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Rational Design of an Optical Sensing System for Multivalent Proteins Xuedong Song and Basil I. Swanson* Chemical Science and Technology Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 Received June 24, 1998. In Final Form: December 2, 1998 A generic design principle for detection of multivalent interactions is described. A phospholipid bilayer consisting of natural and pyrene-derivatized phosphatidylcholines is used as both a supporting biomimetic surface and part of a signal transduction element. The pyrene excimer formed in the surface can act as fluorescence donor, and DABCY/BODIPY-FL covalently attached to receptor (GM1) can act as acceptors. Aggregation of the acceptor-tagged receptors resulting from multivalent binding of CT induces a decrease in efficiency of fluorescence quenching of the pyrene excimer by DABCY or energy transfer from pyrene excimer to BODIPY-FL. In the case using fluorescent acceptors that can undergo distance-dependent fluorescence self-quenching, combination of the lower energy transfer efficiency from the excimer and the acceptor’s self-quenching capabililty make acceptor fluorescence go down even further by the binding. This scheme can achieve signal amplification and high surface density of the optical transduction elements, which, in return, require relatively small surface area.
Carbohydrate-protein interactions have attracted tremendous attention in recent years due to the realization of their importance in cell functions.1-5 Although monosaccharide-protein interactions are relatively weak, high affinity and specificity of protein-carbohydrate interactions are achieved through cooperative multivalent binding.1,5,6 This multivalency can be established by several possible ways, either alone or together:5 (a) ligand multivalency; (b) an extended binding region capable of interaction with more than just a single monosaccharide residue of an oligosaccharide; (c) subunit multivalency, the clustering of several identical binding sites by formation of protein oligomers or a protein with multiple binding subunits. For example, concanavalin A7 and peanut lectin8 with four subunits, each having a binding site, bind selectively with glucose (and manoside) and galactose, respectively, through type c, while cholera toxin with five subunits binds strongly with pentasaccharide of ganglioside GM1 through both type b and c.9 The type a interaction has been used to design bioassays for the detection of carbohydrates.10 We recently reported two general optical transduction methods for detection11 of multivalent proteins by taking advantage of type c interactions between a carbohydrate (GM1) and a protein (cholera toxin (CT)). In this communication, we couple a novel optical sensing strategy with type c interaction for detection of target (1) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321. (2) Liang, R.; Loebach, J.; Horan, N.; Ge, M.; Thompson, C.; Yan, L.; Kahne, D. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10554. (3) Fujimoto, T.; Shimizu, C.; Hayashida, O.; Aoyama, Y. J. Am. Chem. Soc. 1998, 120, 601. (4) Sharon, N. Trends Biochem. Sci. 1993, 18, 221. (5) Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637. (6) Mortell, K. H.; Weatherman, R. V.; Kiessling, L. J. Am. Chem. Soc. 1996, 118, 2297. (7) Williams, T. J.; Plessas, N. R.; Goldstein, I. J.; Lonngren, J. Arch. Biochem. Biophys. 1979, 195, 145. (8) Lotan, R.; Skutelsky, E.; Danon, N. J. Biol. Chem. 1975, 250, 8518. (9) (a) Merritt, E. A.; Sarfaty, S.; Vandenakker, F.; Lhoir, C.; Martial, J. A.; Hol, W. G. S. Protein Sci. 1994, 3, 166. (b) Kuziemko, G. M.; Stroh, M.; Stevens, R. C. Biochemistry, 1996, 35, 6375. (10) Ballerstad, R.; Schultz, J. S. Anal. Chim. Acta 1997, 345, 203. (11) (a) Song, X.; Nolan, J.; Swanson, B. I. J. Am. Chem. Soc. 1998, 120, 4873-4874. (b) Song, X.; Nolan, J.; Swanson, B. I. J. Am. Chem. Soc. 1998, 120, 11514-11515. (c) Song, X.; Swanson, B. I. submitted to Anal. Chem.
