Selective interactions of concanavalin A at lipid membranes on the surface of an optical fiber

Taianfa, Vol. 37, NO. 8, pp. 801-807, MO

0039-9140/90$3.00+ II.00 Copyright 0 1990Pergamon Press plc

Printed in Great Britain. All rights reserved

SELECTIVE I~E~CTIONS OF CONCANAVALIN A AT LIPID MEMBRANES ON THE SURFACE OF AN OPTICAL FIBER ULRICH J. KRULL, R. STEPHPN BROWN, BRUCE D. HOUGHAM and IVAN H. BROCK Chemical Sensors Group, Department of Chemistry, Erindale College, University of Toronto, 3359 Mississauga Road North, Mississauga, Ontario, Canada L5L lC6 (Received 12 May 1989. Revised 18 December

1989. Accepted 12 January 1990)

Summary-An optical con@uration was developed for sampling fluorescence coupled into an optical fiber from evanescent wave excitation of fluorescent materials at a lipid membrane on a quartz fiber surface. Selective interactions of pyrene-labor concanavalin A located on a phosp~tidyl cholin~holes~rol lipid membrane with fluorescein isothiocyanate-labelled dextran in bulk aqueous solution were monitored by the intrinsic fluorescence sensing configuration. Monosialoganglioside, G,,, was employed as a receptor in a phospholipid membrane on an optical fiber for selective measurement of pyrene-labelled concanavalin A in solution. Quantitative measurement was hindered by non-selective adsorption of cancanavalin A, but the potential for use of a lipid membrane in a fluorometric biosensor was established.

In development of electrochemical and optical biosensors’*2 lipid membranes have great potential as surfaces which can support a wide range of biological materials such as enzymes, antibodies and receptors, in a matrix within which they remain biochemically active. The high degree of biochemical activity, coupled with the extreme thinness of the membrane provides for relatively large and very rapid interfacial response signals. An example is the selective complexation of polysaccharides by the lectin concanavalin A (Con A) at bilayer or monolayer lipid membranes.3 Con A is a globular protein containing saccharide, ion (co-factor) and hydrophobic binding sites,4*5with dimensions about 4.0 x 3.9 nm, as determined by crystal structure studies.6 Con A is non-selectively adsorbed on bilayer and monolayer lipid membranes, and can bind polysaccharides such as dextran or glycogen to form aggregates which tend to alter the physical structure of the lipid matrix within a few tenths of a second.‘,’ The selective interactions of Con A at lipid membranes provide an interesting model for testing electrochemical and optical detection. Optical methods such as fluorometry are inherently sensitive and less affected by procedural artifacts than are electrochemical methods, which may suffer from problems such as mixed potentials and charging currents. In particular, total internal reflectance fluorometry is a sensitive technique for detecting

the presence of biochemical species at a solid-liquid interface. The solution of Maxwell’s wave equations for total internal reflection within a waveguide indicates that the exponential decay of the electric field intensity of electromagnetic radiation extends beyond an interface defined by two materials of different refractive index (if the refractive index of the waveguide, n,, is greater than that of the external coating, FZ~),~~”producing so-called evanescent radiation. Under these conditions the external electric field intensity Z, at a distance normal to the surface is given by Z = Z(B)exp( --22&J

(1)

where Z(e) is the electric field intensity at the interface and dr is the value of z at which Z = Z(Q/e. The value of dP for a radiation wavelength 1 is ’ = *i(n+ini;B

- n:)‘i2

(2)

where 0 is the angle of incidence of the totally internally reflected electromagnetic radiation on the interface. The use of evanescent excitation for an intrinsic optrode con~g~ation, in which a lipid membrane is deposited on the surface of an optical fiber, offers the advantages of mechanical stabilization of the membrane, increased effective path-length for optical excitation (particularly on a multimode optical fiber) and analytical 801

