Bioconjugate Chem. 2007, 18, 999−1003
999
Binding of Streptavidin with Biotinylated Thermosensitive Nanospheres Based on Poly(N,N-diethylacrylamide-co-2-hydroxyethyl methacrylate) M. Colonne, Y. Chen, K. Wu, S. Freiberg, S. Giasson, and X. X. Zhu* De´partement de Chimie, Universite´ de Montre´al, C.P. 6128, Succursale Centre-ville, Montre´al, QC, H3C 3J7, Canada. Received September 29, 2006; Revised Manuscript Received February 19, 2007
Thermosensitive polymer nanospheres based on N,N-diethylacrylamide and 2-hydroxyethyl methacrylate (HEMA) have been prepared, characterized, and conjugated with biotin. The thermosensitivity of poly(N,N-diethylacrylamide) was enhanced by the incorporation of HEMA up to about 40 mol %. Atomic force microscopic images show that these particles can be closely packed even without the surface charges as in the latex particles. Biotinylation reduces the thermosensitivity of the copolymer nanospheres. The biotinylated hydrogel nanospheres showed a reduction in size upon binding with streptavidin, indicating the formation of a less hydrophilic conjugate. No aggregation of the biotinylated particles due to the cross-linking effect of streptavidin was observed. This size change could be reversed by the addition of free biotin to the system. The interaction is specific, and no such changes were observed when streptavidin was replaced by bovine serum albumin.
INTRODUCTION Biologically reactive polymer hydrogels represent an extension of the temperature- or pH-activated systems (1). Miyata et al. reported on the preparation of hydrogels conjugated with an antibody that exhibited a reduction in volume in response to a specific antigen by forming cross-links in the gel network that can be reversed upon the addition of free antigen (2). Thermosensitive hydrogels are known to swell in water below the lower critical swelling temperature (LCST) due to hydrogen bonding with water (3, 4). Above the LCST, hydrogen bonds with water are disrupted, resulting in a shrinkage of the gels. Such hydrogels can be made into nanospheres by emulsion polymerization, and the size of spheres can be modulated by temperature (5, 6). Bead size modulation by biochemical interactions may lead to biosensor applications. Using charged polystyrene particles which can form colloidal crystal arrays (CCA) induced by electrostatic repulsion, Asher and co-workers built biological sensing systems by embedding them in thermosensitive gels and monitoring the changes of interparticle distances (7-10). Polymerized CCA of charged particles based on thermosensitive poly(N-isopropylacrylamide) (PNIPAM) were also embedded in polyacrylamide matrix to induce a fast response to changes in temperature (within nanoseconds) (9). The high and specific affinity between streptavidin and biotin (association constant, Ka ≈ 1015) is well-known, and their coupling has been used for the rapid isolation of biomolecules such as proteins (11, 12) or for the preparation of biosensors (13-17). Streptavidin (60 kDa) is a tetrameric protein that can bind four biotin molecules. Hoffman, Stayton, and co-workers used linear thermosensitive polymers such as PNIPAM or poly(N,N-diethyl acrylamide) (PDEA) to conjugate genetically engineered streptavidin and then immobilized them on magnetic microbeads (18-22). Depending on the pH and temperature, the polymers could block biotin binding (22). Steric restriction could change the nature of streptavidin binding and cause streptavidin to bind only two biotin molecules instead of four (18). The preparation of fiberoptic biosensors (14) was also realized by attaching a biotinylated polymeric thin film to the * Corresponding author. Fax: 514-340-5290; E-mail: julian.zhu@ umontreal.ca.
end-face of an optic fiber. It was shown that the system linked to avidin can support biotinylated antigens or antibodies that can be used to detect an anti-cholera toxin antibody with satisfactory performance (14). The change in hydrophilicity of the thermosensitive polymers can be caused not only by a temperature change but also by the interactions between conjugated biological molecules such as streptavidin and biotin. We have prepared a model system with noncharged thermosensitive polymeric nanospheres that can be biotinylated so that they can respond to the presence of streptavidin. PDEA was selected as the polymer matrix with varying amounts of 2-hydroxyethyl methacrylate (HEMA) as functional comonomers. The copolymer nanoparticles were conjugated with biotin, and the binding with streptavidin and the specificity of the binding have been investigated.