proteins with multiple binding subunits. This method can be adapted for any interaction involved in coreceptors as well. In our recent report,11 two general optical transduction schemes for detection of multivalent interactions were developed through distance-dependent fluorescence selfquenching and resonant fluorescence energy transfer with high specificity and sensitivity. In these systems, a bilayer of natural phospholipids (β-palmitoyl-γ-oleoyl-L-R-phosphatidylcholine (POPC)), either in aqueous solution or on solid support, is constructed to mimic cell membrane surfaces in which optically tagged receptors (GM1) can anchor and diffuse laterally. The binding of the protein (CT) brings multiple receptors, and therefore the tagged fluorophores, into a close distance to induce either fluorescence self-quenching or energy transfer. Although the resonant energy transfer scheme is quite sensitive, specific, and fast when using flow cytometry or fluorimetry, it has limitations in microsensor applications, owing to the relatively small number of chromophores in the bilayer on the expected small surface area for waveguide transducers. In our new signal transduction method presented here, we apply a biomimetic bilayer surface of a synthetic, fluorophore-tagged phospholipid mixed with a natural phospholipid (POPC). In this case, the surfaces act not only as a supporting surface but also a part of signal transduction elements. It is expected that the bilayer of the mixed phospholipids still retains structural and dynamic properties of a biomimetic surface so that the fluorophore-labeled receptors anchored in the surface can be laterally mobile. The pyrene excimers in the bilayer act as fluorescence donors, and 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCY) or 4,4difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-5-indacene-3propionic acid (BODIPY-FL) (see Figure 1 for their structures), covalently attached to the receptor (GM1), acts as a fluorescence acceptor. The reason for choosing pyrene as the fluorescence donor is its high tendency to form excimers,12 which gives strong excimer fluorescence with a large Stokes shift (about 135 nm) and relatively long fluorescence lifetime (>50 ns).12 The significant overlap of the pyrene excimer fluorescence with the absorption spectra of DABCY and BODIPY-FL along with the long fluorescence lifetime of the excimer makes it
10.1021/la980758k CCC: $18.00 © 1999 American Chemical Society Published on Web 06/12/1999
Optical Sensing System for Multivalent Proteins
Figure 1. (a) Structures and acronyms of the molecules used in this communication. (b) Schematic illustration of the signal transduction mechanism: (top) acceptor-tagged receptors are distributed homogeneously in the outer leaflet of the bilayers of natural and donor-derivatized phospholipids to result in weak donor and strong acceptor (in the case of fluorescent acceptor) fluorescence due to fluorescence quenching or energy transfer; (bottom) aggregation of the acceptor-tagged receptors resulting from CT binding reduces the energy transfer efficiency and/or promotes fluorescence self-quenching of the acceptors to cause the increase of donor fluorescence and decrease of acceptor fluorescence simultaneously.
Figure 2. Normalized absorption/excitation and/or emission spectra of DABCY-GM1, P-PC, and BFL-GM1 in different media.
possible for an efficient energy transfer from the excimer to either acceptors to occur (Figure 2). Given the long fluorescence lifetime and rapid lateral diffusion, it is possible for one acceptor molecule to quench the fluorescence of many donor molecules, so that a signal transduction amplification can become reality for microsensors based on waveguide. (12) Zachariasse, K. A. In Photochemistry on Solid Surfaces; Anpo, M., Matsuura, T., Ed.; Elsevier: New York, 1989; p 48.
