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Magnetic Bio/Nanoreactor with Multilayer Shells of Glucose Oxidase and Inorganic Nanoparticles Ming Fang,† Patrick S. Grant,† Michael J. McShane,† Gleb B. Sukhorukov,‡ Vladimir O. Golub,§ and Yuri M. Lvov*,† Institute for Micromanufacturing, P.O. Box 10137, Louisiana Tech University, Ruston, Louisiana 71272, Max Planck Institute for Colloids and Interfaces, Potsdam/Golm, Germany, and Advanced Materials Research Institute, University of New Orleans, New Orleans, Louisiana 70148 Received March 13, 2002. In Final Form: May 29, 2002 Organized multilayers of nanoparticles (9-, 20-, and 45-nm-diameter silica or 12-nm magnetite) and glucose oxidase (GOx) were assembled in alternation with oppositely charged polyelectrolytes on 420-nm latex particles. Stepwise growth of the multilayer films on latex was confirmed by microelectrophoresis and transmission electron microscopy. The inclusion of silica layers on latex yields a higher surface area, resulting in greater GOx adsorption and thereby increasing the catalytic activity of the bioreactor. The bioactivity was proportional to the core surface area and also to the number of GOx layers in the shells. Also, the presence of magnetic nanoparticles allows self-stirring of the nanoreactors with a rotating magnetic field and enhances its productivity. The ensemble of GOx and fluorescent dyes in the shells demonstrated the correlation between Ru-bpy fluorescence and glucose concentration in solution.
Introduction The layer-by-layer (LbL) self-assembly process has been widely applied to nanocomposite thin films.1-7 The method involves the alternate adsorption of oppositely charged species and relies on the reversal of the surface charge of the film after deposition of every component. The LbL assembly process elaborated for planar solid supports has also been applied to colloid particles with diameters ranging from 100 nm to up to tens of micrometers.8-14 An example of amazing control over structure, these demonstrations have shown the capability to take nanoscale materials and process them further, with modifications occurring on the nanoscale. This enabling technology is providing a foundation upon which novel functional materials, with advantages of high surface area to volume ratio, are being developed for applications ranging from electronic to biomedical. As an example of this process (the one followed in the work reported here), a polycation solution is added to a suspension of colloid particles and, after adsorption †
Louisiana Tech University. Max Planck Institute for Colloids and Interfaces. § University of New Orleans. ‡
(1) Decher, G. Science 1997, 227, 1232. (2) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (3) Tsukruk, V.; Rinderspacher, F.; Bliznyuk, V. Langmuir 1997, 13, 2171. (4) Liu, Y.; Wang, A.; Claus, R. Appl. Phys. Lett. 1997, 71, 2265. (5) Mamedov, A.; Kotov, N. Langmuir 2000, 16, 5530. (6) Sikar, K.; Revzin, A.; Pishko, M. Anal. Chem. 2000, 72, 2930. (7) Forzani, E.; Otero, M.; Perez, M.; Teijelo, M, Calvo, E. Langmuir 2002, 18, 4020. (8) Caruso, F.; Caruso, R. A.; Mo¨hwald, H. Science 1998, 282, 1111. (9) Lvov, Y.; Price, R.; Singh, A.; Selinger, J.; Spector, M.; Schnur, J. Langmuir 2000, 16, 5932. (10) Sukhorukov, G.; Donath, E.; Lichtenfeld, H.; Knippel, E.; Knippel, M.; Budde, A.; Mo¨hwald, H. Colloids Surf., A 1998, 137, 253. (11) Sukhorukov, G. In Novel Methods to Study Interfacial Layers; Miller, R., Mo¨bius, D., Eds.; Elsevier: Amsterdam, 2001; pp 383-414. (12) Schu¨ler, C.; Caruso, F. Makromol. Rapid Commun. 2000, 21, 750. Caruso, F.; Schu¨ler, C. Langmuir 2000, 16, 9595. (13) Lvov, Y.; Caruso, F. Anal. Chem. 2001, 73, 4212. (14) Lvov, Y.; Antipov, A.; Mamedov, A.; Mo¨hwald, H.; Sukhorukov, G. Nano Lett. 2001, 1, 125.