EXPERIMENTAL PROCEDURES Materials and Chemicals. All chemicals were purchased from Aldrich-Sigma. Tetrahydrofuran (THF) was purified and dried prior to use. Biotin, streptavidin, bovine serum albumin (BSA), scandium triflate, potasium persulfate (KPS), sodium dodecyl sulfate (SDS), and 2-(4′-hydroxyazobenzene)benzoic acid (HABA) were used without further purification. N,N′Methylene bis(acrylamide) (BA) was recrystallized from methanol. N,N-Diethylacrylamide (DEA) was prepared from diethylamine and acryloyl chloride (4, 23) and vacuum-distilled before use. HEMA was also vacuum-distilled before use. Cellulose sacks for dialysis (MW > 12 000) and Millipore Isopore membrane filters (2.0 µm) were purchased from Sigma. Water was purified by a water purification system from Millipore. Preparation of Nanospheres and Biotinylation. The preparation of the biotinylated polymer nanospheres is shown in Scheme 1. Emulsion polymerization was done in a 500 mL three-necked flask with mechanical agitation at a constant rate of about 240 rpm. The monomers (DEA and HEMA), BA, and SDS were added to 200 mL of stirred Milli-Q water followed by nitrogen purging for 30 min at 70 °C. KPS, dissolved in 15 mL of Milli-Q water, was added to the solution. The mixture contained 1.5 mol % BA, 0.040 g SDS, and 0.120 g of KPS. A total amount of 26.5 mmol of monomers was used with HEMA
10.1021/bc060302b CCC: $37.00 © 2007 American Chemical Society Published on Web 04/13/2007
1000 Bioconjugate Chem., Vol. 18, No. 3, 2007 Scheme 1. Preparation of the Biotinylated Polymer Nanospheres
Colonne et al. Table 1. Composition and Thermosensitive Properties of Poly(DEA-co-HEMA) Nanospheres HEMA/DEA molar ratio sample 100% DEA 2% HEMA 9% HEMA 15% HEMA 27% HEMA 37% HEMA 48% HEMA 54% HEMA 100% HEMA
feed
finala
0/100 2.5/97.5 5/95 10/90 20/80 30/70 40/60 50/50 100/0
0/00 2/98 9/91 15/85 27/73 37/63 48/52 54/46 100/0
LCST (°C) size changeb (%) 28 28 28 25 23 23 20 25
66.7 68.9 70.2 75.9 70.1 77.8 71.0 66.7 7.7
a Measured by elemental analyses of the samples. b Particle size variation between 10 and 50 °C vs size at 10 °C.
content varying from 0 to 100 mol %. The reaction was continued for 4 h and allowed to cool to room temperature before filtering the milky suspensions through 2.0 µm Millipore Isopore membrane filters. The filtrate was centrifuged for 40 min at about 15 000 rpm (relative centrifuge force (RCF) ) 32 583 × g). The polymer spheres were collected and purified by dialysis for 1 week in Milli-Q water. To conjugate biotin to the nanospheres containing HEMA, biotin (50 mg, 0.205 mmol) and scandium triflate (20 mg, 0.04 mmol) were stirred in an 8% solution of the spheres containing 0.205 mmol HEMA at 70 °C for 4 days. The resulting solution was centrifuged (15 000 rpm, RCF ) 32 583 × g) at 20 °C for 20 min, rinsed with Milli-Q water, and dialyzed for 1 week. For the binding studies, typically, 1 mg of streptavidin (17 nmol) was added directly to the biotinylated spheres in water (10 mL) in a cuvette and mixed for 30 min without filtration. Sample Characterization and Binding Studies. To determine the amount of biotin attached on the nanospheres, the avidin-HABA assay (24-26) was used by monitoring the absorbance values at 500 nm for the avidin-HABA reagent and for the solution after addition of biotinylated nanospheres. UV-visible spectra were collected on a Cary 300 Bio UVvisible spectrophotometer. A calibration curve was generated by measuring the change in UV absorbance upon the gradual addition of free biotin of known concentration sequentially into the avidin-HABA reagent. The equivalent amount of free biotin available on the biotinylated nanospheres was calculated by comparing the absorbance change to that of the calibration curve. Dynamic light scattering (DLS) was performed using a Brookhaven BI-200SM light scattering instrument setup with a Science/Electronics temperature controller and a 532 nm green JDS laser. Samples were prepared by suspending 1-2 drops of polymer spheres in about 20 mL of Milli-Q water followed by filtration through a 2 µm filter. To ensure data reproducibility, measurements were taken 25 min after the temperature was
Figure 1. Thermosensitivity of the poly(DEA-co-HEMA) nanospheres as studied by DLS. HEMA enhances the thermosensitivity of the beads containing DEA, even if the homopolymer of HEMA itself does not exhibit thermosensitivity.
