Bioconjugate Chem. 2004, 15, 747−753
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Reversible Meso-Scale Smart Polymer-Protein Particles of Controlled Sizes Samarth Kulkarni,† Christine Schilli,‡ Axel H. E. Mu¨ller,*,‡ Allan S. Hoffman,*,† and Patrick S. Stayton*,† Department of Bioengineering, University of Washington, Seattle, Washington 98195 and Makromolekulare Chemie II, Universita¨t Bayreuth, D-95440 Bayreuth, Germany. Received November 20, 2003; Revised Manuscript Received May 4, 2004
Functionalized beads and particles in the size range of tens to hundreds of nanometers (nano- to meso-scale) are finding increased applications in the bioanalytical field. We show here that conjugates of streptavidin and the temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm), synthesized with low polydispersities by reversible addition-fragmentation chain transfer (RAFT) polymerization, rapidly formed mesoscale polymer-protein particles above the lower critical solution temperature (LCST). The average hydrodynamic diameters of these particles could be controlled between 250 nm to 900 nm by the choice of conjugate concentration and polymer molecular weight, and/or through control of the rate of temperature change. Once formed, the biohybrid particles were found to be stable for >16 h at the controlled size, unlike the free PNIPAAm which continued to aggregate and grow over time into very large and polydisperse aggregates. The reversibility between the smart polymer-protein particles and the free polymer-protein conjugates opens potential uses in traditional diagnostic formats and in microfluidic formats where the differential diffusive and physical properties might be exploited for separations, analyte concentration, and signal generation.
INTRODUCTION
There is a continuing need for better upstream processing of sample fluids, control over molecular recognition events, and better sensitivity in the bioanalytical field. Important developments in the synthesis of microand nanoparticles with controlled physical and chemical properties are providing new opportunities for this field (1-3). Controlled structures such as micelles, cylindrical rods, and vesicles self-assemble in aqueous phases from amphiphilic block copolymers or synthetic peptides and can be subsequently derivatized with functional biomolecules (4, 5). There has also been considerable progress recently in the synthesis of metal-based and inorganic nanoparticles that exhibit interesting optical properties and which can be functionalized with a variety of synthetic and biomolecular coatings (6, 7). “Smart” beads are another interesting class that exhibit a reversible change in their properties in response to signals such as temperature or pH. A variety of synthetic techniques have been described to construct such beads, typically based on incorporating a stimuliresponsive polymer into a cross-linked bead or particle, or by grafting a stimuli-responsive polymer onto the cross-linked bead or particle (8, 9). Our own group has been developing biohybrid materials that utilize the stimuli-responsiveness of the smart polymer chain to control the molecular recognition activity of proteins or * To whom correspondence should be addressed. P.S.S.: tel 206-685-8148, fax 206-685-8256, e-mail stayton@ u.washington.edu; A.S.H.: tel 206-543-9423, fax 206-543-6124, e-mail
[email protected], A.H.E.M.: tel +49-921553399, fax +49-921-553393, e-mail
[email protected]. † University of Washington. ‡ Universita ¨ t Bayreuth.
