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Langmuir 1998, 14, 4138-4142
Optical Absorption Study of the Biotin-Avidin Interaction on Colloidal Silver and Gold Particles Murali Sastry,*,† Neeta Lala,† Vijaya Patil,† S. P. Chavan,‡ and A. G. Chittiboyina‡ Materials Chemistry Division and Organic Chemistry Technology Division, National Chemical Laboratory, Pune 411 008, India Received January 15, 1998. In Final Form: May 28, 1998 The biotin-avidin reaction is well studied and is often used as a prototypical interaction in the development of immunoassays. In this paper, this reaction is studied on the surface of colloidal silver and gold particles as a first step in the development of a sol-based assay. More specifically, silver and gold colloidal particles were biotinylated by self-assembly of a biotin disulfide molecule, and the reaction of the surface-modified colloidal particles with avidin molecules was followed using optical absorption spectroscopy. The specific interaction of avidin, a tetrameric protein, with biotin leads to cross-linking of the colloidal particles (“flocculation”) and a consequent growth of a long wavelength absorption peak. The degree of flocculation was quantified using a semiempirical flocculation parameter, and the dependence of this parameter on the extent of biotinylation of the colloidal particle surface as well as the concentration of avidin in solution was studied to determine the optimum working conditions of the sol. The silver sol required electrostatic stabilization of the biotin-capped particles through the simultaneous incorporation of a charged bifunctional molecule, 4-carboxythiophenol, in the capping monolayer while the gold sol was stable with biotin capping. Both biotinylated silver and gold sols showed a visible color change on addition of avidin. However, changes in the optical absorption spectra were more marked for the silver sol.
Introduction There is much current interest in the area of metal colloids following the work of Brust et al.1 wherein it was demonstrated that alkanethiol molecules could be selfassembled on colloidal gold particles. This has led to the exciting possibility of modification of the chemical and physical properties of colloidal particles through the use of terminally functionalized capping molecules.2,3 The terminal functionality provides an additional degree of freedom that may be used for covalently binding the colloidal particles to metal surfaces,4 for electrostatically assembling the particles in Langmuir-Blodgett films5 and in other organic composites6 as well as in the controlled aggregation of colloidal particles using interactions between complementary DNA nucleotides attached to the particles.7 The latter approach based on the use of specific * To whom correspondence may be addressed. Present address: Department of Materials and Nuclear Engineering, University of Maryland, College Park, MD 20742-2115; fax, 301-314-7136; e-mail;
[email protected]. † Materials Chemistry Division. ‡ Organic Chemistry Technology Division. (1) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801. (2) (a) Brust, M.; Fink, J.; Bethell, D.; Sciffrin, D. J.; Kiely, C. J. Chem. Soc., Chem. Commun. 1995, 1655. (b) Weisbecker, C. S.; Merritt, M. V.; Whitesides, G. M. Langmuir 1996, 12, 3763. (c) Hostetler, M. J.; Green, S. J.; Stokes, J. J.; Murray, R. W. J. Am. Chem. Soc. 1996, 118, 4212. (d) Green, S. J.; Stokes, J. J.; Hostetler, M. J.; Pietron, J.; Murray, R. W. J. Phys. Chem. B 1997, 101, 2663. (e) Ingram, R. S.; Hostetler, M. J.; Murray, R. W. J. Am. Chem. Soc. 1997, 119, 9175. (3) (a) Sastry, M.; Mayya, K. S.; Bandyopadhyay, K. Colloids Surf., A 1997, 127, 221. (b) Mayya, K. S.; Patil, V.; Sastry, M. Langmuir 1997, 13, 3944. (4) Colvin, V. L.; Goldstein, A. N.; Alivisatos, A. P. J. Am. Chem. Soc. 1992, 114, 5221. (5) (a) Mayya, K. S.; Patil, V.; Sastry, M. Langmuir 1997, 13, 2575. (b) Sastry, M.; Mayya, K. S.; Patil, V.; Paranjape, D. V.; Hegde, S. G. J. Phys. Chem. B 1997, 101, 4954. (c) Mayya, K. S.; Sastry, M. J. Phys. Chem. B 1997, 101, 9790. (6) (a) Sastry, M.; Patil, V.; Mayya, K. S. Langmuir 1997, 13, 4490. (b) Patil, V.; Sastry, M. J. Chem. Soc., Faraday Trans. 1997, 93, 4347.