stabilized. At each temperature, 8 measurements were taken to determine the average diameter of the particles, and each temperature cycle was repeated at least 3 times per sample. The size variation due to measurement error is considered to be within 5% as confirmed by multiple measurements carried out by using the same sample on various heating and cooling cycles. The LCST of the aqueous system of the particles is defined as the midpoint of the inflection of the size change during heating. Diffraction measurements were taken on a home-assembled spectrophotometer (Gamble Technologies USB2000) of wavelengths ranging from 350 to 1000 nm with a tungsten halogen source and an R200-7 VIS/NIR reflection probe. Measurements were taken after sample centrifugation at 15 000 rpm (RCF ) 32 583 × g) for 30 min at 20 °C and ensuring that the reflectance probe was held 90° to the bead-containing cuvette. Atomic force microscopy (AFM) was performed on a Digital Instruments Nanoscope III (Dimension 3100) in tapping mode with image set point at 75% of the pop-off value.
RESULTS AND DISCUSSION Preparation and Thermosensitivity of the Nanospheres. Table 1 shows the list of the nanospheres prepared and the resulting compositions as obtained by elemental analysis. Colloidal stability is evident below a HEMA content of 48 mol %, but precipitation occurred over a period of several days with a higher HEMA content. At high HEMA contents (88 and 100 mol %), the samples precipitated readily, although dilute suspensions can be maintained. DLS results at different temperatures are shown in Figure 1. It is interesting to note that the nanospheres made of PDEA alone, known to be thermosensitive, exhibited lower thermosensitivity than some of the copolymers with HEMA, although the HEMA homopolymer beads do not show any size change with
Technical Notes
Bioconjugate Chem., Vol. 18, No. 3, 2007 1001
Figure 3. AFM images of hexagonally ordered poly(DEA-co-HEMA) nanospheres with 2% HEMA (size 394 nm measured by DLS at 20 °C) beads after spin-casting. (A) Height image. (B) Phase image showing a thin layer of polymer mass forming a ring around the core of the particles.
Figure 2. Poly(DEA-co-HEMA) nanospheres with different HEMA contents can diffract visible light at specific wavelengths as a result of the well-packed nanospheres of uniform size. Size of particles (measured by DLS at 20 °C): 15% HEMA, 744 nm; 9% HEMA, 571 nm; 2% HEMA, 556 nm.
temperature (Figure 1). The size differences (between 50 and 10 °C) of the thermosensitive nanospheres also increased with the HEMA content up to 37 mol % HEMA (Table 1). It is clear that HEMA enhances significantly the thermosensitivity of the PDEA beads at lower HEMA contents (even at just 2 mol % HEMA). At higher HEMA contents (above 50%), however, the opposite effect on thermosensitivity of the copolymers was observed. The results indicate an enhanced hydrogen bonding in the presence of small quantities of HEMA in the copolymer, with corresponding enhancement in the thermosensitivity of the gel. An increase in the HEMA content may result in a more rigid structure that is harder to contract upon the disruption of hydrogen bonds, which means that the enhancement of the thermosensivity was not accompanied by an enhanced hydrophilicity of the copolymers. The LCST of the spheres (Table 1) shows a general decreasing trend with increasing HEMA content to 48 mol %. Self-Assembly of the Nanospheres. The centrifugation of the nanospheres (white color for a stable suspension) yielded samples that were iridescent when the HEMA content was less than 40 mol %. The color of the sample is dependent on the viewing angle. This indicates that the beads are packed in a regular fashion meeting conditions of the Bragg’s law in the visible light wavelength range. Diffraction spectra obtained for various polymers are shown in Figure 2, and constructive interference is observed at specific wavelengths. Self-assembly of the nanoparticles is also clearly seen by AFM (Figure 3). Upon spin-casting of the mixture, the beads assembled into hexagonally ordered arrays. AFM images of 37% HEMA beads showed that these soft beads are compressed from their expected spherical diameters. In solution, the particles are spherical with a diameter of approximately 450 nm at room temperature (obtained by DLS, Figure 1), but the dried samples form disclike particles with an average diameter of approximately 425 nm and a height of about 85 nm (data not shown). In an attempt to increase the spherical character of the particles after spin-casting and drying, beads with a higher degree of cross-linking (10% cross-linking agent) and 2.5% HEMA in the feed were synthesized. It was hoped that increased cross-linking density might reduce the amount of particle deformation upon drying. Figure 3 shows both the height and phase images of the particles. However, the AFM images still show they were in the form of discs (ca. 420 nm in diameter and ca. 110 nm in height). The phase image in Figure 3B shows
Figure 4. (A) The UV-visible spectra of the avidin-HABA complex, showing a decrease in the absorbance of the complex as free biotin concentration changed from 0 to 15 µM. The inset shows the calibration curve of the changes in absorbance as a function of biotin concentration (26). (B) UV-visible spectra of the avidin-HABA complex without (solid line) and with (dotted line) added biotinylated poly(DEA-coHEMA) nanospheres containing 37% HEMA.
