Transduction of the reaction between urea and covalently immobilized urease by fluorescent amphiphilic membranes

898

Thin Solid Films, 244 ( 1994) 898.- 904

Transduction of the reaction between urea and covalently immobilized urease by fluorescent amphiphilic membranes John D. Brennan,

K. M. R. Kallury

and U. J. Krull*

Chemicul Sensors Group. Erindale Campus, University of Toronto, 3359 Mississauga Road N., Mississauga, Ont., L.5L iC6 (Canada)

Abstract Amphiphiles with chain lengths of 12 and 16 carbons having a triethoxy chlorosilane group at one terminus were covalently immobilized by reaction with hydroxyl groups on the surfaces of planar quartz wafers and optical fibres. The enzyme urease was covalently immobilized onto either carboxylic acid or amine functionalities at the other terminus of the immobilized amphiphiles, resulting in a surface coverage of about 60% of a close-packed monolayer of protein. A small amount (2-3 mol.“/o) of the fluorescent probe nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) was partitioned into the membranes from an aqueous suspension following immobilization of the urease. Addition of urea to coated wafers or optical fibers placed into aqueous solutions resulted in substantial changes of fluorescence intensity from both the carboxylic acid and amine functionalized membranes. A 20 uM change in the concentration of urea could be detected, with a limit of detection of 40 uM of urea. The sensitivity degraded ten-fold over a period of 7 days when the samples were stored in buffer at a temperature of 4 “C. An investigation into the mechanism of the fluorescence response revealed that local alterations of pH at the surface of membranes due to enzymatic hydrolysis of urea resulted in changes in the extent of ionization of both the membrane and the urease. The resulting changes in the electrostatic interactions between the membrane and the enzyme produced alterations in the rotational mobility of the amphiphiles and fluorophores which affected the self-quenching of NBD-PE.

1. Introduction In cases where amphiphilic membranes contain a small amount of a fluorescently labelled phospholipid such as nitrobenzaoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE), selective binding of substrates with proteins at the surface of the membranes can result in modification of fluorescence intensity [ 1, 21. Generation of a fluorescence signal from this system is based on the sensitivity of the emission intensity of NBD-PE to local environmental structure and mobility. Perturbation of the structure or electrostatic fields within the membrane resulting from selective chemical reactions can alter the quantum yield of the probe through changes in self-quenching [ 3,4], and may provide optical signals suitable for the development of biosensors [2]. To be of practical utility, membranes must provide sufficient ruggedness to permit use over an extended period of time (several months) without severe alteration of the response characteristics. In the case of transducers based on fluorescent lipid membranes, this entails stabilization of the assembly onto an optically transparent solid support such as a quartz wafer or

*Author

to whom

correspondence

0040-6090/94/$7.00 SSDI 0040-6090(93)04076-5

should

be addressed

optical fiber. The stabilization method must allow the membrane to retain characteristics of molecular mobility and fluidity which are essential for transduction of selective reactions. Previous work in our group has shown that sensitivity to pH could be obtained from fluorescent membranes that were stabilized onto quartz surfaces by covalent linkage of individual amphiphiles onto a surface [2]. The reaction to form the self-assembled membranes involved displacement of chlorides on terminal silane moieties by hydroxyl groups at the surface of a substrate to form a silyl ether. Such membranes permitted subsequent incorporation of fluorescent species such as NBD-PE by selective partitioning from aqueous solution. It was shown that the exterior of the membrane could be composed of either carboxylic acid [2] or amine [5] functionalities to provide the membrane with sensitivity to pH variations. Covalently immobilized membranes ( CIMs) containing NBD-PE can provide transduction of enzyme-substrate reactions if the products alter the local pH at the headgroup region. For example, the reaction of urea with urease that was linked to the surface of CIMs by carbonyl diimidazole has been studied [5], and it was shown that these membranes retained molecular mobility and fluidity that was sufficient for the transduction

i; 1994 ~

Elsevier Sequoia.