recognition induced self-assembly of colloidal particles in conjunction with the beautiful color changes that accompany such an aggregation process in colloidal silver and gold particles8 shows promise for the development of simple diagnostic tools of biological analytes. In this paper, we examine in detail changes in the optical properties of biotin (vitamin H)-capped silver and gold colloidal particles in the presence of avidin molecules in solution. The well-known high-affinity, specific binding of biotin to avidin (Ka ∼ 1015 mol-1)9 has been used to induce the aggregation of the colloidal particles, thus simulating other specific surface recognition processes such as antigen-antibody reactions. Avidin is a tetrameric protein, which coordinates to the biotin ligands from different colloidal particles and thereby leads to crosslinking of the particles. Such a flocculation of the particles induced by specific interaction with avidin results in the growth of a long wavelength component in the optical absorption spectrum2b,3,10 and a clearly observable change in the color of the sol. It is clear that a sol-based diagnostic technique would be better adapted for use in the field than the more sophisticated immunoassay techniques, which make use of microgravimetric,11 amperometric,11c,12 fluorescence,13 and chemiluminescence14 principles. Presented below are details of the investigation. (7) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607. (8) Mulvaney, P. Langmuir 1996, 12, 788. (9) (a) Green, N. M. Adv. Protein Chem. 1975, 29, 85. (b) Blankenburg, R.; Meller, P.; Ringsdorf, H.; Salesse, C. Biochemistry 1989, 28, 8214. (c) Herron, J. N.; Muller, W.; Paudler, M.; Riegler, H.; Ringsdorf, H.; Suci, P. A. Langmuir 1992, 8, 1413. (10) Blatchford, C. G.; Campbell, J. R.; Creighton, J. A. Surf. Sci. 1982, 120, 435. (11) (a) Ebersole, R. C.; Ward, M. D. J. Am. Chem. Soc. 1988, 110, 8623. (b) Ebersole, R. C.; Miller, J. A.; Moran, J. R.; Ward, M. D. J. Am. Chem. Soc. 1990, 112, 3239. (c) Blonder, R.; Levi, S.; Tao, G.; Ben-Dov, I.; Willner, I. J. Am. Chem. Soc. 1997, 119, 10467. (12) Electrochemical Sensors in Immunological Analysis; Ngo, T. T., Ed.; Plenum Press: New York, 1987.
S0743-7463(98)00075-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/30/1998
Biotin-Avidin Interaction
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Chart 1
Experimental Section 5a
The gold and silver colloidal particles5b were prepared as described elsewhere. The silver and gold colloidal particles had dimensions of 70 ( 12 and 130 ( 30 Å, respectively. The pH of the as-prepared silver sol was ca. 9 while the gold sol pH was close to 3. The pH of both sols was adjusted to ca. 7 to achieve physiological pH conditions using dilute H2SO4 and ammonia solutions. The colloidal particles were then capped with the disulfide biotin molecule (Chart 1) by mixing to 9 mL of the sol 1 mL of the biotin solution in absolute ethanol. Different capping concentrations of biotin in the hydrosol were obtained by varying the concentration of biotin in the ethanolic solution and not by altering the amount of biotin solution added to the sol. The above procedure ensures that the colloidal particle density in the different hydrosols is identical. This is an important control parameter crucial to accurate quantification of the kinetics of avidin-induced flocculation in the sols. The disulfide biotin molecule was synthesized using the procedure outlined below. To a stirred solution of 0.01 g (0.04 mM) of biotin15 and 0.0063 g (0.4 mM) of 2-hydroxyethyl disulfide in acetonitrile, 0.0084 g (0.4 mM) of dicyclohexylcarbodiimide16 was added at room temperature. After vigorous stirring for 12 h, the crude product was obtained by evaporating the solvent from the reaction mixture and was further purified by column chromatography (15% MeOH/CHCl3) to yield the biotin disulfide molecule used in this study (Chart 1). In the case of silver particles, capping with biotin alone did not yield stable sols under the pH conditions mentioned above. To improve the stability of the silver sol, the biotin molecules were coadsorbed with an aromatic bifunctional molecule, 4-carboxythiophenol (4-CTP) by adding ethanolic solutions of the mixture (4-CTP and biotin) to the sols as mentioned earlier. The ionized carboxylic acid groups are expected to provide electrostatic stabilization of the particle size distribution.3 The relative capping concentrations of 4-CTP and biotin