Scheme 1. Illustration of the Layer-by-Layer Assembly of Polyelectrolyte, Nanoparticles (Silica or Magnetite), and Enzyme (Glucose Oxidase) on Latex Particles
saturation, the particles are separated from the unadsorbed polycation in solution by centrifugation. At this stage, the particles are coated with a polycation layer. Thereafter, anionic nanoparticles are deposited in the same manner and, if desired, additional polycation/enzyme layers are added by further alternate adsorption cycles (see Scheme 1). This LbL deposition of organized enzyme multilayered shells on latex particles is a promising approach for creating tiny reactors for biocatalysis applications, as has recently been demonstrated for glucose oxidase, peroxidase, urease, and β-glucosidase.11-14 Such organized enzyme/polyion multilayers, with enzyme layers arranged in a predetermined order within semipermeable polymeric shells, present a new class of bio/nanoreactors. The degree of order and complexity that can be imparted to these systems via the flexible LbL technique makes them distinct from the more traditional liposome- or copolymer-shelled enzymatic reactors. This work describes an expansion of capabilities for bio/ nanoreactors through inclusion of inorganic and magnetic nanoparticles within multilayer polymer/enzyme films. The aim is to develop methods to provide enhanced functionality such as catalytic activity and manipulation by producing higher surface area and magnetic shells. The results described are part of our overall strategy to create unique and complex biocolloids, with emphasis here on tailored enzyme self-assembly using nanoparticle layers on latex microcores. Examples of potential applications of
10.1021/la025731m CCC: $22.00 © 2002 American Chemical Society Published on Web 07/13/2002
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these general technologies include therapeutic devices, reactors for efficient catalysis, and biosensors. The LbL ensembles of ordered glucose oxidase multilayer shells on 420-nm-diameter latex spheres were prepared. Layers of nanoparticles with different diameters (9-nm, 20-nm, and 45-nm silica or 12-nm magnetite) were assembled on latex spheres, and enhanced enzymatic activity of glucose oxidase was registered in all cases. Deposition of magnetite nanoparticles provided a magnetic momentum that allowed the nanobioreactors to be selfstirred and separated with an external magnetic field. In further development, we composed the shell from glucose oxidase, an oxygen-sensitive ruthenium compound, and a reference fluorophore for testing as a glucose sensor. In this arrangement, glucose oxidase consumes oxygen in the presence of glucose, and the fluorescence intensity of the dye increases. Monitoring of fluorescence intensity allows an indirect measurement of glucose concentration. Experimental Section Materials. Glucose oxidase (GOx, G-6641, type II-S, from Aspergillus Niger, Sigma) was used at a concentration of 2 mg/ mL in 10 mM phosphate buffer saline (PBS) at pH 7.4. Poly(ethyleneimine) (PEI, Aldrich, MW 250 000), poly(dimethyldiallylammonium chloride) (PDDA, Aldrich), and sodium poly(styrenesulfonate) (PSS, Aldrich, MV 70 000) were used in PBS solutions at concentrations of 1 and 3 mg/mL, respectively. An aqueous dispersion of carboxylated polystyrene latex was obtained from Microparticles Gmbh, Germany. The silica particles (diameters of 9, 20, and 45 nm) were obtained from Nissan Chemical Inc., Japan. Magnetite nanoparticles (Fe3O4/Fe2O3, mean diameter of 14 nm) were from Mediport Kardiotechnik GmbH, Germany. They were prepared by coprecipitation of ferrous and ferric salt solutions by concentrated ammonium hydroxide and stabilized by monolayers of cis-9-octadecenoic acid.15 The dye (dianisidine), glucose, and peroxidase were from Sigma. Tris(2,2′-bipyridyl)dichloro-ruthenium(II) hexahydrate (Ru-bpy, Sigma, MW 748.63) was used at a concentration of 0.57 mg/mL in 0.2 M Trizma buffer at pH 7.6. Fluorescein isothiocyanate bound to dextran (FITC, Sigma, MW 2 000 000) was used at a concentration of 1.2 mg/mL in 0.2 M Trisma buffer at pH 7.6.
Results and Discussion Assembly of Polyion/Nanoparticle/Enzyme Multilayers on Quartz Crystal Microbalance (QCM) Resonators. To elaborate, a nanoparticle/GOx multilayer assembly on a flat support was monitored by the QCM (USI-System, Japan).16,17 In the measurements, gold electrode resonators were immersed in a polyion solution for a given period of time (15 min), removed, and dried. Then, the frequency change (which is proportional to the adsorbed mass) was measured. The resonators used were coated with evaporated gold electrodes (0.16 cm2) on both sides, and their resonance frequency was 9 MHz.17 The weakly positively charged Ru-bpy was premixed with PSS, 0.57 and 2 mg/mL, and alternated in the assembly with 2 mg/mL PDDA. FITC-dextran was premixed with PDDA, 1.2 and 2 mg/mL, and alternated with PSS. The premixing of the dyes was necessary for multilayer formation due to poor efficiency in direct assembly. The assembly regimes were elaborated with QCM monitoring and then used for the assembly on latex cores. (15) Buske, N. Prog. Colloid Polym. Sci. 1994, 95, 175. (16) Lvov, Y. In Protein Architecture: Interfacial Molecular Assembly and Immobilization Biotechnology; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker: New York, 2000; pp 125-168. (17) Ariga, K.; Onda, M.; Lvov, Y.; Kunitake, T. Chem. Lett. 1997, 25.
