloroform, methanol and acetone in that order. Surface 
characterization was effected by XPS and ellipsometry. 
b) Treatment of the silanized surface with glutaric anhydride--the above 
substrate 3a was suspended in THF (25 ml) and glutaric anhydride 4 (500 
mg) was added. The mixture was refluxed under nitrogen for 12 hours, the 
substrate removed from the solution and washed extensively with alcohol. 
The resulting surface was characterized by XPS and ellipsometry. 
2. Formation of the carboxydecyl dimethylsilylated surface 2 
A. From 11-triethoxysilyl undecanoic acid methyl ester 8 (FIG. 1) 
a) Silanization of silica substrates with the methyl ester 8 
The substrate was suspended in toluene containing the methyl ester 8 (2% 
solution) at room temperature under nitrogen overnight. It was then 
removed and washed thoroughly with dichloromethane and dried under vacuum 
for several hours. 
b) Hydrolysis of the methyl ester function--after sodium hydroxide, sodium 
carbonate, potassium t-butoxide were found to cleave the surface-to-silane 
siloxane bond, it was attempted to use a non-basic approach. The methyl 
ester-containing substrates were refluxed for 24 hours with lithium iodide 
(200 mg) in DMF (20 ml). The substrates were recovered and washed 
thoroughly with distilled water and vacuum dried before XPS and 
ellipsometric analysis. Alternatively, the methyl ester moiety can be 
removed by refluxing with trimethylchlorosilane (5 ml) and sodium iodide 
(500 mg) for six hours. 
B. From 11-triethoxysilyl undecanoic acid t-butyldimethylsilyl ester (9a): 
a) silanization of silica substrates with the ester 9a--this reaction was 
carried out at room temperature under nitrogen in toluene solution as 
described under the methyl ester 8. Surface analysis was done by 
ellipsometry and XPS. 
b) hydrolysis of the t-butyldimethylsilyl ester moiety--the hydrolysis of 
the silyl ester was accomplished by suspending the substrate from reaction 
a) in aqueous methanolic hydrochloric acid (1:1, 10%, 10 ml) for three 
hours. The substrate was washed copiously with water and dried. Surface 
analysis was done by the usual techniques. 
Immobilization of Urease on the Carboxyl-Functionalized silanized surfaces 
1 & 2 
A. Direct Immobilization 
The carboxylic surface 1 or 2 (100 mg) was suspended in distilled water and 
treated with EDC (5 mg) for 12 hours, the supernatant liquid decanted off 
and the substrate treated with urease (1 mg) in distilled water (1 ml) for 
a period of 48 hours at 5.degree. C. The supernatant liquid was carefully 
drawn off and its enzymatic activity determined by spectrophotometry after 
dilution to 10 ml. The substrate was thoroughly washed with distilled 
water and kept under water in the refrigerator. 
B. Activation with N-Hydroxysuccinimide and immobilization. 
a) Treatment of the surfaces 1 and 2 with NHS--the carboxylic surfaces 1 
and 2 were suspended in THF (5 ml) and DCC (10 mg) in the same solvent was 
added. The mixture was stirred under nitrogen at room temperature for 24 
hours. The substrates were washed thoroughly with methanol and then with 
distilled water. 
b) Reaction of the NHS-activated substrates with urease--the activated 
substrate (100 mg) was suspended in distilled water (1 ml) and urease (1 
mg) added. The mixture was set aside for 48 hours at 5.degree. C., the 
supernatant liquid carefully drawn off and tested spectrometrically for 
residual enzyme activity. The substrate was washed well with water and 
stored in the fridge. 
C. Activation with carbonyldiimidazole prior to immobilization. 
a) Treatment with CDI--the carboxylic substrates 1 and 2 were suspended in 
THF (5 ml) and CDI (20 mg) was added. After standing for two hours at room 
temperature, the substrates were washed with THF and used immediately. 
b) Reaction of the CDI-activated substrates with urease--the CDI treated 
substrates (100 mg) were suspended in water (1 ml) and urease (1 mg) 
added. The mixture was allowed to stand for 12 hours at 5.degree. C., the 
supernatant was removed carefully, diluted ten times and analysed 
spectrophotometrically. The substrates were washed with water and stored 
in the refrigerator. 