to reversibly control smart bead aggregation in microfluidic channels (10, 11). The polymer component serves as an antenna and a switch to reversibly control the activity and aggregation state of smart polymerprotein conjugates or smart polymer-bead conjugates. It was recently shown that one mechanism by which protein switching activity can be designed involves reversible particle formation and dissolution that is driven by the polymer phase transition (12). The free polymer-protein conjugates in solution were in an onstate, and the switching activity (i.e. off-state) was found to be dependent on the orientation of the protein active site relative to the polymer in the aggregated particle state. These initial findings motivated the current study of the physical properties of the smart polymer-protein particles. Better control of the size and formation kinetics of these smart meso-scale particles that form and dissolve upon exposure to specific stimuli should provide design opportunities for bioanalytical and diagnostic technologies. EXPERIMENTAL PROCEDURES
Materials. N-Isopropylacrylamide (Aldrich, 97%) was recrystallized twice from benzene/hexane 3:2 (v:v) and dried under vacuum prior to use. 1,4-Dioxane (Merck) was refluxed over potassium for 3 days and then distilled. Azobis(isobutyronitrile) (AIBN, Fluka) was recrystallized from methanol and dried under vacuum prior to use. 1-Biotinamido-4-[4′-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC) was obtained from Pierce, IL. Recombinant streptavidin was expressed in Escherichia coli and purified as previously described (13). Synthesis of PNIPAAm with Functional SH End Group. PNIPAAm with narrow molecular weight distribution was synthesized using the reversible addition-
10.1021/bc034215k CCC: $27.50 © 2004 American Chemical Society Published on Web 06/30/2004
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fragmentation chain transfer (RAFT) polymerization technique as described previously (14). The chain transfer agent (CTA), benzyl 1-pyrrolecarbodithioate, was synthesized as described (15). RAFT polymerization was performed at 60 °C using the benzyl CTA, AIBN as initiator, and the monomer under a nitrogen atmosphere in 1,4-dioxane as a solvent. For the polymerization series conducted at different benzyl CTA concentrations, stock solutions were prepared with a monomer concentration of 1.8 mol/L, an initiator concentration of 6.90 mmol/L, and CTA concentrations of 3.92 × 10-2, 1.96 × 10-2, and 4.90 × 10-3 mol/L. CTA (0.915-0.114 g; 3.92-0.49 mmol) was dissolved in 1,4-dioxane (98 mL), and the solution was degassed by three freeze-thaw evacuation cycles. AIBN (0.115 g, 0.70 mmol) was dissolved separately in 1,4-dioxane (2 mL) and degassed by three freeze-thaw evacuation cycles. The monomer (20.37 g, 0.18 mol) was added via a Schlenk tube under nitrogen to the solution of the chain transfer agent in dioxane. After the monomer was completely dissolved and the mixture heated to 60 °C (temperature of oil bath), the initiator solution was injected with a syringe. All polymerization reactions were carried out under nitrogen atmosphere and aliquots were withdrawn at different time intervals. The aliquots were immediately immersed in liquid nitrogen and subsequently freeze-dried. Residual monomer was removed by sublimation. The dried samples were dissolved in THF containing 0.25% tetrabutylammonium bromide for GPC analysis. The dithiocarbamate end groups of the synthesized polymers were hydrolyzed under basic conditions to yield the corresponding thiol-terminated polymers. The polymer was dissolved in a mixture of MeOH/aq 28% NaOH (7:3) for 4 h. Ethylenediaminetetra(acetic acid) (EDTA) was added to the solution to prevent oxidation of the resultant thiol groups. The reaction mixture was acidified with 88% formic acid, MeOH was evaporated, and the residue was freeze-dried. Lower Critical Solution Temperature (LCST) Measurement of PNIPAAm. The LCST was determined as the temperature at 10% of the maximum absorbance at 600 nm. PNIPAAm at a concentration of 0.25 wt. % in deionized H2O was used. The data were collected using a UV-vis spectrophotometer with a jacketed cuvette holder to control the temperature of the sample. A heating rate of 0.5 °C/min was used, and absorbance values were measured every 30 s. Biotinylation of PNIPAAm and Complexation with Streptavidin. The PNIPAAm-SH was biotinylated using a functionalized biotin, 1-biotinamido-4-[4′-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). Briefly, BMCC was dissolved in DMSO at a concentration of 2.4 mg/mL. Four milligrams of PNIPAAm was dissolved in 875 µL of phosphate buffer (PB), 50 mM, pH 7.0, just prior to the reaction. BMCC solution (75 µL) was added to the PNIPAAm solution and the reaction proceeded overnight at RT. The excess BMCC was removed by desalting on a PD-10 column (Pierce) using PB, 50mM, pH 7.0, as an eluent. The efficiency of biotinylation of the PNIPAAm was determined using the neutravidinHABA assay as described by Lackey et al (16). The biotinylated PNIPAAm (b-PNIPAAm) was physically mixed with streptavidin in PB, 50 mM, pH 7.4, and incubated overnight for conjugation. The b-PNIPAAm and tetrameric streptavidin were mixed stoichiometrically at a 4:1 ratio. Dynamic Light Scattering. Sizing measurements were performed using a Brookhaven BI90Plus instrument equipped with a 535-channel correlator. A 656 nm laser source was used as the incident beam, and mea-
Kulkarni et al.