802

ULRICX-I J. KRULL

sampling that is restricted to the chemically selective interface.” Biochemical interfaces have been studied fluorometrically by evanescent excitation techniques. These investigations have established aspects of quantitative selective binding, and the kinetics and mechanisms of binding processes such as those involving imm~ochemical systems.‘2-14Relatively little work has been done at optical wavelengths for investigation of lipid membranes and associated protein-mediated selective interactions. Recent experimental work has confirmed that lipid membranes containing fluorescent lipid species at concentrations of l-2 mole% can be detected ~uoromet~~lly after their deposition on an optical fiber by Langmuir-Blodgett monolayer transfer,” but the fluorescence intensity is rather low, so the technique may not be suitable for development of transducers based on fluorescent lipid membranes. This paper reports an investigation of optimization of collection of fluorescence radiation from lipid membranes deposited on quartz optical fibers. The selective complexation of Con A with saccharide residues was chosen to illustrate acquisition of fluorescence signals from a chemically-selective lipid monolayer, the use of a lipid membrane as a structural support for the adsorbed selective protein, and the use of an irreversibly bound receptor site supported in the lipid matrix, in the form of the saccharide residue of a glycolipid for complexation with the lectin. EXPERIMENTAL

Reageptts The materials used for lipid membrane preparation were egg phosphatidyl choline (EPC), cholesterol (C) (Avanti Biochemicals, Birming ham, AL) and monosialoganglioside from bovine brain, Gu,, 95% (Sigma Chemical Co., St. Louis, MO), and were used without further pu~fication. Vesicular solutions of EPCjC were formed by preparing an ethanolic solution of the lipid, evaporating the ethanol to leave a dry film, and suspension of this in an aqueous buffer consisting of 10mM Tris, 10e4M calcium chloride and manganese chloride at pH 7.0. The EPC/C molar ratio was selected, and sufficient buffer was added to achieve a total lipid concentration of 1.3 mg/ml. Vesicles of EPC/C/GM1 in molar ratio 51312 were prepared by a similar procedure to give a total lipid concentration of

et ui.

1.4 mg/ml. Similar solutions of EPC/C and EPC/C/Gkll in hexane were prepared for formation of monolayers on a Lanker-Blodgett trough (Lauda Model 1974, Sybron Brinkman, Toronto). Before the dip casting the fiber surfaces were treated with a solution consisting of 0.1% v/v octad~lt~chlorosilane (Aldrich Chemical Company, Milwaukee, WI), 80% hexadecane, 12% carbon tetrachloride and 8% chloroform. Irene-buty~l concanavalin A, (Py-Con A), and fluorescein iso~i~yanate dextran (FITC-dextran, m.w. 4000), were used as received (from Molecular Probes, Eugene, OR) and were dissolved in the aqueous buffer used for the vesicular work. All solvents were reagent grade, and water with a resistivity of at least 18 MiZ. cm was obtained from a Mill&Q cartridge filter system. Apparatus Vesicle aggregation induced by selective and non-selective binding events was monitored by measurement of the absorbance at 483 nm of aqueous solutions in a l-cm path-length fusedsilica cuvette with a DU-50 spectrophotometer (Joann). Vesicular solutions were dispersed with a Vibra-Cell Model 250 probe-tip sonicator (Sonics and Materials, Inc., Danbury, CT) set at 40 W power, after suspension of the vesicles in aqueous solution and before the experiments were started. Fluorescence from the optical fibers (silica core fibers, 400 pm, Tasso, Montreal, Canada) was induced by a nitrogen laser (LN 103, Photochemical Research Associates, London ON, Canada), and the emitted radiation was processed by a monochromator (Bentham M300, Gptikon, Waterloo, ON, Canada) and photomultiplier tube detector (R928, Hamamatsu). The output from the photomultiplier was passed to a gated-integ~tor~boxcar averager (Stanford Research Systems) which was operated with a sampling gate width of 40 nsec. The fluorescence acquisition equipment was operated in the two configurations shown in Fig. 1. Procedures Vesicle experiments, These experiments were done by standard procedures’5~‘6 to establish that lipid membranes containing GM1would be capable of selectively binding Con A. The lightscattering experiments monitored the agglomeration of vesicles caused by cross-linking mediated by the lectin.