that each particle has a core size of ca. 420 nm surrounded by additional ring of ca. 120-130 nm in thickness increasing the diameter to ca. 670 nm. Therefore, this moderate increase in the degree of cross-linking did not help the particles to retain their spherical form upon centrifugation and dehydration. The Biotinylated Spheres and Their Interaction with Streptavidin. The chemical conjugation of biotin to the polymer nanospheres (formation of an ester bond) can be evidenced by the reduced thermosensitivity of particles as shown in the example of the spheres containing 37% HEMA (Figure 5). Before biotinylation, the particles exhibited a sharp reduction in size from ca. 900 to 200 nm in water when the temperature was raised from 10 to 50 °C. After biotinylation, the change in size becomes much smaller (from ca. 800 to 450 nm). The results suggest a reduction in hydrophilicity upon biotinylation. Biotin coupling to the bead surface may render the outer layer of particle more hydrophobic, causing reduced thermosensitivity. The amount of biotin attached on the polymer particles was quantified by the avidin-HABA assay (24-26). Figure 4 shows the UV-visible spectra of the avidin-HABA complex and the
1002 Bioconjugate Chem., Vol. 18, No. 3, 2007
Colonne et al.
Figure 6. Size variation of biotinylated poly(DEA-co-HEMA) nanospheres containing 37% HEMA with different amounts of added streptavidin to 10 mL of a sample containing the nanospheres at three different temperatures.
Figure 5. Thermosensitivity of the poly(DEA-co-HEMA) nanospheres (A) before and (B) after biotinylation, and in the presence or absence of streptavidin. “P” indicates the polymer particles containing 37 mol % HEMA.
spectroscopic changes of the complex upon the addition of biotinylated particles. The absorbance of the avidin-HABA complex at 500 nm decreased when biotinylated particles were added, since HABA (dissociation constant Kd ≈ 10-6 M) was replaced by biotin, which has a much higher binding affinity to avidin (Kd ≈ 10-15 M for avidin complex with free biotin) (26). On the basis of the calibration curve in Figure 4A, the amount of available biotin on the biotinylated particles was calculated (ca. 9.8 nmol/mL of the particle solution). The value can be further converted to ca. 2.0 µmol/g of particles. The amount of biotin detected may only correspond to those accessible for the avidin-HABA test, which may underestimate the total amount of biotin attached. The amount of biotin estimated in this test is still meaningful, since only the accessible biotin molecules can participate in the subsequent interaction studies. This amount corresponds only to 0.1% of the HEMA groups on the polymer spheres. Figure 5A shows a blank experiment with unbiotinylated particles containing 37% HEMA. The addition of free streptavidin to these particles did not cause any appreciable change of their thermosensitivity, indicating the absence of any interactions. When streptavidin was added to a solution of biotinylated beads (Figure 5B), however, the change in the thermosensitivity curves is clearly shown. The size of these particles was reduced upon the interaction between steptavidin and biotin throughout the entire temperature range, with the largest reduction in size at temperatures below the LCST. The overall complex became less thermosensitive with a smaller size change than the original particles before the binding. Figure 6 shows that the change in particle size became more pronounced with subsequent additions of streptavidin due to increased biotin-streptavidin interactions. At the relatively low concentrations of the spheres used in this study, we did not observe any aggregation of the particles that may be caused by the cross-linking effect of streptavidin for the nanospheres. The reduction in size is due to reduced hydrophilicity of the particles upon binding with streptavidin,
similar to the process experienced by the thermosensitive particles upon heating. Even at such low biotinylation levels, the presence of streptavidin was still clearly sensed by the biotinylated spheres. A high level of immobilized biotin may be desirable for a more efficient binding; care should be taken here since increased biotinylation may also reduce the thermosensitivity of the spheres (19) and hence its sensitivity toward the protein. It is interesting to note that the binding between the protein (streptavidin) and the biotinylated particles was reversible. Figure 5B shows that the addition of free biotin to the streptavidin-containing system actually restored the particle size and their thermosensitivity to a state similar to that before their binding with streptavidin. Clearly, streptavidin has a higher affinity to free biotin than to the biotinylated particles. This reversibility indicates that such a system can even be reused