Synthesis of Bifunctional Phosphatidylcholines and Their Covalent Binding 
to Carboxy Functionalized Silanized Substrates and to Enzymes. 
A. Preparation of protected omega-functional fatty acid reagents. 
a) 12-trifiuoroacetoxy dodecanoyl chloride--12-hydroxydodecanoic acid (2.2 
g) was dissolved in THF (25 ml) and the solution treated with 
trifluoroacetic anhydride (3 ml) in the presence of a few drops of 
pyridine. The mixture was stirred overnight at room temperature under 
nitrogen and then subjected to vacuum to remove solvent and other volatile 
organics. The residue was extracted with ether, the organic layer washed 
with water, dried and concentrated on a rotary evaporator to furnish the 
trifluoroacetate as a colourless oily liquid (CIMS, MH.sup.+, m/z 313). 
The acid chloride of the above acetate was obtained by stirring it in THF 
solution with thionyl chloride for two hours. The solvent and excess 
reagent were removed under vacuum and the residue used as such for the 
next step (FTIR: .sup..nu. C=O 1805 cm.sup.-1 and 1740 cm.sup.-1). 
b) 11-N-t-butoxycarbonylamino-undecanoic acid--this N-protected acid was 
obtained by reacting 11-aminoundecanoic acid (10 mmoles) dissolved in 0.1M 
potassium hydroxide (till neutral) with di-t-butylcarbonate (1.2 molar 
equivalents) at 0.degree. C. with stirring for two hours. The solution was 
rendered slightly acidic with acetic acid and the precipitated N-BOC 
derivative filtered off, washed with plenty of water and dried in a vacuum 
desiccator (yield 80%)--FTIR: .sup..nu. C=O 1728, 1708 and 1665 cm.sup.-1. 
B. Synthesis of 
1-(12-trifluoroacetoxydodecanoyl)-sn-glycero-phosphatidylcholine (15) 
Glycerophosphorylcholine cadmium chloride complex (1 mmole) was treated in 
aqueous methanol with silver carbonate and the precipitated inorganic 
material was filtered off. The filtrate was concentrated under vacuum and 
the residual free lipid dried by repeated evaporation with dry benzene. 
The dried free glycero-phosphorylcholine (1 mmole) was reacted with 
12-trifluoroacetoxy-dodecanoyl chloride (1.1 mmole) in pyridine medium at 
0.degree. C. for 24 hours. The pyridine was removed under vacuum and the 
residue extracted with chloroform. Drying and concentration on the rotary 
evaporator furnished the title lyso lipid 15 (FIG. 3) which was 
characterized by proton NMR. 
C. Synthesis of 
1-(12-trifluoroacetoxydodecanoyl)-2-(11-BOC-aminoundecanoyl)-sn-glyceropho 
sphorylcholine 16. 
The above lyso lipid (0.8 mmole) was stirred in dichloromethane solution 
with 11-(N-BOC-amino)-undecanoic acid (1 mmole), DCC (1 mmole) and DMAP (1 
mmole) for 48 hours under nitrogen. The mixture was concentrated on a 
rotary evaporator and the residue extracted with ether to remove 
ether-solubles. The ether solubles were treated with chloroform and the 
chloroform solution passed through Rexyn I-300 (trademark). The lipid 16 
(FIG. 3) was further purified by chromatography over silica gel. The 
product was characterized by proton NMR. 
D. Deprotection of the N-BOC group on the sn-2 chain of the choline 16. 
The diacyl lipid 16 was stirred in dichloromethane solution at 
0.degree.-5.degree. C. with trifluoroacetic acid (1 ml) for two hours. The 
product 17 (FIG. 3) was isolated by concentration under vacuum and used 
immediately for the next step. 
E. Covalent binding of the bifunctional lipid 17 to the carboxylic supports 
1 and 2. 