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reserved

J. D. Brennan et al. / Reaction of urea and covalently immobilized w-ease

of the selective enzymatic reaction into a fluorescence signal. The mechanism of fluorescence transduction of selective enzymatic reactions by CIMs has not been elucidated. An understanding of the processes involved in the generation of the fluorescence signal may allow the system to be optimized in terms of parameters such as the fluidity of the membranes, the quantity of fluorophore, and the amount of protein linked to the surface. This work examines the origins of the fluorescence signals caused by the selective interaction of immobilized urease with urea at the surface of CIMs.

2. Experimental details 2.1. Chemicals Urease (EC 3.5.1.5) type IV from jack bean (activity of 105 000 unit gg’), urea and tetramethylurea were purchased from Sigma Chemical Company (St. Louis, MO), and were used without further purification. The fluorescent probe N-( 7-nitrobenz-2-oxa- 1,3-diazole-4yl) dipalmitoylphosphatidylethanolamine (NBD-PE) was purchased from Avanti Polar Lipids (Birmingham, AL). The chemicals used for preparation of covalently immobilized membranes have been described previously [2, 51. The activating agents used for the immobilization of proteins were carbonyldiimidazole (CDI) and cyanuric chloride (Aldrich, Milwaukee, WI). All water was obtained from a Milli-Q 5 stage cartridge purification system (Millipore Corp., Mississauga, Ont.) and had a specific resistance not less than 18 MR cm. All other chemicals were of reagent grade. Quartz wafers were purchased from Heraeus-Amersil Inc. (Sayerville, NJ). Plastic clad silica optical fibers (400 urn diameter) were purchased from Tasso Inc. (Montreal, Que.), and the plastic was removed to expose the silica core. 2.2. Equipment The instrumentation used for fluorescence microscopy and pH titrations of membranes that were deposited on planar wafers or optical fibers is described elsewhere [5, 61. The fluorescence spectrometer was an SLM 4800, and used concave holographic monochromators of 2 nm mm-’ dispersion and 8 nm bandwidth for spectral collection. The samples consisting of monolayers on planar wafers were irradiated at an angle of 30 “C. X-ray photoelectron spectroscopy (XPS) of covalently stabilized monolayers was conducted using a Leybold LH200 spectrometer ( Leybold-Hareaus, Cologne, Germany) [ 51. Ellipsometry was performed with a Rudolph Research Auto EL II null reflection ellipsometer (Rudolph Research Corporation, Flanders, NJ) utilizing a wavelength of 632.8 nm and an incident angle of 70”.

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2.3. Procedures 2.3.1. Preparation of covalently immobilized membranes coated with urease

The procedures for preparation of covalently immobilized membranes containing either C- 16 carboxylic acid moieties or C-12 alkylamine moieties have been described previously [2, 51. Briefly, preparation of carboxylic acid membranes began with 16-OH hexadecanoic acid which was converted to the methyl ester and was then reacted with dimethyldichlorosilane. The preparation of amine monolayers began with 12nitrododecanoic acid which was converted to an acid chloride by reaction with thionyl chloride and was then reacted with y-aminopropyltriethoxysilane. In each case, the silylated species was dissolved in toluene and a quartz wafer was suspended in this solution for a period of 3 h. The ester was converted back to the carboxylic acid by reduction with LiI. The nitro group was converted to an amine by reduction with Zn/H+. The procedure used for linkage of urease onto the C-12 alkylamine membranes via either CD1 or cyanuric chloride has been described previously [5]. Immobilization of urease onto the carboxylic acid membrane began by suspending the sample (total surface area 6.4 cm*) in THF (5 ml) and adding 20 mg of CDI. The samples were allowed to stand at room temperature for 2 h and were then washed with THF. The samples were immersed in water (1 ml), and urease ( 1 mg) was added. The mixture was allowed to stand for 12 h at 5 “C. The samples were washed with water and stored dry at -20 “C until used. 2.3.2. Incorporation of NBD-PE into CIMs and titration of immobilized membranes The procedures used to incorporate NBD-PE into covalently immobilized carboxylic acid and alkylamine membranes have been described previously [5, 61. In all cases the NBD-PE was partitioned into the membrane from an aqueous suspension at a pH of 7.4 and a temperature of 4 “C. The procedure used for titration of covalently immobilized membranes on either planar wafers or optical fibers was identical to that described previously [2, 51, and involved direct addition of acid, base or urea to stirred solutions containing the CIMs.