surements were performed at a 90° angle. Samples were filtered through 0.2 µm cutoff mixed cellulose ester filters (Whatman), and 75 µL sample volumes were used for measurement. PB, 50 mM, pH 7.4, was used as a diluent to achieve various concentrations of the conjugate. A heater within the cuvette holder of the instrument was used to heat or cool the sample to various temperatures. The temperature of the sample was measured using a thin wire thermocouple (Cole Palmer). Measurements were performed at several temperatures below and above the LCST of the PNIPAAm. For each conjugate concentration, fresh samples were prepared on different days and measurements were performed in triplicate. The intensity autocorrelation function (ACF), G(2)(τ), was measured in the self-beating mode and can be expressed by the Siegert relationship:
G(2)(τ) ) B(1 + f 2|g(1)(τ)|2) where τ is the decay time, B is a measured baseline, f is the coherence factor, and g(1)(τ) is the normalized firstorder electric field time autocorrelation function E(τ). g(1)(τ) is related to the line-width distribution G(Γ) by:
|g(1)(τ)| )
∫0∞ G(Γ)exp(-Γτ)dΓ
Using the cumulant analysis (17),
|g(1)(τ)| ) exp(-Γτ)[1 + (µ2/2!)τ2 - (µ3/3!)τ3 + ...] the average line width 〈Γ〉 is obtained. This yields the apparent diffusion coefficient, Dapp ) 〈Γ〉/q2, which can be extrapolated to zero scattering vector, q, to obtain the translational diffusion coefficient, D. The hydrodynamic radius, Rh, is then determined using the Stokes-Einstein relation:
Rh ) kBT/(6πηoD) where kB, T, and ηo are the Boltzmann constant, the absolute temperature, and solvent viscosity, respectively. For relatively narrow decay-rate distributions, the second moment, µ2, designated as the polydispersity index, can be used to estimate the relative distribution width. Assuming a log-normal size distribution for the particles, the full-width at half-maximum (fwhm) was calculated from Rh and µ2 using:
fwhm ) 2Rhsqrt(µ2) The reliability of size measurements by the cumulant analysis was verified by applying other established models to the obtained correlation function. The models chosen were the nonnegatively constrained least squares (NNLS) (18, 19) and CONTIN (20, 21). Root-mean-square deviations for each of the model fits were calculated and used for preliminary comparison of model fits. Stability of the Nanoparticles. To determine the stability of the particles, a 100 µL sample of PNIPAAmstreptavidin conjugate was heated to 42.5 °C in the cuvette as described above. An airtight lid on the cuvette was used to minimize evaporation. Size measurements were taken at different time points up to 1000 min. As a control, a b-PNIPAAm sample, without the streptavidin was used. This PNIPAAm sample had the same molar concentration as that of the polymer (both bound and unbound) in the conjugate solution. Particle Formation and Dissolution Kinetics. Since sizing measurements cannot be performed within
Controlled and Reversible Nanoparticles from Smart Polymers
small time intervals, the scattering intensity, I, in counts per second (CPS), was used as an index of nanoparticle formation. As the PNIPAAm-streptavidin conjugates transform from free soluble conjugates to particulate form above the LCST, the scattering intensity increases. This scattering intensity is measured to analyze the kinetics of formation of the particles. The cuvette containing the conjugate sample was cooled to 23 °C and then placed in the heated cuvette holder of the DLS instrument maintained at 42.5 °C. The intensities were measured at a sampling interval of 5 µs as the temperature increased and finally equilibrated at 42.5 °C. For particle dissolution studies, the conjugate sample incubated at 42.5 °C in a cuvette was subsequently placed in the jacketed cuvette holder maintained at 25 °C. Intensities were measured at a sampling interval of 5 µs as the sample cooled. The temperature profile for the sample was recorded using a thermocouple with a sampling interval of 15 s. Dependence of Nanoparticle Size on Concentration and Molecular Weight of PNIPAAm. Nanoparticle hydrodynamic diameters were measured for the bioconjugates of streptavidin with PNIPAAm of molecular weights (number-averaged molecular weight, Mn) 2.9 kDa, 4.8 kDa, 14.9 kDa, and 25.9 kDa, respectively, to determine the dependence of size on molecular weight of the polymer. The concentration dependence was determined by using concentrations of 9 µM, 3 µM, 1 µM, 500 nM, and 100 nM for each bioconjugate. The TukeyKramer multiple comparisons statistical test was used to analyze the data. Effect of Heating Rate. Size measurements were made at three different heating rates for the 14.9 kDa PNIPAAm-streptavidin conjugate at a 1 µM concentration. The cuvette containing the conjugate was cooled to 23 °C and then placed in the heated cuvette holder of the DLS instrument maintained at 33, 38.5, and 42.5 °C to get different heating rates. The temperature of the conjugate solution in the cuvette was measured using a thermocouple with a sampling time of 15 s. The heating rates were measured by calculating the derivative of the temperature-time curve at the respective LCST values. Heating rates of 0.04 °C/s, 0.12 °C/s, and 0.16 °C/s were obtained for the 32.5, 38.5, and 42.5 °C block temperatures, respectively. RESULTS