The above lipid 17 was reacted with the NHS-activated supports 11 (FIG. 1) 
in chloroform medium overnight at room temperature under nitrogen. The 
resulting substrate was washed thoroughly with methanol to remove any 
unbound lipid and characterized by ellipsometry. 
F. Deprotection of the trifluoroacetoxyl group on the sn-1 chain. 
The above substrate 18 with the covalently bound lipid was treated with 
sodium bicarbonate (5% aq., 20 ml) for three hours at room temperature. 
The resulting substrate 19 was washed well with water. 
G. Oxidation of the omega-hydroxyl in 19. 
The substrate 19 was suspended in potassium permanganate (5% aq., 10 ml) 
and warmed at 50.degree. C. on a water bath for three hours. The substrate 
(20) was recovered and washed extensively with water and characterized by 
XPS. 
H. Activation of the compound 20 with NHS and condensation with urease. 
The carboxylic support 20 (100 mg) was treated with NHS (100 mg) in 
chloroform for 12 hours at room temperature in the presence of DCC (100 
mg). The resulting substrate was washed extensively with methanol and 
resuspended in distilled water (1 ml). Urease (1 mg) was added and the 
mixture set aside for 48 hours at 5.degree. C. The clear supernatant was 
carefully recovered, diluted ten times and analysed spetrometrically. The 
substrate 21, with both the lipid and enzyme attached, was washed well 
with water and stored in a refrigerator. 
Lipid and Urease Attachment to the Carboxylic Supports 1 and 2 by an 
Alternate Route Utilizing Phosphatidylethanolamines. 
a) Covalent binding of 1-palmitoyl-sn-glycerophosphatidyl-ethanolamine to 
NHS-activated carboxylic supports 11: the NHS-activated substrates 11 
(FIG. 2) were suspended in chloroform and reacted with 
1-palmitoyl-sn-glycerophosphatidylethanolamine (10 mg) for 12 hours at 
room temperature. The substrate 23 (FIG. 4) was recovered and washed well 
with methanol, dried and characterized by XPS. 
b) Condensation of 23 with sebacoyl chloride--the substrate 23 was 
suspended in THF and treated with sebacoyl chloride (20 mg) in the 
presence of a few drops of triethylamine and stood overnight under 
nitrogen. The resulting substrate was suspended in sodium bicarbonate (1% 
aq., 10 ml) for two hours and washed with water to yield 24. 
c) Activation of the carboxyl of 24 and coupling with urease--the substrate 
24 was activated in the usual manner with NHS in a chloroform solution. 
The resulting substrate was suspended in 1 ml water containing urease (1 
mg) for 48 hours at 5.degree. C. and the supernatant liquid was removed 
from the substrate carefully for analyzing residual enzyme 
spectrophotometrically. The substrate 25 or 26 was recovered and stored in 
a refrigerator. 
Spectrophotometric Measurements 
Calibration curves correlating the concentration of the enzyme urease with 
the absorbance of the pH sensitive indicator dye bromocresol purple 
present in the substrate solution were initially obtained. For this 
purpose, a standard solution of urease (1 mg in 100 ml distilled water) 
was prepared and 2 ml of this solution mixed with 1 ml of substrate 
solution in a UV cell. The absorbance of this mixture at 588 nm was read 
off after allowing it to stand for 15 minutes. The procedure was repeated 
with 0.5 ml increments of the urease solution up to a maximum of 7 ml. A 
blank experiment without the enzyme was also done to ascertain the 
absorbance of the substrate solution at the same concentration and 
wavelength 588 nm. Each of the silica substrates on which the urease was 
immobilized (two without the lipid and two with the lipid) was suspended 
in 1 ml of water and 1 ml of the substrate solution was added. After 
standing for 15 minutes, the solution was diluted to 10 ml with water and 
its absorbance read off. 
The absorbances of the filtrates from the enzyme immobilization reactions 
were also determined in a similar fashion after diluting them to 10 ml 
(including the volume of the substrate). The concentration of the 
non-immobilized enzyme was obtained from the calibration curve recorded. 