3. Results and discussion 3.1. Characterization of CIA4s coated with immobilized urease The presence of urease was confirmed by ellipsometry and XPS measurements. The ellipsometry results for the C-16 carboxylic acid membranes and the C-12 alkylamine membranes that were immobilized onto silicon wafers indicated that the thickness of these mem-

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J. D. Brennan et al. 1 Reaction of urea and covalently immobilized urease

branes was 2.8 f 0.1 nm and 2.2 ) 0.1 nm respectively. Addition of urease resulted in an increase in thickness of 5.7 f 0.2 nm and 4.4 Ifr0.2 nm respectively. This increase is much smaller than the dimensions of the enzyme (about lO.Onm x 10.0 nm x ll.Onm) [7]. It is likely that the value is somewhat low due to the incomplete coverage of the surface with urease, since the thickness is an average over an area of roughly 1 mm2. This thickness corresponds to a surface coverage of approximately 60% for the carboxylic acid membranes and of about 50% for the alkylamine membranes. XPS spectra were collected for both carboxylic acid and alkylamine membranes, both before and after addition of urease. The XPS results suggested that the carboxylic acid membranes were 5.3 f 0.5 nm thick, based on the ratio of the C( 1s) and Si(2p) signals corresponding to the monolayer and substrate respectively. These values were calculated according to the method of Andrade [8]. This thickness corresponds to the presence of roughly two monolayers of the carboxylic acid, assuming a thickness of 2.8 nm for a single monolayer. Addition of urease to the C-16 carboxylic acid membrane resulted in the presence of a peak corresponding to nitrogen, and an increase in the C( 1s) signal. The ratio of the N( 1s) peak of urease to the Si(2p) peak of the substrate suggested that the urease layer was 6.4 + 0.6 nm thick, indicative of a surface coverage of about 60”%70”/;1 of a close-packed monolayer. The XPS results for the alkylamine membrane suggested that the thickness of the membrane was 8.5 k 0.6 nm. This value corresponds to the presence of about four monolayers of the alkylamine, assuming a thickness of 2.2 nm per monolayer. Introduction of urease onto the w-aminated quartz surface by either the CD1 or the cyanuric chloride linkage method resulted in a 37O/;,increase in the N( 1s) binding energy peak intensity, and an 88”/11decrease in the Si(2p) binding energy peak from the quartz. The thickness of the urease layer was calculated by comparing the total (C( 1s) + N( 1s)) signal from the overlayer to the Si(2p) peak of the substrate. Using this method, the total thickness value of the membrane and urease was calculated to be 15.1 f 1.3 nm, indicating that the urease layer had a thickness of 7.6 f 1.8 nm. This value corresponds to a surface coverage of 70”/0of a close-packed monolayer of urease. NBD-PE was added to the membrane/protein system after immobilization of protein to avoid interactions between the activating agents and the NBD-PE which contains a potentially reactive amine. XPS studies of both the carboxylic acid and alkylamine samples that contained NBD-PE suggested that there was about 2-3 mol.% of NBD-PE present in the membranes, based on the intensity of the P(2p) and N( 1s) signals

corresponding NBD-PE.

to the phosphate and nitro group of the

3.2. Detection of urea using CIA4s coated with immobilized urease Urease was chosen for these investigations since it is of interest to the field of biosensing, and because it is easily immobilized by reaction of amine or carboxylic acid moieties on the immobilized amphiphiles with the eta-amino functionalities on lysine residues of the enzyme via a carbonyl diimidazole or cyanuric chloride linkage [9, lo]. Additions of urea were performed to prepare a response curve (Fig. l(a)) for a carboxylic acid/urease membrane. When the pH was set to an initial value of 7.0, the membrane provided a sensitivity to changes of concentration of urea of the order of 100 uM with a limit of detection of 200 nM urea. The concentrationresponse curve shown in Fig. l(b) was obtained from an alkylamine/urease membrane that was immobilized by the CD1 method on an optical fiber, and indicates that the monolayer could be used to detect changes of concentration of urea of the order of 20 uM with a limit

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600

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Fig. I. Response of fluorescence intensity to changes in the concentration of urea at pH 7.4: (a) urease covalently linked to a carboxylic acid monolayer on a quartz wafer via carbonyl diimidazole, RZ = 0.990; (b) urease covalently linked to an alkyl amine monolayer on a quartz fiber via carbonyl diimidazole, R* = 0.996.