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Nanoparticle/Enzyme Multilayer Formation on Latex Spheres. For the multilayer shell formation, ∼1011 latex particles in 200 µL of solution were added to a 2-mL centrifuge tube followed by the addition of polyions, nanoparticles, and GOx to give the following shell architectures: {PEI/PSS}2 + {PEI/nanoparticles}0-4 + {PEI/GOx}1-2, where the number of nanoparticle layers (silica of different diameters or magnetite) varied from zero to four. After addition of each species, 20 min was allowed to elapse so that saturation adsorption of the components on the colloid particles was reached. The coated latex spheres were then centrifuged at 12 000g at 12 °C (an Eppendorf 5804R refrigerated centrifuge was used), and the supernatant containing the unabsorbed species was removed. This procedure was repeated four times after every adsorption step. Time of the component adsorption was selected based on experience and observations collected during QCM experiments. Microelectrophoresis. Enzyme/nanoparticle multilayer assembly was followed qualitatively by measuring the surface potential of coated particles using a ZetaPlus photon correlation spectroscopy and microelectrophoresis instrument (Brookhaven Instruments) by taking the average of 10 measurements after every deposition step. All measurements were performed in air-equilibrated 1 mM KCl solution. Transmission Electron Microscopy (TEM). TEM measurements were performed with a Philips CM12 electron microscope operated at 120 kV. TEM samples were prepared by depositing a diluted suspension onto a carbon-coated copper grid. The extra solution was blotted off, and the grid was allowed to air-dry for 1-2 min. Glucose Oxidase Activity Assay. To measure the activity of the GOx multilayers, 2.4 mL of 0.21 mM o-dianisidine solution in 50 mM sodium acetate buffer (pH 5.1), 0.5 mL of 10% (w/v) β-D-glucose solution, and 0.1 mL of a peroxidase solution (containing 60 units mL-1) were mixed in a cuvette and air-equilibrated until the absorbance at 500 nm was constant. UV absorbance was measured with a HP Agilent-8453 spectrophotometer. GOx-coated latex was added, and absorbance data were recorded continuously for 4 min, starting immediately after mixing. For each experiment, the same number of enzyme-coated particles was added to this test solution. The assay is based on production of H2O2 in the enzymesubstrate reaction; the peroxide is in turn consumed by peroxidase to result in catalytic oxidation of dianisidine, resulting in dark red coloration (maximum absorbance at 500 nm). Assembly of Polyion/Nanoparticle/Enzyme Multilayers on QCM Resonators. To elaborate self-assembly conditions for all components, we optimized deposition of glucose oxidase, PEI, silica, and magnetite on QCM electrodes. We produced the same component architecture which is planned for bio/nanoreactors on the electrodes. The QCM frequency shift (decrease of the frequency), caused by the deposition of material on the resonator, is related to the adsorbed mass and layer thickness of the material by the Sauerbrey relation.2,18 For the 9-MHz resonators used in this work, the relationship between QCM frequency shift (∆F, Hz) and adsorption mass (M, g) is ∆M ) -0.87 × 10-9 ∆F. Due to the densities of silica (2.2 g/cm3), magnetite nanoparticles19 (5.17 g/cm3), and GOx (1.3 g/cm3), the relationship coefficients of (18) Sauerbrey, G. Z. Phys. 1959, 155, 206. (19) Handbook of Chemistry and Physics; Weast, R., Ed.; CRC Press: Cleveland, 1974; p B-99.
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Figure 2. Zeta potential of the coated latex particles vs the number of adsorption steps for shell compositions of {PEI/PSS + (PEI/20-nm silica)2 + (PEI/GOx)3} (squares) and {PEI/PSS + (PEI/12-nm magnetite)2 + (PEI/GOx)3} (circles).