Since the enzyme used for the immobilization reactions in each case was 1 
mg, subtraction of the concentration of the non-immobilized urease from 
this gives the amount of urease immobilized on the substrates. 
XPS Characterization 
The surfaces were analyzed by means of X-ray photoelectron spectroscopy. 
The results are presented in Table I. 
Instrumentation 
The positive FAB mass spectra were recorded on a VG 70-250S 
double-focussing mass spectrometer operating at 8 kV and equipped with a 
VG 11-250 data system. The FAB beam employed xenon atoms of about 8 kV 
generated by an Ion Tech saddle field gun. Nitrobenzyl alcohol was used as 
the matrix for the spectra. 
Ellipsometric measurements were made on an Auto EL-2 Ellipsometer, Rudolph 
Research, Flanders, N.J., using a He-Ne laser with a wavelength of 6328 
.ANG. and as incident angle of 70.degree.. Data were analyzed on a HP85 
computer. A refractive index of 1.5 for the surface silane film was 
assumed in the calculations. 
X-ray photoelectron spectra were recorded on a Leybold LH200 machine with 
excitation by non-monochromatized Mg K.sub.a radiation. An excitation 
voltage of 1253.6 eV and a detector voltage of 2.65 eV along with an 
emission current of 25 .mu.A were utilized. Take-off angles of 90.degree. 
were employed for both low and high resolution experiments. The spot size 
employed was 4.times.7 mm. Pass energies of 192 eV and 48 eV were made use 
of for broad and narrow region scans respectively. The intensities 
reported were corrected for Scofield factors. 
Spectrophotometric measurements were carried out on a Hewlett Packard 8452A 
diode array spectrophotometer at 2 nm resolution using the software 
supplied by the manufacturer. 
Results and Discussion 
Generation of Carboxy-Functional Silanized Surfaces 
Silica has been selected as the support for the immobilization of enzymes 
in the present invention, as a representative of the silicon-based carrier 
materials such as quartz, controlled pore glass and oxidized silicon chips 
which are extensively used in biosensory applications. It is well-known 
that inorganic carriers have an advantage over organic polymeric materials 
owing to their greater compression resistance and stability of surface 
structure. Further, granular supports such as porous glass or silica, 
utilized for affinity chromatography, are characterized by their 
resistance to acids and organic solvents and to microbial attack, in 
addition to their rigidity, thermal stability and outstanding hydrodynamic 
properties. 
Some serious disadvantages with the silica-type materials for linkage to 
biomolecules is their limited binding capacity, non-specific adsorption 
and denaturation of proteins due to the surface silanol groups. These 
shortcomings could be eliminated by derivatization of the surface, 
especially through the silanization reaction. The most commonly employed 
reagent for this purpose is gamma-aminopropyltriethoxysilane (abbreviated 
as APTES. listed in the Chemical Abstracts under 3-triethoxysilyl 
1-propanamine). The surface amino groups thus generated could be linked to 
the carboxylic groups on the protein in the presence of a carbodiimide 
reagent. Alternately, these surface amino moieties could be cross-linked 
with the amino groups on proteins with reagents like glutaraldehyde, 
cyanuric chloride or a diisocyanate. Of all these procedures, the 
glutaraldehyde-mediated immobilization of enzymes is the most commonly 
utilized one. However, there are several shortcomings associated with this 
protocol which are yet to be addressed. First and foremost, it is known 
that APTES forms multilayer structures on hydroxylic substrates. The 
overall structure is dependent upon factors like the amount of surface 
water, curing temperature, nature of the solvent used for silanization, 
presence of catalysts and pH of the reaction medium, to mention a few of 
these factors. It has also been demonstrated that the amino moieties form 
hydrogen-bonded structures resulting from the partial or total proton 
transfer from the substrate or silane silanol groups. These authors showed 
in a 1988 paper that nearly 75% of the amino groups are involved in 
hydrogen-bonded structures by XPS studies. Further, the mechanism of 
cross-linking of amino groups by glutaraldehyde is still poorly understood 
and controversial, since a mixture of products is always produced 
including Schiff bases, secondary amines, pyridinium-type cross-linkers 
and many other speculative structures. Moreover, the activity of the 
immobilized enzymes with the three-carbon spacer supplied by APTES is very 
low, due to interaction with the support surfaces. 