were varied to determine an optimum concentration ratio that would yield a stable sol as well as sufficient biotinylation of the colloidal particle surface for avidin detection. After the optimum 4-CTP/biotin ratio was determined for the silver sol, the reaction of the biotinylated silver and gold sols with avidin was studied as a function of avidin concentration in the hydrosol. Avidin (egg white) was purchased from Sisco Research Laboratories (SRL, No. 0148154) and used as-received. In this case as well, 1 mL of aqueous avidin solution of different concentrations was added to 9 mL of the biotinylated sol to vary the avidin concentration in the hydrosol thereby ensuring constant colloidal particle density in all the cases studied. The (13) Diamandis, E. P.; Christopoulos, T. K. Anal. Chem. 1990, 62, 1149A. (14) Hage, D. S.; Kao, P. C. Anal. Chem. 1991, 63, 586. (15) Biotin was synthesized using a procedure developed in-house: Ravindranathan, T.; Chavan, S. P.; Tejwani, S. P. A New and Improved Process for (+)-Biotin, US Patent No. 5, 274, 107, 1993. (16) Smith, M.; Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1958, 80, 6204.
Figure 1. Optical absorption spectra of the silver sol capped with (A) 10-4 M biotin, (B) 10-5 M biotin, and (C) 10-6 M biotin. The dashed line is the spectrum of the uncapped silver sol. The measurement times from capping the silver sol are indicated next to the respective curves.
biotin functionalization of the colloidal particles and the reaction with avidin were followed in time using optical absorption spectroscopy carried out on a Hewlett-Packard 8542 diode array spectrophotometer operated at 2 nm resolution. Results and Discussion The changes in the optical properties of biotinylated colloidal particles on reaction with the protein avidin are due to aggregation of the colloidal particles arising from cross-linking by the tetrameric protein molecule. It is important, therefore, to quantify the aggregation process and thereby be able to critically assess the optimum biotin capping concentration as well as the avidin concentration for use in a possible sol-based assay. Such optical absorption sensitive aggregation processes can be conveniently quantified using a semiempirical parameter, termed the “flocculation parameter”, first introduced by Weisbecker et al.2b and partially modified by Sastry et al.3b The flocculation parameter is defined as the integrated absorbance between the transverse surface plasmon resonance maximum and a long wavelength cutoff that encompasses the longitudinal plasmon resonance.2b In the case of gold, the integration interval is 525-750 nm, whereas for the silver sol of this study, the interval has been taken to be 395-750 nm. To provide a more sound physical basis to the flocculation parameter, we have modified the procedure marginally to include normalization of the optical absorption spectra at the transverse resonance maximum followed by subtraction of the integrated extinction of the uncapped sol.3b This yields a modified flocculation parameter that would give small values for stable sols. The definition of this semiempirical parameter is not arbitrary. It is well-known that as silver and gold colloidal particles aggregate into stringlike structures, there is growth of a higher wavelength component that shifts to the red and increases in intensity as the aggregation proceeds.10 The long wavelength component (the longitudinal plasmon resonance) arises due to coupling of the plasma modes of the individual clusters and therefore lifts the 3-fold degeneracy of the transverse and longitudinal modes of vibration.10 The flocculation parameter as defined above would correctly track any aggregation process occurring in the sols. Figure 1 shows the optical absorption spectra recorded from the silver sol as a function of time after capping with
4140 Langmuir, Vol. 14, No. 15, 1998