Synthesis of PNIPAAm with Narrow Molecular Weight Distributions. Using the RAFT technique, PNIPAAm of different molecular weights with narrow molecular weight distributions were synthesized. A range of four molecular weights (Mn) were chosen for conjugation to streptavidin: 25.9 kDa (polydispersity ) 1.08), 14.8 kDa (polydispersity ) 1.09), 4.8 kDa (polydispersity ) 1.16), and 2.9 kDa (polydispersity ) 1.16). The dithiocarbamate end group on the polymer from the chain transfer agent was cleaved to generate a functional thiol end group that was used for end-specific biotin conjugation. The LCST values, as defined by 10% change in absorbance at 0.2 mg/mL polymer concentration, for the 25.9 kDa (31 °C) and 14.9 kDa (31.4 °C) polymers were similar, but those for the 2.9 kDa (26.8 °C) and 4.8 kDa polymers (29.0 °C) were slightly lower. A linear dependence of LCST values versus reciprocal molecular weight values was previously described for the dithiocarbamateterminated polymers (22). Biotinylation of PNIPAAm and Complexation with Streptavidin. Biotinylation efficiencies calculated
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Figure 1. Schematic representation of the formation of PNIPAAm-streptavidin smart nanoparticles. Below the LCST, the PNIPAAm is hydrophilic and the bioconjugate remains in solution. Above the LCST, the hydrophobicity of the PNIPAAm directs the aggregation of the bioconjugates into nanoparticles of defined sizes. The hydrophilic streptavidin stabilizes the nanoparticles and prevents further aggregation.
Figure 2. Stability of the nanoparticles formed from the 14.9 kDa PNIPAAm-streptavidin conjugate at 42.5 °C and a concentration of 3 µM is shown. The particles fomed by the bioconjugate (b) are stable in size for over 16 h. In contrast, aggregates of b-PNIPAAm (0) at the same concentration do not have a hydrophilic moiety to stabilize the size and aggregate over time into larger particles. Error bars represent ( 1 standard deviation over the three measurements.
by the HABA assay yielded average values of 57% for the 25.9 and 14.9 kDa polymers, 62% for the 4.8 kDa polymer, and 60% for the 2.9 kDa polymer. The biotinylation efficiency was used to calculate the amount of polymer to be added to streptavidin for complexation. We have shown previously that a maximum of two biotinylated polymers are bound per tetrameric streptavidin due to steric hindrance (23). In these studies, a ratio of 2.4 biotinylated PNIPAAm polymer chains to streptavidin tetramer was used. Stability and Kinetic Properties of StreptavidinPNIPAAm Particle Formation. The polymer-streptavidin conjugates are soluble at temperatures below the LCST of PNIPAAm, and no sizing measurements could be made due to the low level of scattering intensities. As the conjugate was heated to temperatures above the LCST of PNIPAAm, the polymer becomes hydrophobic and drives the aggregation of the conjugates into uniform and stable particles (Figure 1). The stability of the nanoparticle size for the 14.9 kDa conjugate at a fixed concentration of 3 µM is shown in Figure 2. Once formed, the particles did not aggregate further and had the same average size after 16 h (Tukey-Kramer test, p < 0.05). In contrast, PNIPAAm-biotin alone continued to aggregate into larger particles over this time period (>1 µm diameter after 1 h). Sizing measurements could not
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Kulkarni et al. Table 1. Particle Sizes of Streptavidin-PNIPAAm Bioconjugatesa concentration Mn of PNIPAAm conjugate to streptavidin PNIPAAm-25.9 kDa-Stav Dh (nm) fwhm (nm) PNIPAAm-14.9 kDa-Stav Dh (nm) fwhm (nm) PNIPAAm-4.8 kDa-Stav Dh (nm) fwhm (nm) PNIPAAm-2.9 kDa-Stav Dh (nm) fwhm (nm)
Figure 3. The kinetics of formation of particles by the 14.9 kDa PNIPAAm-streptavidin conjugate at a concentration of 1 µM as measured by light scattering intensity is shown. The scattering intensity (s) is measured as the temperature of the conjugate is ramped from 23 °C to 42.5 °C. As the particles are formed, the scattering intensity, measured in kilocounts per second (kCPS), increases in a highly cooperative manner. The increase in sample temperature (- - -) with time is overlaid onto the kinetic trace. The LCST of the PNIPAAm (- - -) is shown on the temperature axis.