To overcome the above problems posed by APTES, it is now proposed, 
according to this invention, to design silanizing agents that would 
unequivocally form well-defined, preferably monolayer-level, structures on 
hydroxylic surfaces. In addition, it will be more advantageous to have a 
terminal carboxylic moiety on the silane rather than an amino group, since 
the former does not require cross-linking agents which can create 
complications and could be coupled directly to enzymes. According to the 
invention, it was considered appropriate to develop 
carboxyl-functionalized silanized substrates. These can be generated by 
two different techniques. One consists of initially silanizing a 
hydroxylic substrate with an aminoalkyldimethylsilane and then 
derivatizing the amino moiety of the surface-bound silane with a 
dicarboxylic anhydride to generate a spacer-arm carrying a terminal 
carboxyl group. The second approach involves the synthesis of an 
omega-ester functionalized alkyl chain-substituted alkoxysilane which can 
be initially linked to a hydroxylic support through the alkoxyl and then 
the terminal ester could be hydrolyzed to a carboxylic moiety under mild 
conditions, not detrimental to the surface-siloxane bond. 
As illustrated in FIG. 1., both of the above strategies have been adopted 
to build carboxyalkyl substituted silanized substrates for the purposes of 
the present invention. In these experiments, 
gamma-aminopropyldimethylethoxysilane was selected as the supplier of the 
surface amino group owing to the fact that it can only form one surface 
siloxane bond with virtually no chance for cross-linking and therefore no 
multi-layer build-up. However, more drastic conditions were found to be 
necessary with this silane to effect silanization of hydroxylic substrates 
like silica or quartz, compared to APTES. This is due to Steric factors 
presented by the two methyl groups on the silicon atom. 
The best conditions were found to be, refluxing a substrate with this 
reagent for six hours in toluene, followed by thorough washing with a 
variety of solvents. The next step is the introduction of the carboxyl, 
which could be achieved by refluxing the silanized substrate 3a (FIG. 1 ) 
with glutaric anhydride in THF for several hours to form a five-carbon 
spacer arm with a terminal carboxylic group (surface 1, FIG. 1). 
The alternate approach involves the synthesis of the ester-terminated 
triethoxysilanes 8 and 9a (FIG. 1) starting from 10-undecylenic acid 6. 
This unsaturated acid was converted into either its methyl ester 7 or 
t-butyldimethylsilyl ester 9. Both 7 and 9 could be hydrosilylated with 
triethoxysilane in the presence of chloroplatinic acid catalyst to 
introduce the terminal triethoxysilyl moiety. The hydroxylic substrates 
silica or quartz utilized in the current work were silanized with 8 or 9a 
at room temperature in toluene. The t-butildimethylsilyl ester could be 
readily hydrolysed with dilute hydrochloric acid to yield the carboxylic 
surface 2 (FIG. 1). On the other hand, the normally applicable basic 
hydrolytic conditions did not work for hydrolysing the methyl ester 
surface 5a, since under these conditions, the siloxane bond to the surface 
was also cleaved. However, refluxing with either lithium iodide in DMF or 
trimethylchlorosilane/sodium iodide mixture was found to smoothly 
dimethylate the ester moiety of 5a into the carboxylic surface 2. 
Immobilization of urease on the carboxy-functionalized silane surfaces 1 
and 2. 
The amino groups on the lysine residues on the urease are the targets for 
coupling with the carboxylic substrates 1 and 2. Three different 
approaches were followed to effect this coupling, as illustrated in FIG. 