10-4 M (Figure 1a), 10-5 M (Figure 1b), and 10-6 M (Figure 1c) of the biotin molecule. The spectrum of the uncapped sol (dashed lines) is shown for comparison in Figure 1 while the time of measurement is indicated next to the respective curves. The biotin disulfide molecule is expected to oxidatively dissociate (as has been observed for dialkyl disulfides)17 and form a thiolate linkage with the silver (and gold) particle surface yielding two separate biotin functional groups in the process. That the thiolate linkage of the biotin molecule has been achieved can be seen from the reduction in the plasmon resonance on capping (Figure 1). However, the more interesting point to note is the appearance and time-dependent red shift of a long wavelength component after biotinylation of the silver particle surface, which is most pronounced in the 10-4 M capping case (Figure 1a). This is a clear indication of flocculation of the silver colloidal particles induced by the surface-bound biotin and is an undesirable feature if the sol is to be used to detect avidin, which would induce further flocculation. The biotinylated colloidal silver particles shown in Figure 1 completely precipitated out of solution within 24 h of capping. In this context, we mention the work of Ahern and Garrell18 who studied protein-metal colloidal particle interactions using surfaceenhanced Raman spectroscopy and optical absorption spectroscopy. The result germane to this study is the observation of Ahern and Garrell of flocculation of silver colloidal particles on capping with biotin,18 in agreement with our findings. We would like to mention here that an estimate of the amount of uncoordinated biotin in solution after capping the colloidal particle surface could not be obtained. However, given the large energy of chemisorption (ca. 30 kcal/mol)19 and the fact the biotin molecule is highly hydrophobic, we believe that the equilibrium between chemisorbed and free biotin molecules in solution would strongly favor surface capping. On the basis of the optical absorption spectra recorded after 40 min, which shows the largest damping of the surface plasmon resonance for the 10-4 M capping case (compare Figure 1a-c), it can be inferred that the surface capping increases as the concentration of biotin in solution is increased. The probability for cross-linking of the silver colloidal particles would consequently increase with surface coverage as is observed in the optical absorption spectra, which shows the most prominent longitudinal mode for the 10-4 M capped sol. In this sense, the surface capping is mirrored by the starting biotin capping concentration. To stabilize the silver sol, capping of the colloidal particles was done by coadsorption of the biotin molecule with 4-CTP as mentioned in the Experimental Section. Figure 2 shows the optical absorption spectra recorded as a function of biotin/4-CTP concentration ratio in the ethanolic solution (overall concentration of 10-5 M, concentration ratios indicated next to the curves)20 at time t ) 0 (full lines) and t ) 24 h (dotted lines). The inset shows a plot of the flocculation parameter calculated at t ) 6 h as a function of relative percentage biotin concentration. From the figure it is observed that all the sols show some degree of flocculation after 6 h of capping, which, as expected, is most pronounced for the 10:1 capped (17) Nuzzo, R. G.; Zegarski, B. R.; Dubois, L. H. J. Am. Chem. Soc. 1987, 109, 733. (18) Ahern, A. M.; Garrell, R. L. Langmuir 1991, 7, 254. (19) Leff, D. V.; Ohara, P. C.; Heath, J. R.; Gelbart, W. M. J. Phys. Chem. 1995, 99, 7036. (20) The ratio of biotin/4-CTP in solution is not expected to reflect the actual concentration ratio on the colloidal particle surface. Our approach to obtaining a stable sol may be viewed as being purely empirical.
Sastry et al.
Figure 2. Optical absorption spectra of silver sols capped with different concentrations of biotin. The overall capping concentration was 10-5 M and the relative concentrations of biotin/ 4-CTP are indicated next to the respective curves. The solid lines refer to spectra recorded immediately on capping of the sols, whereas the dotted lines refer to spectra measured 6 h after capping. The inset shows the variation of the flocculation parameter with relative percentage biotin concentration in the sol capping monolayer measured 6 h after capping the sol.
Figure 3. Optical absorption spectra recorded from a silver sol capped with 5:1 biotin/4-CTP ratio of the molecules (overall concentration ) 10-5 M) after addition of 10-6 M avidin to the sol: dotted line, as-prepared silver sol; curve A, silver sol capped with 5:1 biotin/4-CTP measured 6 h after capping the sol; curve B, sol shown as curve A immediately after addition of 10-6 M of avidin; curve C, the biotinylated silver sol 6 h after addition of 10-6 M avidin. The inset shows the variation of the flocculation parameter calculated for the silver sol as a function of relative biotin percentage concentration 6 h after addition of 10-6 M avidin to the sol.