if necessary. Results in Figure 5 also suggest that the streptavidin binding to the biotinylated particles may be disrupted above the LCST (as shown by the size change of the particles), which was suggested previously (22). An experiment was performed to study the size variation of the beads at three different temperatures when increasing amounts of streptavidin were added to a sample containing 37% HEMA (Figure 6). The profiles are rather similar below the LCST of the gels. It is shown that the size change becomes more pronounced when the amount of streptavidin reaches about 0.6 mg (9 nmol) in 10 mL of the aqueous mixture which contains 2 mg of solid polymer particles (the amount of available biotin is 4 nmol). It is known that 1 molecule of streptavidin can bind up to 4 molecules of biotin. Therefore, this much lower stoichiometric ratio indicates that steric hindrance may have inhibited the binding of the immobilized biotin on the particles to streptavidin and that not all the binding sites were occupied. To verify the specificity of the interaction between the biotinylated nanospheres and streptavidin, BSA, with a similar molecular weight (66 000 Da) to that of streptavidin, was also added to a solution of the beads. No size change of the nanospheres occurred, which indicates that the complexation is specific to the coupling between biotin and streptavidin.
CONCLUSION It is clear that the hydrophilicity of the biotinylated hydrogel nanospheres changes upon binding with streptavidin. The biotin loading on the polymer spheres was relatively low, but already effective in triggering a response of the polymer spheres upon interaction with the protein. This is rather similar to the dehydration of the thermosensitive polymers in water when the temperature is raised, caused by the disruption of hydrogen bonds. The size change of the particles is more pronounced at
Bioconjugate Chem., Vol. 18, No. 3, 2007 1003
Technical Notes
lower temperatures when the particles are most hydrated. The binding is reversible upon the addition of free biotin to the mixture, indicating its higher binding affinity in the solution in comparison to the biotinylated beads. It may be interesting to selectively introduce extra HEMA surface groups to the particles by a delayed addition of HEMA during the polymerization. This may help in the optimization of this model sensor system. This work sets an example to attach other bioactive molecules in a similar fashion to noncharged soft hydrogel spheres for the development of useful biosensing systems with specificity.
ACKNOWLEDGMENT Financial support FQRNT of Quebec, NSERC of Canada, and the Canada Research Chair program is gratefully acknowledged.
LITERATURE CITED (1) Bae, Y. H., Okano, T., and Kim, S. W. (1991) “On-off” thermocontrol of solute transport. I. Temperature dependence of swelling of N-isopropylacrylamide networks modified with hydrophobic components in water. Pharm. Res. 8, 531-537. (2) Miyata, T., Asami, N., and Uragami, T. (1999) A reversibly antigenresponsive hydrogel. Nature (London) 399, 766-769. (3) Liu, H. Y., and Zhu, X. X. (1999) Lower critical solution temperatures of N-substituted acrylamide copolymers in aqueous solutions. Polymer 40, 6985-6990. (4) Nichifor, M., and Zhu, X. X. (2003) Copolymers of N-alkylacrylamides and styrene as new thermosensitive materials. Polymer 44, 3053-3060. (5) McPhee, W., Tam, K. C., and Pelton, R. (1993) Poly(N-isopropylacrylamide) lattices prepared with sodium dodecyl sulfate. J. Colloid Interface Sci. 156, 24-30. (6) Pelton, R. H., Pelton, H. M., Morphesis, A., and Rowell, R. L. (1989) Particle sizes and electrophoretic mobilities of poly(Nisopropylacrylamide) latex. Langmuir 5, 816-818. (7) Asher, S. A., Holtz, J., Liu, L., and Wu, Z. (1994) Self-assembly motif for creating submicron periodic materials. Polymerized crystalline colloidal arrays. J. Am. Chem. Soc. 116, 4997-4998. (8) Reese, C. E., Baltusavich, M. E., Keim, J. P., and Asher, S. A. (2001) Development of an intelligent polymerized crystalline colloidal array colorimetric reagent. Anal. Chem. 73, 5038-5042. (9) Holtz, J. H., and Asher, S. A. (1997) Polymerized colloidal crystal hydrogel films as intelligent chemical sensing materials. Nature (London) 389, 829-832. (10) Asher, S. A., Alexeev, V. L., Goponenko, A. V., Sharma, A. C., Lednev, I. K., Wilcox, C. S., and Finegold, D. N. (2003) Photonic crystal carbohydrate sensors: Low ionic strength sugar sensing. J. Am. Chem. Soc. 125, 3322-3329. (11) Furukawa, H., Shimojyo, R., Ohnishi, N., Fukuda, H., and Kondo, A. (2003) Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles. Appl. Microbiol. Biotechnol. 62, 478-483. (12) Garret-Flaudy, F., and Freitag, R. (2001) Use of the avidin (imino)biotin system as a general approach to affinity precipitation. Biotechnol. Bioeng. 71, 223-234.