2. The first approach was the direct one-pot coupling in aqueous medium 
utilizing the water-soluble ethyldimethylaminopropyl carbodiimide 
hydrochloride to initially activate the surface carboxyl of substrates 1 
and 2. The supernatant liquid was drawn off and the diimide-activated 
surfaces were treated with urease in water. The excess unbound enzyme 
present in the solution was estimated by spectrophotometry. The second 
route consisted of initially activating the surface carboxyls 1 and 2 with 
N-hydroxysuccinimide and condensation with urease in aqueous medium in the 
second step. 
The third method involved the activation of the surface carboxyls with 
carbonyldiimidazole and then treatment with urease in aqueous medium. 
Determination of the amount of the non-immobilized enzyme indicated that 
the direct coupling of urease gave about 30% immobilization yield while 
the figures for NHS and CDI activation methods were 60% and 50% 
respectively. 
It was found that with CDI, the intermediate imidazoles 13 (FIG. 2) are 
very unstable and have to be used immediately after formation. On the 
other hand, the NHS esters 11 were reasonably stable over the 24 hour 
period examined and give better yields than the other two methods. Hence, 
for the lipid-immobilization procedures, this NHS-activation method was 
followed. 
Synthesis of bifunctional phospholipids and their covalent binding to 
silanized substrates and to urease. 
The covalent attachment of phospholipids to both substrate and enzyme 
requires the synthesis of lipids with two functional groups, one each on 
the terminal carbon of both acyl chains. In order that they do not react 
with each other or simultaneously with the substrate, both should be 
protected with appropriate protective groups. Moreover, since the head 
groups of phospholipids are extremely sensitive to strongly acidic or 
basic conditions, either the introduction of the omega-protected 
functional acyl chains onto the glycerolphosphocholine skeleton or removal 
of the protective moieties should be carried out under the mildest 
possible conditions. 
The strategy developed according to the present invention to effect dual 
binding of phosphatidylcholines to substrate and enzyme consists of 
introducing an omega-amino moiety onto the sn-2 acyl chain which can be 
linked to the surface carboxylic group and an omega-protected 
hydroxy-substituted acyl chain at the sn-1 position which can be oxidized 
to a carboxyl and bound to an amino group on the enzyme (after the surface 
binding of the sn-2 chain). In other words, the two functionalities on the 
lipid consist of an amine which can be linked to the surface and a 
carboxyl which can be linked to the enzyme. Selective acylation of a 
glycerophosphocholine at the sn-1 position can be carried out by 
controlling the reaction temperature (0.degree. C.) and the amount of the 
acylating agent. Furthermore, attachment of the sn-2 chain of the lipid to 
the substrate is preferable compared to the sn-1 chain since in the 
L-.alpha.-configuration of the lipid, the substrate-sn-2 chain linkage 
would leave both the sn-1 chain and the head group oriented away from the 
substrate surface, a condition favourable for enzyme binding and for 
effective functioning of the lipid in preserving the activity of the 
enzyme, in analogy with the orientation of natural biomembranes. 
FIG. 3 illustrates the eight-step synthetic sequence to effect the binding 
of the phosphocholine class of lipids to the substrate and to the enzyme. 
Condensation of glycerophosphorylcholine with 
12-trifluoroacetoxydodecanoyl chloride at 0.degree. C. yields the lyso 
lipid 15 which is further acylated with 11-t-butoxycarbonylamino 
undecanoic acid in the presence of a diimide to furnish the diacyl 
bifunctional lipid 16. Making use of the stability of the trifluoroacetyl 
protective group to mildly acidic reagents and the lability of the BOC 
group to the same, the latter is removed from 16 to generate the sn-2 
terminal free amino group 17 (FIG. 3). This free amino group is bound to 
the surface carboxyl of either substrate 1 or 2 to form the 
phospholipid-bound substrate 18. In the next stage, deprotection of the 
trifluoroacetyl group on the sn-1 chain is carried out under mildly basic 
conditions and the liberated free hydroxyl 19 is oxidized to carboxyl 20 
with neutral permanganate. The carboxyl was activated to its NHS-ester and 
coupled with urease in aqueous medium to give the silane-lipid-enzyme 
structure 21. 