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When the acceptor-tagged GM1 of a proper percentage (4-20%) are homogeneously distributed in the surface, a relatively weak fluorescence of the pyrene excimer compared with that without the acceptor-tagged receptors should be observed due to the quenching by the acceptors. In the case of fluorescent acceptors such as BODIPY-FL, strong acceptor fluorescence should be expected even though only the donor is excited. The aggregation of the receptors, and thereafter the fluorescence acceptors, caused by multivalent binding of proteins will reduce the overall energy transfer efficiency and promote distancedependent self-quenching of the acceptor with a consequence of an increase in excimer fluorescence and a fluorescence decrease for the fluorescent acceptors such as BODIPY-FL-GM1. The schematic illustration of the signal transduction is shown in Figure 1 (see ref 9b for the structure of the pentasaccharide moiety of GM1). In the case using a fluorescent acceptor which can undergo distance-dependent fluorescence self-quenching, two sets of data for one system as parameters for the presence of the target can be obtained to improve sensitivity and reliability.14 In most cases, bilayer vesicles of the mixture of pyrenederivatized phosphatidylcholine (P-PC) and POPC ([POPC]/[P-PC] ) 2/1) are used as a supporting matrix and fluorescence donating layer. For such a bilayer, the fluorescence intensity of the excimer at peak (482 nm) is about twice as large as the intensity of the highest peak (395 nm) attributed to pyrene monomer.15 Introduction of only 10% of nonfluorescent DABCY-GM1 (versus P-PC) into the outer leaflet of the bilayer surface causes a 70% fluorescence decrease of the excimer, while more than 80% fluorescence of the excimer is quenched by only 5% DABCY-GM1 incorporated into both leaflets of the bilayer. This result suggests the formation of enclosed bilayer structures and efficient fluorescence quenching of the excimers not only in the same layer but also in the other layer. It is estimated that each BADCY can quench the excimer fluorescence of more than 10 pyrene molecules if two-thirds of the pyrenes or more are assumed to form the excimers (it is a reasonable assumption based on the relative fluorescence intensity of excimer/monomer ) 2/1).16 In the case of DABCY-GM1 (10% versus P-PC) in the outer leaflet of the bilayer vesicles of POPC/P-PC (2/1), the presence of CT (approximately one-fifth of DABCY-GM1) induces a fluorescence increase of the pyrene excimer up to approximately 45%, while the excimer fluorescence increases up to 60% with DABCYGM1 in both leaflets of the bilayers (Figure 3). Similar to the method using the fluorescence self-quenching mechanism reported previously,11 the sensitivity (less than 0.1 nM for CT) and detection range depend on the concentration of DABCY-GM1 as well as the ratio of [POPC]/[PPC]/[DABCY-GM1]. If the ratio of [POPC]/[P-PC] is too (13) About 12% fluorescence self-quenching is observed for BF-GM1 in the outer leaflet of the POPC bilayer with comparable surface density when enough CT is added. (14) On one hand, the aggregation of the fluorescence acceptors induced by the multivalent binding reduces the energy transfer efficiency from the fluorescence donor to the acceptor so that the donor fluorescence increases and acceptor fluorescence decreases when only the donor is excited. The ratio of the donor fluorescence intensity over acceptor fluorescence intensity can be taken as one set of indicators for the presence of the multivalent protein. On the other hand, if the acceptor is excited, the acceptor fluorescence drops due to fluorescence selfquenching induced by the acceptor aggregation. The degree of the acceptor fluorescence quenching can also be taken as a set of parameters for the presence of the target protein. The self-quenching of the acceptor fluorescence induced by the aggregation should further reduce the energy transfer efficiency to boost the fluorescence response. (15) See ref 12 for emission spectra of the pyrene monomer in organic solvents.
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Figure 3. Relative fluorescence intensity at 482 nm (excitation at 345 nm) of pyrene excimer for POPC/P-PC bilayers with DABCY-GM1 in both leaflets of the bilayers as a function of CT.