sol. However, the presence of 4-CTP on the silver surface stabilized the 1:1 and 5:1 sols over time periods of up to a couple of months during which no further changes in the optical absorption spectra were observed. The 10:1 sol, however, precipitated within 24 h of storage and, therefore, would not be suitable in the development of a sol-based assay. Figure 3 shows the optical absorption spectra recorded from a silver sol capped with 5:1 biotin/4-CTP, and as before, the overall concentration of the capping molecules was 10-5 M. The dotted line is the absorption spectrum
Biotin-Avidin Interaction
Figure 4. Optical absorption spectra recorded from the 5:1 biotin/4-CTP capped sol 6 h after addition of different amounts of avidin to the sol. The concentration of avidin is indicated next to the respective curves. The inset shows the variation in the flocculation parameter with avidin concentration for the 5:1 biotin/4-CTP capped silver sol calculated 6 h after addition of avidin.
recorded from the as-prepared sol, curve A is the spectrum recorded after capping with biotin and 4-CTP and allowing the flocs of silver colloidal particles to stabilize (ca. 6 h), curve B is the spectrum from the biotin- and 4-CTP-capped sol recorded immediately after addition of 10-6 M avidin, and curve C is the spectrum recorded 6 h after addition of avidin to the biotinylated silver sol. It is clear that addition of avidin to the biotinylated silver sol induces immediate cross-linking of the colloidal particles and a strong (quite easily visible) change in color of the sol. To determine the efficiency of cross linking with degree of biotinylation of the silver particle surface, similar measurements were carried out on silver sols capped with 1:1 and 2:1 biotin/4-CTP molecules as well. The inset of Figure 3 shows the flocculation parameters calculated for the silver sols capped with different amounts of biotin, 6 h after addition of avidin to the sol. It is clearly seen from the inset of Figure 3 that maximum flocculation (crosslinking) of the silver colloidal particles induced by avidin in the solution occurs at a 5:1 biotin/4-CTP concentration on the silver particle surface indicating that this concentration is close to optimum for detection of avidin. Using the 5:1 biotin/4-CTP surface-modified silver sol, we further investigated the efficiency of detection of avidin by following the variation in the flocculation parameter with concentration of avidin in solution. Figure 4 shows some representative optical absorption spectra recorded 6 h after addition of different amounts of avidin to the 5:1 silver sol. It is clearly seen that considerable flocculation has occurred at an avidin concentration of ca. 2 × 10-6 M. The inset of Figure 4 shows the flocculation parameter of the 5:1 silver sol as a function of avidin concentration in solution calculated 6 h after addition of the protein. The solid curve in the inset has been drawn to aid the eye and has no physical significance. A maximum in the flocculation parameter occurs at 2 × 10-6 M avidin, with a large decrease on either side of this concentration. This result together with the finding illustrated in Figure 3 that maximum cross-linking of the colloidal particles occurs at a surface coverage of 5:1 biotin/4-CTP are the salient features of this study and determine the optimum working conditions of the sol. We discuss these results briefly below. There are two conditions to be satisfied for efficient cross-linking of the biotinylated silver particles on reaction
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with avidin. The first is based on the density of biotin groups on the colloidal particle surface. It has previously been reported that the separation between biotin groups in self-assembled monolayers 21a as well as in Langmuir monolayers21b critically affects the rate of binding of streptavidin (a tetrameric protein with biotin binding similar to avidin) from solution. In other words, packing of the biotin functional groups on the surface must be commensurate with the separation between the coordinating regions of the protein molecule, avidin. A critical surface coverage of biotin groups is therefore important for significant avidin binding to the surface. In the case of colloids, this aspect is complicated further by the particle surface curvature and may thus marginally influence the geometry of coordination of the protein to biotin. The other condition is that there should be a sufficiently large number of uncoordinated biotin groups on the surface that permit cross-linking of the silver colloidal particles. The enhanced flocculation of the silver particles at a surface coverage of 5:1 biotin/4-CTP and avidin concentration of 10-6 M (Figure 3, inset) indicates that the separation between the biotin groups is consistent with the structure of the protein. We would like to point out that controlling the biotin concentration on the silver particle surface was not completely in our handssit was necessary to stabilize silver colloidal particles with 