In order to ascertain the effect of a surface lipid with a reverse 
configuration to that described above, a different strategy was developed 
to bind a phosphatidylethanolamine to the substrate through the head group 
and bind the enzyme to the sn-2 acyl chain. This protocol is illustrated 
in FIG. 4. It consists of initially binding 
1-palmitoyl-sn-glycerophosphatidylethanolamine 22 to either of the 
NHS-activated surfaces 11 to furnish the head group-attached surfaces 23, 
which were subsequently treated with sebacoyl chloride to introduce the 
omega-carboxy functionality on the sn-2 chain (compound 24). These 
terminal carboxyls were activated with NHS and coupled with urease to 
furnish the silane-lipid-enzyme surfaces 25 and 26, respectively. 
In this strategy, the amino moiety of the phosphatidylethanolamine is 
utilized as one of the two required functionalities and the second one is 
formed on the sn-2 chain. Therefore, a monofunctional 
phosphatidylethanolamine serves the same purpose as a bi-functional 
phosphocholine. 
Spectrophotometric measurements 
The results of the spectrometric measurements are presented in Table II. 
The data indicates that a C-11 spacer arm facilitates the immobilization 
of more urease than a C-6 arm (surfaces 2 and 1, respectively). The 
proportion of active enzyme is also larger with the longer spacer 
arm-carrying surface. However, the most striking feature is that the 
lipid-containing enzyme-linked surfaces exhibit a higher active enzyme 
content (10-20% more) compared to immobilized enzyme surfaces without the 
lipid and the proportion of the active enzyme remains more or less 
constant after 48 hours standing. Further, even if the substrates were 
heated in an oven to 100.degree. C. for 15 minutes, they were found to 
still retain the same amount of active enzyme. 
Change of pH from 4 to 8 also did not seem to have any significant effect 
on the activity of the lipid-bound enzyme, as observed by preliminary 
experiments within the scope of this invention. 
The results obtained in this work clearly demonstrate that enzymes linked 
to silanized substrates through phospholipid intermediate cross-linkers 
retain almost their entire activity in the solid phase even after several 
days of storage. The covalent linkage of the phospholipids has been found 
to eliminate leakage problems associated with physisorbed phospholipids, 
experienced by earlier workers, and to provide a stable lipid-bound 
enzyme-based biosensory device. It can be concluded that phospholipids 
exert a stabilizing influence on immobilized enzymes to a considerable 
extent. 
While the experiments conducted to validate the present invention were 
limited in the choice of substrates, spacer compounds, lipids and 
bioactive substances, it will be appreciated by those skilled in the art 
that the invention lends itself to a broader interpretation. 
In particular, it is possible to use any solid substrate as defined 
hereinabove as long as the substrate features specific functional groups 
enabling the attachment of spacer compounds. While the functional groups 
tested herein were carboxylic and amino, it is feasible to utilize the 
other functional groups and adjust the functionality of the spacer 
compounds accordingly. For example, surface aldehyde functionalities, 
generated by the periodate oxidation of a polysaccharide support, could be 
coupled to an amino group on a long chain diamine used as a spacer. 
Analogously, an epoxy or a halo functionality introduced onto the surface 
of a synthetic polymer could be linked to an amino moiety on a diamine 
spacer. 
With respect to spacer molecules, alkoxysilanes with long alkyl chains 
(7-18 carbons) carrying terminal carboxylic moieties can be successfully 
utilized for immobilizing bioactive substances on hydroxy-functionalized 
supports either directly or through phospholipids. Where non-hydroxylic 
polymeric materials are used as supports, other spacers like long chain 
diamines or dicarboxylic acids or omega-mercapto carboxylic acids can be 
made use of. 
The main features in the invention, viz. phospholipid-stabilized 
biomolecules in the immobilized state, could be utilized for a variety of 
other purposes such as drug delivery systems, solid-state 
hormonal/steroidal formulations, enzyme-linked immunosorbent assays, 
contact lenses and other bioreactor applications. 