J. D. Brennan et al. / Reaction of urea and covalently immobilized urease

of detection of 40 uM urea. Addition of tetramethylurea or KC1 to either system produced no changes in the fluorescence intensity. Addition of urea to samples that had the urease denatured by heating at 90 “C for 2 h also gave no fluorescence response. Addition of urea to samples that had no protein present or had BSA adsorbed to the membrane also provided no changes in fluorescence intensity. These experiments confirmed that the catalytic hydrolysis of urea was responsible for the signals that were observed. A fluorimetric assay of activity was conducted for urease that was immobilized onto the C-16 carboxylic acid membrane. The results indicated that the enzyme activity decreased substantially over a period of 48 h, and was effectively absent after a period of 7 days when the sample was stored dry or in phosphate buffer. The results suggest that the covalently immobilized membranes do not significantly stabilize the activity of the immobilized urease, indicating that such membranes may not be useful for the development of practical sensors. The use of ionizable phospholipids for preparation of the supporting membrane should be attempted, since such membranes have been shown to result in the retention of 95% of the activity of immobilized urease over a period of at least 7 days when the sample is stored dry [ll]. 3.3. Mechanism

of fluorescence coated with immobilized urease

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molecule is 100 nm*). Given the turnover number for the urease-urea reaction, a total of 2.24 x lOI hydroxide ions (3.7 x lo-’ moles) will be produced per second at the interface initially, providing a high concentration of basic species in the small volume occupied by the interface. Calculation of the exact pH at the interface for any given concentration of urea in bulk solution is not possible, given the fact that many parameters, such as the amount of active enzyme at the interface, the effects of stirring and the influence of the charged interface on diffusion coefficients, are not known. The opposite response of carboxylic acid membranes with urease to that of alkylamine membranes with urease suggests that the two membranes should have opposite responses to changes of pH (in the absence of urea). A titration of a covalently immobilized C-16 carboxylic acid membrane into which NBD-PE had been partitioned, and with urease linked by the CD1 method, revealed a fluorescence response to pH as shown in Fig. 2(a). The fluorescence intensity decreased as pH was increased from 4.0 to 9.0, and the signal was reversible as pH was cycled. Titrations were also performed to investigate quantitatively the pH response of alkyl amine monolayers that were coated with urease.

response for CIMs

The products of the reaction of urea with urease indicate that the pH of solution shifts towards more basic conditions as the reaction proceeds. Figure 1 indicates that increases in the concentration of urea caused decreases in the fluroescence intensity of the carboxylic acid membrane that contained urease, but caused increases in the fluorescence intensity of alkylamine membranes that contained urease. All changes of fluorescence intensity occurred over a period of lo-20 s after addition of urea, and the intensity remained at a new, steady value after that time. Urea is uncharged, and therefore the concentration of the species at the membrane-aqueous interface should be similar to that in bulk solution. The turnover number of urease [ 121 is 1 x lo4 s-‘, indicating that the urea at the membraneaqueous interface would be rapidly converted to base. As the reaction proceeds, the pH at the interface initially becomes basic, and a diffusion gradient for hydroxide ion is set up from the interface back into solution. Based on the actual time of response, it appears that over a period of several seconds a steady state of pH is reached due to diffusion of urea and hydroxide ions. Assuming that the membrane has a surface coverage of 70% urease (from XPS studies), there will be about 2.2 x 10” molecules of urease on the surface (assuming the molecular area per urease

Fig. 2. Response of fluorescence intensity to changes in pH for (a) urease covalently linked to a carboxylic acid monolayer on a quartz wafer oia carbonyl diimidazole; (b) urease covalently linked to an alkyl amine monolayer on a quartz fiber via carbonyl diimidazole.