(13) Kumpumbu-Kalemba, L., and Leclerc, M. (2000) Electrochemical characterization of monolayers of a biotinylated polythiophene: Towards the development of polymeric biosensors. Chem. Commun. 1847-1848. (14) Marks, R. S., Novoa, A., Thomassey, D., and Cosnier, S. (2002) An innovative strategy for immobilization of receptor proteins on to an optical fiber by use of poly(pyrrole-biotin). Anal. Bioanal. Chem. 374, 1056-1063. (15) Glidle, A., Yasukawa, T., Hadyoon, S, C., Anicet, N., Matsu, T., Nomura, M., and Cooper, J. M. (2003) Analysis of protein adsorption and binding at biosensor polymer interfaces using X-ray photon spectroscopy and scanning electrochemical microscopy. Anal. Chem. 75, 2559-2570. (16) Shim, M., Kam, N. W. S., Chen, R. J., Li, Y., and Dai, H. (2002) Functionalization of carbon nanotubes for biocompatibility and biomolecular recognition. Nano Lett. 2, 285-288. (17) Lee, K-B., Yoon, K. R., Woo, S. I., and Choi, I. S. (2003) Surface modification of poly(glycolic acid) for biomedical applications. J. Pharm. Sci. 92, 933-937. (18) Ding, Z., Fong, R. B., Long, C. J., Stayton, P. S., and Hoffman, A. S. (2001) Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield. Nature (London) 411, 59-62. (19) Bulmus, V., Ding, Z., Long, C. J., Stayton, P. S., and Hoffman, A. S. (2000) Site-specific polymer-streptavidin bioconjugate for pHcontrolled binding and triggered release of biotin. Bioconjugate Chem. 11, 78-83. (20) Malmstadt, N., Hyre, D. E., Ding, Z., Hoffman, A. S., and Stayton, P. S. (2003) Affinity thermoprecipitation and recovery of biotinylated biomolecules via a mutant streptavidin-smart polymer conjugate. Bioconjugate Chem. 14, 575-580. (21) Kulkarni, S., Schilli, C., Mu¨ller, A. H. E., Hoffman, A. S., and Stayton, P. S. (2004) Reversible meso-scale smart polymerprotein particles of controlled sizes. Bioconjugate Chem. 15, 747753. (22) Ding, Z., Long, C. J., Hayashi, Y., Bulmus, E. V., Hoffman, A. S., and Stayton, P. S. (1999) Temperature control of biotin binding and release with a streptavidin-poly(N-isopropylacrylamide) sitespecific conjugate. Bioconjugate Chem. 10, 395-400. (23) Shea, K. J., Stoddard, G. J., Shavelle, D. M., Wakui, F., and Choate, R. M. (1990) Synthesis and characterization of highly crosslinked poly(acrylamides) and poly(methacrylamides). A new class of macroporous polyamides. Macromolecules 23, 44974507. (24) Green, N. M. (1975) Avidin. In AdVances in in Protein Chemistry, Vol 29, pp 85-133, Academic Press, New York. (25) Green, N. M. (1970) Spectrophotometric determination of avidin and biotin. Methods Enzymol. 18, 418-424. (26) Qi, K., Ma, Q. G., Remsen, E. E., Clark, C. G., and Wooley, K. L. (2004) Determination of the bioavailability of biotin conjugated onto shell cross-linked (SCK) nanoparticles. J. Am. Chem. Soc. 126, 6599-6607. BC060302B