Figure 4. Dissolution kinetics for the 14.9 kDa PNIPAAmstreptavidin conjugate at a concentration of 1 µM is shown. The scattering intensity is measured as the sample is cooled from 42.5 °C to 25 °C. As the particles dissolve, the scattering intensity (s), measured in kilocounts per second (kCPS), decreases. The decrease in sample temperature (- -) with time is overlaid onto the kinetic trace. The LCST of the PNIPAAm (‚‚‚) is shown on the temperature axis.
be made beyond 400 min for the PNIPAAm-biotin as the size exceeded the range of the instrument. The particle formation kinetics, measured by the increase in scattering intensity as the conjugate was heated from 20 °C to 42.5 °C, are shown in Figure 3. The transition from soluble conjugates to particles was found to be rapid (within 20 s) and occurs in a narrow temperature range at the critical point. The time lapse of ca. 100 s observed after the temperature of the solution surpasses the LCST suggests that there is an induction period with early nucleation events that fall below the resolution of this observation technique. After reversal of the temperature stimulus, the scattering intensity decreased by more than 90% within 2 min, consistent with the dissolution of the particles in this time period (Figure 4). In Figure 4, it is seen that the dissolution of the particles commences as soon as the sample is removed from the 42.5 °C heat block and exposed to the atmosphere. This anomaly could be due to dissolution of particles exposed to the surface, where cooling is faster than in the bulk (where temperature was measured). Particle Size Controlled by Concentration and Molecular Weight. At temperatures above the LCST,
9 µM
3 µM
1 µM
0.5 µM
0.1 µM
719 51 744 53 975 198 449 124
670 92 711 157 751 256 305 149
426 131 571 141 381 78
336 123 381 124 375 126
236 81 271 75 243 85
a Size data for the particles formed from PNIPAAm-streptavidin conjugates of different molecular weights and different concentrations is shown. The particles were formed by heating the sample to 42.5 °C. Dh represents the average hydrodymamic diameter, and fwhm represents the relative width of the size distribution. Sizing data for the 2.9 kDa PNIPAAm conjugate could not be obtained at lower concentrations due to insufficient scattering from the particles. Values represent the average of three measurements for each molecular weight and concentration.
Figure 5. Dependence of the average hydrodynamic particle diameter on conjugate concentration is shown for the 14.9 kDa PNIPAAm-streptavidin conjugate at 42.5 °C. Size measurements were performed 20 min after particle formation. Error bars represent ( 1 standard deviation over three measurements with freshly prepared samples. The average hydrodynamic diameters increase with concentration.