J. D. Brennan et al. / Reaction of’ urea and coaalenrly immohilixd urcw.se

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TABLE 1. Slope of response of fluorescence pH: the pH is shifting from 4.0 to 9.0 Membrane

intensity

Slope of response

Carboxylic Carboxylic

acid acid + urease

Positive Negative

(Fig. I(a)) Alkylamine Alkylamine

+ urease

Negative Positive

(Fig.

to changes

of

to pH

l(b))

For this system, the fluorescence intensity increased as pH was increased from 4.0 to 9.0, as shown in Fig. 2(b). These results were consistent with the fluorescence response observed during addition of urea to the membranes that contained urease. The fluorescence responses observed from carboxylic acid membranes that were coated with urease were opposite to the responses obtained on addition of acid to covalently immobilized carboxylic acid monolayers that did not contain urease [2], suggesting that the electrostatics of the membrane/protein system were different from those of membranes that had no protein present. Such an opposite response was also observed between amine membranes, and amine membranes that were coated with urease. These trends are summarized in Table 1. A determination of how the presence of protein affects the response of the underlying membrane is necessary to establish the mechanism responsible for changes of fluorescence intensity with pH. Three factors must be considered: (1) the immobilization of a protein onto the membrane before addition of NBD-PE may alter the final location of the probe and may therefore influence the fluorescence response characteristics of the system; (2) the reagents used to activate the membrane for covalent linkage of proteins may alter the structure of the headgroups of the membrane and thus alter the fluorescence response to changes of pH; (3) most proteins are negatively charged at neutral pH and become neutral and then positively charged as the pH becomes more acidic, so that the protein itself may dominate the electrostatic characteristics and thus the fluorescence signals from a protein/membrane system. Each of these factors is considered below. The location of the NBD-PE probe in the membrane was examined using fluorescence microscopy and spectroscopy. Images obtained from wafers that were incubated in water that contained NBD-PE showed regions of variable fluorescence intensity with no discrete boundaries between bright and dark regions. Instead, there were areas of intermediate brightness at the borders between light and dark areas. If it is assumed that the NBD-PE partitions into regions of low packing

density within the membrane, then the lack of discrete boundaries between bright and dark regions may suggest that membranes containing proteins have discrete regions of high and low packing density, but gradual transitions between these regions, perhaps as a result of the protein being edge active and thus partitioning to the phase boundaries. Overall, the images suggest that the protein influenced the distribution of the fluorophore within the membrane, and perhaps altered the structure of the underlying membrane. A fluorescence spectrum collected from a sample consisting of a C-16 carboxylic acid membrane that was coated with urease showed an emission maximum at 540 nm. This was similar to the emission maximum for the probe in Langmuir-Blodgett monolayers of stearic acid deposited on quartz which were submerged in water. This suggests that the probe was in an environment of intermediate polarity, ruling out direct association of the probe with the aqueous environment or with the hydrophobic regions of the enzyme. It must be noted that neither the images nor the spectra can provide conclusive evidence about the location of the probe. The results do, however, support the supposition that the NBD-PE is associated with the membrane. For both the alkylamine and carboxylic acid membranes, linkage of urease to the headgroups results in a reversal of the fluorescence response to changes of pH (see Table 1). The converse fluorescence response is probably not the result of an alteration of the structure of the headgroups in the membrane when CD1 is used as the activating agent. The dimensions of a single urease molecule are 10 nm x 10 nm x 11 nm, and thus each urease would cover an area of approximately 100 nm’. The amphiphile has a molecular area of about 0.25 nm2 for either membrane, and thus the amphiphile:urease ratio is 400: 1. If the urease reacts with only one amphiphile, then 399 headgroups out of 400 would retain the carboxylic acid or amine functionality (or more if there is not 100% coverage of the membrane by the protein, as in this work), indicating that essentially all headgroups remain unreacted. The XPS results confirm that regeneration of headgroups is achieved in the final step of the covalent immobilization of the urease by CDI. This rules out direct alteration of the headgroups by the linking agents as a plausible mechanism to explain the observed fluorescence response, and suggests that the protein (either alone or in combination with the membrane) is somehow affecting the signal. The isoelectric point of urease is 5.1 [ 131. Above a pH value of 5.1 the protein is negatively charged, and the charge on the protein will be reduced as the pH is brought from a value of 9.0 to a value of 5.1. The electrostatics therefore resemble those of a carboxylic acid membrane, which provides decreases in intensity as pH becomes more acidic. To investigate the role of the

J. D. Brennan

et al.