4-CTP and this requirement more than anything else determined the maximum possible coverage of biotin attainable without compromising the long term stability of the sol. The fact that the flocculation rate for the 5:1 sol falls rapidly when the avidin concentration is less than 2 × 10-6 M (Figure 4, inset) implies that the surface-immobilized avidin is not of sufficient density to form large flocs capable of appearing as a detectable long wavelength component in the optical absorption spectrum of the sol. The fall in the flocculation parameter at higher concentrations indicates that almost complete coverage of the surface with avidin occurs, preventing cross-linking of the colloidal particles. In the case of the gold colloidal particles, the sol was stable in the presence of biotin alone, thereby obviating the need for additional electrostatic stabilization using 4-CTP. This result is in agreement with the observations of Ahern and Garrell18 who found that while silver colloidal particles flocculated on capping with biotin, gold sols were quite stable with no evidence for cross-linking. Capping of the gold sols with different concentrations of biotin did not result in appreciable changes in the optical absorption spectra, and therefore we have studied the avidin-induced flocculation of the gold sol capped with 10-6 M of biotin, a concentration close to that used in the optimum silver sol described above. The optical absorption spectra recorded from the biotin-capped gold sol measured 6 h after addition of different amounts of avidin to the sol are shown in Figure 5. It is seen that the flocculation of the gold colloidal particles is significant at an avidin concentration of 2 × 10-6 M. The inset of Figure 5 shows the flocculation parameter calculated 6 h after addition of different amounts of avidin to the biotin-capped gold sol. In this case as well, the solid curve in the inset has been drawn to aid the eye. The nature of variation of the flocculation parameter with avidin concentration is quite similar to that obtained for the silver sol with a fairly sharp reduction in the flocculation values on either side of an avidin concentration of 2 × 10-6 M. This indicates that differences in surface curvature between silver and gold particles (the gold particles are nearly twice as large (21) (a) Haussling, L.; Ringsdorf, H.; Schmitt, F. J.; Knoll, W. Langmuir 1991, 7, 1837. (b) Reiter, R.; Motschmann, H.; Knoll, W. Langmuir 1993, 9, 2430.
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Figure 5. Optical absorption spectra recorded from a gold sol capped with 10-6 M of biotin 6 h after addition of different amounts of avidin to the sol. The avidin concentrations are indicated next to the respective curves. The inset shows the variation in the flocculation parameter with avidin concentration in the sol for the 10-6 M biotin-capped gold sol, the flocculation parameter calculated 6 h after addition of avidin.
as the silver particles) do not play a big role in determining either the efficiency of avidin binding to the biotinylated surface or cross-linking of the particles. However, the value of the flocculation parameter at the maximum, that is, at an avidin concentration of 2 × 10-6 M, is much higher for the silver sol, the values being ca. 170 for silver and 45 for the gold sol. Both sols did show a visually detectable color change on addition of avidin, but as expected, the
Sastry et al.
change was more dramatic for the silver sol. This clearly indicates that the silver sol is more sensitive to detection of avidin than the gold sol. To check the specificity of the method, optical absorption spectra were recorded from the biotin-capped silver and gold sols after addition of bovine serum albumin (BSA), a protein that does not bind to biotin. No observable changes in the optical absorption spectra were seen, clearly indicating that the biotin-avidin reaction is responsible for the flocculation and changes in the optical absorption spectra described in detail above. To summarize, it has been shown that the reaction of biotinylated silver and gold colloidal particles with avidin leads to significant changes in the optical absorption spectra of the sols due to flocculation of the colloidal particles. The optimum concentration of the biotin molecule on the colloidal particle surface for detection of avidin was determined through calculation of a semiempirical flocculation parameter. Both biotinylated silver and gold sols showed a visible color change on reaction with avidin with the silver sol showing larger changes in the optical properties. We would like to stress that this is a preliminary study and further work is required to improve on the dynamic range of the biotin-avidin system before such a sol-based immunoassay can be used in the field. However, the initial results are promising, and further work is under way in this direction. Acknowledgment. N.L., V.P., and A.G.C. thank the Council of Scientific and Industrial Research (CSIR), Government of India, for research fellowships. LA9800755