the bioconjugates assemble into stable particles whose size is dependent on the concentration and the molecular weight of the PNIPAAm (Table 1). The dependence of particle hydrodynamic diameter on conjugate concentration for the 14.9 kDa conjugate at 42.5 °C is shown in Figure 5. The particle size increases with the concentration of the bioconjugate for all the molecular weights of PNIPAAm tested (a complete concentration curve could not be obtained for the 2.9 kDa polymer due to insufficient scattering intensity at low concentrations). Furthermore, the particles were stable once formed (>16 h) and further dilution at the elevated temperatures did not significantly affect the size of the particles (data not shown). The hydrodynamic particle sizes also depend on the molecular weight of the PNIPAAm used for conjugation. Figure 6 shows the dependence of particle diameter on PNIPAAm molecular weight for a fixed concentration of the conjugate. Particle sizes were comparable for the 25.9, 14.9, and 4.8 kDa conjugates, with no significant change in size with change in molecular weight (p < 0.05). The 2.9 kDa conjugate had a much smaller size, indicating that below a threshold molecular weight, the nucleation and growth mechanism changes. Table 1 shows particle sizes and full widths at half-maximum (fwhm) for the
Controlled and Reversible Nanoparticles from Smart Polymers
Figure 6. Dependence of the average hydrodynamic particle diameter on molecular weight (number averaged molecular weight) of the PNIPAAm used for conjugation to streptavidin is shown for the 25.9 kDa (b), 14.8 kDa (2), 4.8 kDa (9), and 2.9 kDa ([) conjugates at a concentration of 3 µM at 42.5 °C. Average particle hydrodynamic diameters are comparable for the 25.9 kDa, 14.8 kDa, and 4.8 kDa conjugates but significantly lower for the 2.9 kDa conjugate. Error bars represent ( 1 standard deviation from triplicate sizing measurements made for each conjugate.
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sizes could be formed by varying the heating rate. For example, the size of the 14.9 kDa PNIPAAm-streptavidin conjugate could be tightly controlled to 675 nm (( 20 nm) at a concentration of 1 µM by choosing a heating rate of 0.12 °C/s. Additionally, it was observed that the particles had a wider distribution when formed at lower heating rates. The different heating rates were achieved by incubating the sample at 23 °C and then inserting into heating blocks maintained at different temperatures, resulting in different final temperatures for each sample. Our experimental set up did not allow the sample to be ramped to the same final temperatures at different heating rates. To address the question of relative importance of the final temperature (equilibrium effect) versus the heating rate (kinetic effect) on particle size, the temperatures were changed after formation of the particles. Particles of size 790 nm (( 50 nm) were formed on ramping the temperature to 33 °C at a heating rate of 0.04 °C/s. There was no significant change in the size of these particles by subsequently raising the temperature of this sample to 42.5 °C (data not shown). Similarly, no change in size was observed when particles that were formed by heating to 42.5 °C were cooled to 38 °C (data not shown). This demonstrates that the particle size is largely governed by the kinetics of aggregation at defined concentrations and polymer molecular weights. DISCUSSION
Figure 7. Effect of heating rate on the hydrodynamic particle diameter for the 14.9 kDa PNIPAAm-streptavidin conjugate at a concentration of 1 µM is shown. Different heating rates were achieved by heating the sample incubated at 23 °C to final temperatures of 33, 38, and 42.5 °C corresponding to heating rates of 0.04, 0.12, and 0.16 °C/s, respectively. The size measurements were made 5 min after reaching the final temperature. The average hydrodynamic diameter of the particles decreases with heating rate. Error bars represent ( 1 standard deviation from three measurements.
different polymer molecular weights and concentrations. The fwhm values have been calculated from the second moment µ2 (polydispersity index) and reflect the relative distribution width of the particles. The average values of the polydispersity index ranged from 0.02 to 0.18, indicating narrow distributions of particle size. From the fwhm values, it was observed that the higher molecular weight polymers yielded particles with narrower relative (to size) distribution widths. This indicates that conjugates of higher molecular weight polymers form more uniform particles. Taken together, these data demonstrate that discrete particle sizes between the range of ca. 250 and 900 nm can be designed through appropriate choice of polymer molecular weight and concentration. Effect of Heating Rate on Particle Size. Particle size was observed to be dependent on the heating rate of the conjugate solution (Figure 7). Smaller particles were formed at higher heating rates and nanoparticles of fixed