1Reaction

of urea and

protein on the fluorescence signal directly, a sample was prepared in which the membrane did not have any ionizable groups. In this case the electrostatic characteristics of the protein would control the fluorescence response to changes of pH. The sample was prepared by using cyanuric chloride to link urease onto the surface of a C-12 alkylamine membrane. The chloride groups on cyanuric chloride are rapidly hydrolyzed to hydroxyl functionalities in aqueous conditions, and the new headgroups will undergo the following tautomerization as the pH becomes more basic R-C( OH)=N-R +-+R-(X-NH-R Linkage of cyanuric chloride to the amine groups of the membrane therefore results in a surface in which uncharged headgroups of the membrane have little sensitivity to changes of pH over the range from 9.0 to 4.0. A pH titration was performed for a urease sample that was linked with the cyanuric chloride activating agent and had NBD-PE partitioned from water. This sample showed no changes of fluorescence intensity as the pH was changed between values of 4.0 and 9.0. This suggests that the NBD-PE was not driectly associated with and influenced by the urease, and that the headgroups of the underlying amphiphilic membrane control the fluorescence response of the membrane/protein system. Overall, the results suggest that the responses observed from membranes that contain proteins are not caused by direct electrostatic effects of the protein, or by altered headgroups (i.e. derivatization by activating agents). This leaves a combined electrostatic interaction of both the headgroups and the proteins as the likely mechanism of the fluorescence response. 3.4. A model for intensity with immobilized

changes of CIMs coated

urease

An investigation of the processes responsible for changes in the intensity of NBD-PE in CIMs has shown that the signal is not based on direct effects of pH, polarity, surface potential or surface charge on the emission characteristics of the probe. It has been shown that emission intensity of NBD-PE is strongly influenced by changes in the local concentration of the probe, owing to alterations in the extent of self-quenching [ 141. Self-quenching of NBD-PE has been shown to be based on a combination of static quenching by formation of dimers or aggregates of NBD-PE, and Forster energy transfer to the aggregates, which act as statistical trapsites [ 141.Alterations in the surface charge of CIMs will change the electrostatic interactions between adjacent amphiphiles, which in turn can result in changes in the degree of rotational mobility of the covalently bound amphiphile molecules and NBD-PE. Increased rotational mobility would be expected to result in an increase in the number of statistical trap-sites, and therefore

covalently

immobilized

urease

903

increases in self-quenching [ 141. It is thus necessary to determine how the electrostatic interactions between urease and membranes may change with pH to affect molecular mobility within a membrane. The carboxylic acid membrane containing urease produced decreases in fluorescence intensity as the pH was increased, indicating that there was an increase in the self-quenching of the NBD-PE. It is likely that an increase in the area swept out by the cone associated with rotational mobility is a result of the proteins occupying less surface area due to a reduction in the attractive charge interactions between the protein and the membrane. This situation would occur as a result of the net charge of the protein becoming increasingly negative as the pH is raised, owing to urease having an isoelectric point of 5.1. At the same time, the carboxylic acid headgroups become negatively charged, thus repelling the protein from the surface of the membrane. The repercussions of this situation are an increase in the mobility of the fluorophores and an increase in fluorophorefluorophore interactions (more trap-sites), providing an increase in self-quenching as the pH is increased. For the alkylamine membrane, the protein again becomes more negatively charged as the pH increases from 5.1 to 9.0, but the membrane amphiphiles are positively charged and become neutral as the pH is raised. It is expected that at low pH (around 4.0) the membrane is highly positively charged while the protein is slightly positive, thus a small amount of electrostatic repulsion should exist between headgroups and the protein. Under these conditions the membrane should have some degree of mobility. As the pH is raised to a value of about 8.0, the protein becomes neutral and then negatively charged, while the membrane remains essentially completely ionized and thus positively charged. As the negative charge density of the protein increases, attractive electrostatic interactions between the protein and the membrane increase and the protein therefore interacts with a greater number of headgroups. This would reduce the mobility of the membrane and would cause decreases in self-quenching and increases in intensity, as observed during pH titrations. Above a pH of 8.5 the amine headgroups would become neutralized while the protein remained negative, therefore reducing the electrostatic interactions of membranes with headgroups, and potentially allowing the membrane to become somewhat more mobile again. The fluorescence intensity does level off above a pH value of 8.5, suggesting that this effect did exist.