Selective interactions of concanavalin A Mirror / --m

/e

Lens

II -

Fiber

Fig. 1. Experimental optical configuration used for collection of fluorescence from quartz optical fibers by using (a) detector a for end-on detection of radiation coupled into the fiber; (b) detector b for side-on collection of radiation scattered from the fiber.

The vesicular solutions were sonicated for 20 min at room temperature before the experiments were started. Vesicle solution was mixed with an equal volume of Tris buffer containing Con A at 1.3 mg/ml concentration, and the absorbance of the solution was measured periodically for the next 60 min. Solutions were checked for homogeneous suspension of aggregates before each measurement. Fluorescence experiments. The ability of the equipment to collect fluorescence radiation in the two configurations shown in Fig. 1 was established with a sample consisting of an optical fiber which had been coated with Py-Con A by non-selective adsorption from an aqueous buffered solution containing lo-*M Py-Con A. The ends of all the fibers were polished with a rotary sander and a series of successively finer polishing grits. Non-selective adsorption of Py-Con A was investigated for four fiber surfaces, each 3 cm in length: (a) untreated fiber surfaces exposed by removal of the sheath and cladding with a wire stripper (all fibers were initially cleaned by soaking for 24 hr in Nocromix (Godax, New York), followed by neutralization with sodium hydroxide solution and a water rinse); (b) fiber surfaces which had been coated with a lipid monolayer by Langmuir-Blodgett casting at 7 mm/min from the appropriate lipid mixtures held at a constant surface pressure of 30 mN/m; “*‘* (c) alkylated surfaces prepared by immersion of the fibers in the silane solution for TAL

37,8--D

803

60 min, with vigorous agitation every 10 min; (d) alkylated surfaces modified by deposition of a lipid monolayer. ‘*The hydrophobic alkylation layer deposited on quartz had been observed to be stable for periods of many months and required no specialized storage. Lipid monolayers deposited on quartz or alkylated quartz tend to change spontaneously with time and were used in these experiments within 24 hr of fabrication” Transfer of deposited lipid monolayers through an air-water interface causes massive structural rearrangement and loss from the surface of the substrate. All lipid-coated samples were therefore handled under aqueous solution. All the fibers were supported in a cuvette containing Py-Con A in buffered aqueous solution. Experimentation was continued in some cases by addition of FITC-dextran as an aqueous solution to the sample cuvette to achieve final polysaccharide solution concentrations of 10-6M. RESULTS AND DISCUSSION

The utility of total internal reflection techniques can be understood from equation (2), which indicates that the optical intensity of excitation radiation, and adjustment of penetration depth for sampling near an interface, can be controlled by appropriate selections of excitation wavelength, incident angle and refractive index ratio. Figure 2 provides some examples of theoretical results calculated from equations (1) and (2) for the nitrogen laser source. The experiments described in this work make use of a broad and uncontrolled range of angles of incidence in a multimode distribution at a fixed wavelength (337.1 nm) and a relatively invariant refractive index ratio. The calculations indicate that lipid monolayers (45 nm thick), alkylated surfaces coated with lipid monolayers (7-10 nm) and films incorporating lipid monolayers with Con A and dextran (5-20 nm), will all be exposed to relatively high excitation intensities of the laser radiation provided that the refractive index of the organic layers will be in the range 1.45-1.5. While most descriptions of intrinsic mode optrodes have used an evanescent radiation model, it should be noted that films of many biological materials, such as lipids and proteins, have an index of refraction close to that of the quartz optical fibers (approx. 1.5). If the refractive indices of the waveguide and organic layer were identical, internal reflection would not occur at the fiber/coating interface.

ULRICHJ. KRULL et al.

804

1.2

Refractive

Distance

1.3

1.4

I

1.5

index

(nm)

Fig. 2. Calculated relationships of the evanescent field: (a) penetration depth (d,) and (b) intensity at various distances from a fiber surface for coatings with various indices of refraction, based on equation (2), for a fiber index of refraction of 1.5 and a propagated wavelength of 337 nm.