high, the percentage of pyrene in the form of excimer drops dramatically,17 while the background fluorescence before addition of CT is high for samples with a low percentage of DABCY-GM1. Only 60% increase of the excimer fluorescence upon addition of enough CT to bind all the receptors (about one-fifth of DABCY-GM1) indicates that the excimer fluorescence does not fully recover for the obvious reason that the aggregated acceptor can still quench the fluorescence of the excimers nearby. The advantages of this scheme versus the distancedependent self-quenching for the detection of multivalent proteins are the increase of the fluorescence signal (versus the decrease in the case of self-quenching), potential of signal amplification achieved by the efficient energy transfer, and high surface density of the donor fluorophores. To test the selectivity of this sensing method, albumin is used as a reference. It is found that for a sample containing 0.25 µM POPC, 0.125 µM P-PC, and 25 nM DABCY-GM1 in both leaflets of the bilayer, 2.4 nM CT induces about 50% increase in excimer fluorescence intensity while 15.6 nM albumin causes about 12% increase. The possible cause for the relatively significant nonspecific signal generation for the albumin is the interfering binding of the DABCY or pyrene moieties with the hydrophobic pockets of albumin or the perturbation of the bilayer structures, which determine the efficiency of excimer formation. Construction of a more stable donor layer should reduce such nonspecific signal generation. Unlike the case using a nonfluorescent DABCY as the quencher, simultaneous double color change can be achieved by using a fluorescent acceptor covalently attached to GM1. When BODIPY-FL-GM1 (5% versus P-PC) is incorporated into the outer layer of POPC/PPC ) 2/1 bilayers, a relatively weak fluorescence of pyrene excimer (approximately 40% excimer fluorescence is quenched) and strong fluorescence18 of BODIPY-FL-GM1 (16) The estimation is based on the following experimental data. A series of vesicle samples containing 0.5 µM POPC, 0.25 µM P-PC, and different concentrations of DABCY-GM1 (samples: (1) 0 nM, (2) 5 nM, (3) 10 nM, (4) 20 nM, and (5) 40 nM) were prepared and their fluorescence spectra were measured. It was found that the fluorescence intensity of the samples 2-5 are ca. 70%, 52%, 33%, and 25% of the fluorescence intensity for the sample 1. Taking sample 2 as an example for estimation, each DABCY-GM1 can quench 30% fluorescence of ca. 50 pyrene molecules, which is equivalent to the fluorescence of 15 pyrene molecules. Since approximately two-thirds of pyrenes form excimers, each DABCYGM1 can quench the excimer fluorescence of 10 pyrene molecules. (17) When the ratio of [POPC]/[P-PC] in the vesicles drops from 2/1 to 50/1, the relative intensity of the excimer fluorescence intensity to the monomer fluorescence intensity goes down from 2/1 to 50/1.
Song and Swanson
Figure 4. Fluorescence spectra of POPC/P-PC (0.25/0.125 µM) bilayer with BODIPY-FL-GM1 (6.25 nM) in the outer leaflet after addition of CT. Excitation at 345 nm.
are observed even though only pyrene is excited (at 345 nm), apparently owing to the energy transfer from the excimer to BODIPY-FL-GM1 as anticipated by the good overlap between pyrene excimer fluorescence and absorption spectrum of BODIPY-FL-GM1. In contrast, BODIPYFL-GM1 in POPC vesicles with comparable surface density shows very weak fluorescence when excited at 345 nm. The presence of CT induces aggregation of BODIPY-FL-GM1 in the bilayer surface. On one hand, energy transfer from the excimer to BODIPY-FL becomes less efficient to boost the excimer fluorescence; on the other hand, the combination of less efficient energy transfer and moderate distance-dependent self-quenching13 of BODIPY-FL contributes to the decrease of acceptor emission (Figure 4). Again, higher sensitivity (less than 0.1 nM for CT) can be achieved with lower concentration of BODIPY-FL-GM1 and a larger detection range with a higher concentration of BODIPY-FL-GM1. Similar to the case using DABCY-GM1 as fluorescence acceptor, a relatively small nonspecific signal of albumin is also observed for this system. In summary, we demonstrate a design principle of direct signal transduction for sensitive detection of multivalent binding interaction with fast response (less than five minutes). The system with high surface density of fluorescence donor requires relatively small surface area and can achieve signal amplification through extremely efficient fluorescence quenching or energy transfer. The coupling of distance-dependent energy transfer with fluorescence self-quenching allows double color measurements simultaneously with double excitations. The design principle discussed in this communication should be useful in designing a biosensor based on multivalent proteins and studying the multivalent interaction between lectins and carbohydrates. Acknowledgment. This research is supported by the Laboratory Directed Research and Development program of Los Alamos National Laboratory. The authors thank Dr. John Nolan and David G. Whitten for access of their instruments and for helpful discussions. LA980758K (18) The absorption and emission spectra are identical in the presence and absence of the donor lipid in terms of spectral shape and spectral peaks. But the emission intensity in the presence of the donor lipid is lower than that without the donor lipid.