4. Summary This work has demonstrated that an amphiphilic monolayer may be covalently immobilized onto the

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J. D. Brennan et al. / Reaction of urea and covalently immobilized urease

surface of a quartz wafer or optical fiber with retention of structure. It has further been demonstrated that an enzyme, such as urease, may be covalently linked onto the surface of the membrane with retention of some activity, thereby providing a completely stabilized monolayer/protein system. Incorporation of a fluorophore into this system was achieved, and the distribution and function of the fluorophore were found to be stable over periods of months, even when samples were stored in buffer at room temperature. The urease had a high activity directly after immobilization, but the activity decreased substantially over a priod of 2 days and was effectively lost after 7 days. The denaturation of urease significantly reduced the long-term stability of the membrane-based sensor, indicating that such a sensor is not yet of practical use. Addition of urea to membranes that were coated with covalently linked urease resulted in the production of ammonium and carbonate ions at the surface of these membranes. These ions altered the pH and electrostatic fields at the surface of membranes that contained headgroups that were sensitive to dynamic changes in pH. The mechanism indicated that increased electrostatic attraction between the protein and the membrane resulted in a decrease in the mobility of the NBD-PE, and produced a decrease in the number of trap-sites and therefore an increase in fluorescence intensity. Overall, the speed and reversibility of the analytical measurements confirm the feasibility of using the covalently stabilized protein/amphiphile system as an optical transducer.

Acknowledgments

We are grateful to the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support of this work. J.D.B. would also like to thank NSERC for the provision of a fellowship.

References I J. D. Brennan, R. S. Brown, C. P. McClintock and U. J. Krull, Anal. Chim. Acta, 237 (1990) 253. 2 J. D. Brennan, R. S. Brown, D. Foster, R. K. Kallury and U. J. Krull, Anal. Chim Acta, 255 (1991) 73. 3 D. Hoekstra, Biochemistry, 21 (1982) 2833. 4 R. S. Brown, J. D. Brennan and U. J. Krull, J. Chem. Phys.. submitted for publication. 5 J. D. Brennan, R. S. Brown, A. Della Manna, K. M. R. Kallury, P. A. Piunno and U. J. Krull, Sew. Actuat., C, B/l (1993) 109. 6 J. D. Brennan, R. S. Brown, S. C. Ferraro and U. J. Krull, Thin Solid Films, 203 (1991) 73. 7 E. Jabri, M. H. Lee and R. P. Hausinger, J. Mol. Biol., 27 ( 1992) 934. 8 J. D. Andrade, in J. D. Andrade (ed.), Surjtice and Inter$cial Aspects of Biomedical Polymers, Vol. 1, Swface Chemistry and Physics, Plenum, New York, 1985, p. 105. 9 H. H. Weetall, Methods Enzymol.. 44 (1976) 135. IO P. A. Srere and K. Uyeda, Methods Enzymol., 44 (1976) I I - 19. 11 K. M. R. Kallury, W. E. Lee and M. Thompson, Anal. Chem., 64 (1992) 1062. 12 D. Voet and J. G. Voet, Biochemistry, Wiley, New York, 1990, p. 337. 13 The Merck Index, Merck and Co. Inc., Rahway. NJ, 10th edn., 1983, p. 9667. 14 J. D. Brennan, Ph.D. Thesis, University of Toronto, 1993.