Optical excitation of the organic layer would be due to the coating acting as a physical extension of the quartz waveguide. Even though the evanescent field would than be propagated beyond the coating/ambient (aqueous solution) interface, there would still be a high degree of surface selectivity and bulk solution interference should be minimal. The two configurations shown in Fig. 3 highlight the physical phenomena associated with the two collection strategies shown in Fig. 1. The large diameter of the fibers used in this work means that they act as multimode waveguides with respect to optical transmission. A collection strategy where radiation is collected perpendicular to the surface of the fiber (Fig. 3A) would be efficient in a conventional spectrofluorimetric experiment using a standard lcm cuvette. The fluorescence generated in a TIRF experiment does scatter perpendicularly to the fiber, but can also couple back into the waveguide under certain conditions. Equation (2) represents the exponential decay of electric field intensity across an interface. A thick (relative to d,) and homogeneous fluorescent film exposed to such an evanescent wave would emit

radiation at intensities which would decay exponentially with distance from the interface, since fluorescence intensity is proportional to excitation power. This would generate the equivalent of an external component of a new evanescent wave (longer wavelength), which could couple with the waveguide across the refractive index interface to produce a high electric field strength within the waveguide. 14*lg The presence of such a process could provide for collection of most of the fluorescence by the waveguide, and enable detection to be based on sampling of the fluorescence as shown in Fig. 3B. Evidence has recently appeared indicating that a monolayer lipid membrane or protein film which provides fluorescence in a localized plane can satisfy the conditions of equation (2) and provide significant capture of fluorescence within a waveguide.” The experimental results indicate a considerable difference in the collection efficiencies of the optical configurations shown in Fig. 1. Observation of fluorescence perpendicular to an optical fiber (Fig. lb) coated with Ey-Con A provided a signal magnitude lower by factors up to 100 than that obtained from coupling the fluorescence into the fiber (Fig. la). The efficiency of perpendicular collection was so limited that the distinctive fluorescence profile of the pyrene moiety could not be resolved by the instrumentation used in this work. The same equipment could readily provide a distinctive spectral profile for pyrene when used in the @

Fluarmaont Sample

@

Fluomscont

Sample

Fig. 3. General strategies for collection of fluorescence from waveguides, showing (A) light-scattering and (B) capture of radiation within the light-guide.

Selective interactions of concanavalin A PvCon-A on fiber

0-l 340

I

I

I

.

420

I

I

I

I

I

I

1

500

,

560

I

I

I

I 660

Wavelength (nm)

Fig. 4. Fluorescence intensity measurements for pyrene-concanavalin A adsorbed on the surface of a quartz fiber in the end-on fluorescence collection configuration, (a) after adsorption on the fiber surface and (b) background spectrum from the uncoated fiber.

configuration of Fig. la, as shown in Fig. 4. Note that the intensity of the nitrogen laser source provided adequate spectral separation of the fluorescence from the excitation radiation, even though the laser wavelength was well removed from the wavelength of maximum absorption by the fluorophore. The efficiency of signal collection by coupling the fluorescence into a fiber does not prove that the effect is necessarily due to the process described by equation (2). If the refractive indices of the fiber and organic film were closely matched, then the coupling of fluorescence into the waveguide would be very efficient. In this case the fluorescence would be produced within the waveguide and would remain in the structure if the propagation angle was greater than the critical angle for total internal reflection. Concanavalin A as a receptor Analysis of the adsorption of Py-Con A onto various fiber surfaces indicated no clear preference for non-selective adsorption of the protein by quartz, alkylated quartz or EPC/C-coated fibers. The pH of the solution used in these experiments induces the formation of a quaternary protein structure which takes the form of a tetrameric association of Con A. The complex is physically massive (m.w. N 10’) and contains a minimum of 4 hydrophobic binding sites, as well as numerous areas of relatively high polarity. These attributes combine to provide the protein with a capacity to deposit non-selectively onto many different surfaces. Attachment of the protein at significant concentrations to EPC/C membranes was consistent with previous work,3 and the usefulness of the evanescent