TABLE I 
__________________________________________________________________________ 
X-RAY PHOTOELECTRON SPECTROSCOPIC DATA ON THE CAROXYLIC 
SURFACES 1 AND 2 AND ON SURFACES WITH LIPID AND/OR ENZYME BOUND 
TO THEM 
ELEMENTAL HIGH RESOLUTION DATA 
SURFACE COMPOSITION b.e. 
area 
b.e. 
area 
b.e. 
area 
STUDIED % C 
% N 
% P 
% Si 
% 0 
(eV) 
(%) 
(eV) 
(%) (Ev) 
(%) 
__________________________________________________________________________ 
1 44.7 
3.4 
-- 24.4 
27.5 
285.0 
81.5 
400.8 
100.0 
99.0 
85 
286.9 
12.4 102.5 
15 
288.8 
6.1 
2 63.7 
-- -- 14.1 
22.2 
285.0 
80.4 
-- -- 98.8 
74 
286.7 
13.2 102.9 
26 
288.5 
6.4 
1 + Urease 
63.2 
6.9 
-- 3.2 
26.6 
285.0 
59.0 
400.2 
100.0 
98.9 
87 
286.3 
23.4 102.3 
13 
288.2 
17.6 
2 + Urease 
68.9 
4.1 
-- 12.5 
14.5 
285.0 
71.1 
400.3 
100.0 
98.9 
87 
286.4 
18.4 102.7 
13 
288.2 
10.5 
1 + Lipid 79.7 
2.1 
1.1 
2.2 
14.9 
285.0 
72.1 
400.0 
61.0 
99.2 
60 
286.2 
11.8 
402.2 
39.0 
101.5 
40 
287.0 
8.2 
289.3 
7.7 
2 + Lipid 80.3 
2.1 
1.1 
2.0 
14.5 
285.0 
73.5 
400.0 
52.1 
99.2 
55 
286.3 
17.8 
402.3 
48.9 
102.4 
45 
287.1 
4.6 
288.8 
4.2 
1 + Lipid + Urease 
71.1 
6.7 
1.8 
4.6 
15.8 
285.0 
78.1 
400.1 
71.0 
99.0 
69 
286.3 
10.9 
402.2 
29.0 
101.4 
31 
287.1 
4.2 
288.1 
6.9 
2 + Lipid + Urease 
76.9 
6.7 
1.3 
2.4 
12.7 
285.0 
70.6 
400.0 
69.0 
99.0 
68 
286.4 
20.1 
402.2 
31.0 
102.6 
32 
287.1 
3.2 
288.4 
6.1 
__________________________________________________________________________ 
TABLE II 
__________________________________________________________________________ 
RESULTS OF SPECTROSCOPIC STUDIES 
Assay of Immobilzied active enzyme 
Substrate Total After one hour After 48 hours 
treated with 
Assay of Unbound Enzyme 
immobilization Amount Amount 
1 mg Urease 
Absorption* 
Amount (mg) 
(%) 
yield (%) 
Absorption* 
(mg) (%) 
Absorption 
(mg) (%) 
__________________________________________________________________________ 
1 0.99012 
0.6 60 40 0.54122 
0.268 
67 0.55844.sup.# 
0.028 
7 
2 0.935100 
0.55 55 45 0.65020 
0.351 
78 0.91578.sup.# 
0.054 
12 
1 + Lipid 
0.88746 
0.52 52 48 0.76525 
0.432 
90 0.74133 
0.413 
.86 
2 + Lipid 
0.71686 
0.40 40 60 0.91563 
0.540 
90 0.88708 
0.516 
.86 
__________________________________________________________________________ 
*absorbance of solution diluted ten times 
.sup.# absorbance measured without dilution 
Structure of Substrate 1: --O--Si(CH.sub.3).sub.2 (CH.sub.2).sub.3 
NHCO(CH.sub.2).sub.3 COOH 
Structure of Substrate 2: --O--Si(CH.sub.2).sub.10 COOH