The particle properties of a temperature-responsive polymer-streptavidin conjugate have been investigated in this study. The size of the particles formed above the PNIPAAm LCST could be controlled across an interesting and application relevant mesoscale of ca. 250-900 nanometers by manipulating the polymer molecular weight, the conjugate concentration, and/or the heating rate. Once formed, the particles were remarkably stable with narrow size distributions and yet could be formed or dissolved rapidly. This behavior was not seen with PNIPAAm alone, even at dilute concentrations, as the polymer aggregated into continually larger aggregates over time. The aggregation of PNIPAAm polymers at dilute concentrations into mesoscopic globules above its LCST has also been reported by Gorelov et al. (24). The authors reported that the mesoglobules formed by high molecular weight PNIPAAm (Mn ) 2-9 MDa), at dilute concentrations of 1-5 nM, are stable and do not aggregate further. In our experiments, however, we have observed that at concentrations typical of bioanalytical reactions, aggregates formed from lower Mn PNIPAAm alone are unstable and aggregate continuously (Figure 2). Particles formed from the biohybrid conjugates, however, are stabilized by the charged and hydrophilic streptavidin and remain stable in size even at higher concentrations. The protein thus serves as a hydrophilic capping agent to limit the bioconjugate particle size, similar to surfactants that prevent the superaggregation of similar temperature-responsive polymers (25). Similar observations have been made with colloidal particles of DNAPNIPAAm conjugates where the DNA acts as the hydrophilic shell (26). These experiments also indicate that the formation of the particles is largely a kinetic phenomenon dependent on the concentration and heating rate of the sample. The predominant effect of heating rate over the final equilibrium temperature on particle size demonstrates that once formed, the particles are kinetically stable and changes in equilibrium conditions over the temperature range studied do not alter the particle size. This is also
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substantiated by the fact that dilution after formation of the particles does not significantly change the particle size distribution. The properties of this meso-particle system provide interesting opportunities for the bioanalytical field. The reversibility of the bioconjugate particle formation allows molecular recognition steps to be performed with the free conjugates, while then enabling separation and concentration steps based on particle properties or vice versa. For example, the streptavidin conjugates can be used to capture biotinylated antibodies and their targets in homogeneous solution but can then be filtered as the mesoscale beads after temperature-triggered particle formation. After isolation, the bioconjugates can be released back as free molecular species with simple temperature reversal. Alternatively, the different diffusive properties of the particles might be exploited in microfluidic devices to separate particle-bound targets from freely diffusing contaminants, with the benefit again of being able to regenerate the bioconjugate species at controlled places in the channel device for capture or release (27, 28). In addition to these microfluidic device applications, many lab assays (e.g., PCR) depend on sequential processing steps that could benefit from all reagents being placed in a single complex mixture. The timing of each processing step could subsequently be controlled by sequential signals directing flocculation/aggregation and redispersal of specific biomolecular species. In this application it would be desirable to have the aggregated species turned off, which we have shown can be accomplished by controlling the orientation of the protein active site relative to the polymer (12). Similarly, for bioprocessing applications relying on sequential enzyme reactions, it should be possible to control sequential enzyme activities by bringing them together at specific times to amplify the sequential nature of the reactions. ACKNOWLEDGMENT
The authors thank Dr. M. Schurr (University of Washington) and W. Bernt (Brookhaven Instruments) for useful discussions. The authors gratefully acknowledge NIH grant No. EB00252, DFG grant No. Mu896/13-1, and the NSF-funded Center for Nanotechnology, University of Washington (UIF fellowship to S.K.). LITERATURE CITED (1) Cao, Y. W. C., Jin, R. C., and Mirkin, C. A. (2002) Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297 (5586), 1536-1540. (2) Niemeyer, C. M. (2001) Nanoparticles, proteins, and nucleic acids: Biotechnology meets materials science. Angew. Chem., Int. Ed. 40 (22), 4128-4158. (3) Alivisatos, P. (2004) The use of nanocrystals in biological detection. Nat. Biotechnol. 22 (1), 47-52. (4) Hartgerink, J. D., Beniash, E., and Stupp, S. I. (2002) Peptide-amphiphile nanofibers: A versatile scaffold for the preparation of self-assembling materials. Proc. Natl. Acad. Sci. U.S.A. 99 (8), 5133-5138. (5) Discher, B. M., Won, Y. Y., Ege, D. S., Lee, J. C. M., Bates, F. S., Discher, D. E., and Hammer, D. A. (1999) Polymersomes: Tough vesicles made from diblock copolymers. Science 284 (5417), 1143-1146. (6) Mirkin, C. A., Letsinger, R. L., Mucic, R. C., and Storhoff, J. J. (1996) A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 382 (6592), 607-609. (7) Chan, W. C. W., and Nie, S. M. (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281 (5385), 2016-2018.
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