805

radiation technique was clearly indicated by the elimination of extraneous absorption and fluorescence due to Py-Con A in the bulk aqueous solution or on the sample cuvette surfaces, when the system was operated in the configuration shown in Fig. la. The Py-Con A on the EPC/C membrane was able to complex FITC-dextran, which was added incrementally directly to the solution in the sample cuvette supporting the optical fiber. A response curve for this experiment is shown in Fig. 5 and analysis of the FITC signal demonstrates selective complexation. The results, collected after the test solution had been incubated for 20 min to allow equilibration of interactions between free Py-Con A and FITC-dextran in the solution, are suitable only for analysis of trends since determination of the amount of Py-Con A and FITC-dextran in dynamic equilibrium on the surface of the fiber was not attempted. The Py-Con A is selectively partitioned onto the surface and its concentration there may be over 100 times that in the bulk solution.3 The concentration-response curve for interaction of PyCon A with FITC-dextran shows that selective binding occurs (results corrected for bulk solution concentration of FITC-dextran). The nonlinear response of low sensitivity at 10m6M FITC-dextran may be a result of saturation. Blank experiments using only FITC-dextran indicated that this material was not selectively adsorbed on any of the optical fiber surfaces investigated. GM, as a receptor Glycolipids have been extensively investigated as membrane-intrinsic molecular receptors in model membrane studies of lectin-

.f 0.6 '; Ii L 0.4 w 0.2 n SO

-9

-6

I -I

I -6

I -5

Log(CFITC-Dextrad)

Fig. 5. Concentration-response curve based on increases in relative fluorescence intensity for selective binding of pyreneconcanavalin A (located on a phospholipidcholesterol monolayer) with fluorescein isothiocyanate dextran.

ULRICHJ. KRULLet aI.

806

mediated agglutination.1s*‘6 Gangliosides such as GM, have been used in vesicular form and incorporated into PC/C lipid vesicles to provide surfaces coated with saccharide residues suitable for lectin binding. The agglutination of vesicles by lectin has been monitored by observation of changes of light scattering. A series of experiments using vesicular agglutination was done in this work to demonstrate the ability of GM, to act as a receptor for both Con A and pyrenelabelled Con A. The GM, was present at high molar concentrations (20 mole%) in EPC/C vesicles used at room temperature to ensure that glycolipid diffusion could take place on the surface of the membranes. A series of experiments was designed to investigate aggregation or fusion of vesicles with and without GM, in the absence of any protein, in the presence of non-selective protein (bovine serum albumin, BSA), in the presence of Con A (previously associated with vesicle fusion)2’ and in the presence of Py-Con A. The results of these experiments are summarized in Fig. 6. The trends in light-scattering indicate that little change occurs when BSA is present or when Con A is absent, but some EPC/C vesicle interaction is induced by the presence of the lectin. A greater rate and extent of interaction occurs when GM, is available, and the results confirm that both Con A and pyrene-labelled Con A are selectively complexed by the glycolipid.

g

OS

0

8 0.4 9 0.2

1 0

I

I

I

20

40

60

Time (min)

Fig. 6. Lipid vesicle aggregation determined by light-scattering at 455 nm, with monosialoganglioside as a selective binding agent for concanavalin A. (0) Phospholipid-cholesterol vesicles in the presence of concanavalin A or pyrene-concanavalin A; (0) phospholipid-cholesterol vesicles containing ganglioside in the presence of concanavalin A or pyrene-concanavalin A; (A) lipid vesicles containing the ganglioside G,, ; (V) lipid vesicles containing the protein BSA in the presence of concanavalin A. Variability between vesicle preparations limits absolute comparisons of reaction rates. The results confirm that the presence of ganglioside assists vesicle aggregation in the presence of Con A.

0.30

0.50

0.10

0.90

Average molecular area hm*/molel

Fig. 7. Pressure-area curves showing results from monolayers of A, 50/50 mole% mixture of phosphatidyl choline and cholesterol; B, 70/30 mol% mixture of phosphatidyl choline and cholesterol; C, 50/30/20 mol% mixture of phosphatidyl choline, cholesterol and ganglioside. Monolayers were transferred to quartz fibers at a constant surface pressure of 30 mN/m.

Lipid membranes of EPC/C containing GM1 and devoid of the glycolipid were deposited onto alkylated optical fibers by the LangmuirBlodgett casting technique, and retained in aqueous solution. Experimental compression curves for these monolayers are shown in Fig. 7, and indicate that differences in physical compressibility and therefore structure are observed for the different membranes. Results for incremental additions of Py-Con A to EPC/C membranes indicated non-selective adsorption of the protein on the membrane. The presence of GM, caused a general trend of enhancement of the pyrene signal relative to that for non-selective adsorption. Quantitative correction for the background signal due to non-selective Py-Con A adsorption on the surfaces could not be done by signal subtraction achieved by use of fibers coated with EPC/C but without GM,. The nonselective binding properties of EPC/C membranes and those containing EPC/C/GM, were not equivalent because of differences in lateral structure intrinsic to static monolayers (Fig. 7), variations of lateral structure caused by the deposition technique,‘* and differences in the surface free energy, related to the presence of different functional groups at the membrane-solution interface. Chemically selective membranes that employ proteins as binding agents provide a means of improved correction of non-selective adsorption. A reference signal can be derived from a membrane which contains inactivated protein, but is otherwise chemically

Selective interactions of concanavalin A 1.0 -

-

0.6

-

0.4

-

0.2

-

z

G L

0 -10

cently labelled. This is not necessarily the case, since many different biochemical reactions can perturb the structure of lipid membranes, so a fluorescent lipid membrane could be a generic transducer of selective binding events. These aspects will be the subject of a subsequent communication in this journal.

I

2

.g

0.6

807

Acknowledgements-The I -9

-6 Log

I

I -1

-6

I -5

(CPy-ConAl)

Fig. 8. Concentration-response curve based on relative increases in fluorescence intensity from quartz fibers coated with lipid monolayers containing phosphatidylcholine, cholesterol and ganglioside for selective binding of pyreneconcanavalin A, (results corrected for non-selective adsorption of pyrene-concanavalin A).

identical to the indicator system. It was not possible, however, to denature the GM, used in the present work, to prepare an analogous “inactive” reference membrane. Results for selective Py-Con A adsorption on EPC/C/GM, membranes are shown in Fig. 8 and represent response trends after a 30-min incubation period, with background correction by estimation of contributions to the analytical signal by non-selective adsorption. The results indicate that the lipid membrane does contain an intrinsic selective receptor, but that the analytical system is not very sensitive or reproducible. CONCLUSIONS

Chemically-selective lipid membranes can be prepared at the surface of an optical fiber for investigation of binding interactions by monitoring of fluorescence intensity in an intrinsic sensor configuration. The use of Con A as a selective receptor for an analyte resulted in limitation of the analytical reproducibility and sensitivity, owing to non-selective adsorption on the sensing surface. This clearly identifies one of the serious limitations of fiber optic biosensors when used exclusively for fluorescence intensity measurement at a fixed analytical wavelength. The analytical potential of fluorescence lies in the correction of an analytical signal for noise or isolation of the signal from the noise, by use of simultaneous acquisition of data on wavelength, intensity, lifetime and perhaps polarization. A further limitation exposed in this work is that lipid membranes as used here can provide useful matrices for certain receptors (e.g.,G,, ), but are restricted in scope if the analyte must be fluores-

authors wish to thank the Natural Sciences and Engineering Research Council of Canada, the Canadian Defense Research Establishment, the Ontario Ministry of the Environment and Imperial Gil Canada for financial support of this work. REFERENCES 1. U. J. Krull and M. Thompson, ZEEE Trans. Electron Devices, 1985, 32, 1180. 2. U. J. Krull, R. S. Brown, K. Dyne, B. D. Hougham and E. T. Vandenberg, in Chemical Sensors and Microinstrumentation, R. Murray (ed.), American Chemical Society, Washington, D.C., in the press. 3. U. J. Krull, R. S. Brown, R. N. Koilpillai, R. Nespolo, A. Safarzadeh-Amiri and E. T. Vandenberg, Analyst, 1989, 114, 33. 4. K. D. Hardman and I. J. Goldstein, in Zmmunochemistry of Proteins, M. Z. Atassi (ed.), Vol. 2, p. 373.

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