Figure 1. QCM monitoring (frequency change vs adsorption steps) of polyion/enzyme assembly: {PEI/PSS + (PEI/9-nm silica)3 + (PEI/GOx)2} (a) and {PEI/PSS + (PEI/12-nm magnetite)4 + (PEI/GOx)2} (b).
thickness (L, nm) of their layers and QCM frequency shift differed as follows: ∆L ) -0.012 ∆F (for SiO2),20 ∆L ) -0.005 ∆F (for magnetite), and ∆L ) -0.017 ∆F (experimental scaling for glucose oxidase).2 Figure 1 shows the QCM monitoring for the multilayer constructions. The first three polyion layers were as a precursor film to provide a uniform charge and a smooth surface for subsequent depositions. The average ∆F of PEI/9-nm silica bilayers was 702 ( 31 Hz, which corresponds to a bilayer thickness of 8.4 ( 0.4 nm. This result is in close agreement with the average silica diameter of 9 nm. The average ∆F of PEI/magnetite bilayers was 2561 ( 63 Hz, as Figure 1b showed, which corresponds to a thickness of 13.1 ( 1.0 nm, which agrees with expectations for a PEI/14-nm magnetite bilayer. The average ∆F of PEI/GOx bilayers was 258 ( 21 Hz, and the calculated GOx coverage was 6.0 mg/m2 with a bilayer thickness of 4.4 ( 0.4 nm. This thickness corresponds to a monolayer of GOx, which has the following molecular dimensions: 6.0 × 5.2 × 7.7 nm3.6 Formation of Nanoparticle/Glucose Oxidase Multilayers on Latex Spheres. The assembly of nanoparticle and GOx/PEI multilayers on the latex particles was first followed by microelectrophoresis, which gave us the surface potential of the shelled structures. Figure 2 gives the zeta (ζ) potential of the latex cores coated with polycation (PEI), polyanion (PSS), nanoparticles, and GOx. Initially, the carboxylated latex had a surface potential of -70 mV; PEI adsorption converted it to +40 mV, PSS adsorption made it negative at -50 mV, and so on. Nanoparticles and glucose oxidase (steps 4-12) also converted the surface potential to the negative. Therefore, the zeta potential alternates between negative and positive values, corresponding to the alternate adsorption of cationic and anionic species, respectively. This, together (20) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Langmuir 1997, 13, 6195.
with a regular increase of simultaneously measured core diameters, proves that we succeeded in formation of the shell with a predetermined architecture where nanoparticle layers preceded the enzyme multilayer (Figure 2). The positive charge restoration after PEI adsorption is very stable, while the negative potential provided by the enzyme adsorption slowly declines (steps 10 and 12), probably indicating less perfect GOx coverage at further cycles. This result suggests a potential limitation of the technology for a large (more than five) number of enzyme layers. To increase the surface area of the biocolloids and thus to increase the amount of immobilized GOx, nanoparticles were predeposited on the surface of latex particles. One to four layers of nanoparticles with diameters of 9-45 nm were used as precursors for enzyme deposition. Formation of the nanoparticle shells was visualized by TEM. Figure 3 shows TEM images of the uncoated (initial) 420-nm latex (a) and the same latex sphere coated with {PEI/PSS + (PEI/silica)2 + (PEI/GOx)2} multilayers with different silica diameters (b-d). The uncoated particles exhibited a smooth surface; the presence of PEI/silica shells resulted in increased roughness and surface area, which was varied with particle diameters. The silica nanoparticle distribution on the surface of the latex spheres was observed to be close-packed. The presence of nanoparticles on the surface of the latex spheres makes it difficult to discern the adsorbed enzymes, because they are much smaller and less dense. A similar coating was obtained for 12-nm magnetite layers. In previous work, the catalytic activity of GOx immobilized on latex was studied in dependence on the number of layers of the enzyme on the latex core.11,12About 8% of the particles are lost after depositing every other layer of enzyme on latex cores. Therefore, preparing five GOx layer biocolloids, one will lose 35% of the sample.13 In efforts to improve the biocatalytic effect of such nanoreactors, we modified the latex surface with nanoparticles. Additionally, using magnetite nanoparticles enabled the nanoreactor to be controlled by a relatively weak magnetic field, allowing noninvasive stirring to increase transport rates. To prove that the nanoparticle layer increased the enzymatic activity, control experiments were performed; that is, enzyme was coated on PS particles without the silica nanoparticles’ layers, and the same activity tests were done. Figure 4 shows that nanoparticle underlayers increase bioactivity of the nanoreactors. Bioactivity was determined following the standard Sigma-kit as described in the Experimental Section. Let us first consider the
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Figure 3. TEM images of uncoated latex (a) and latex coated with {PEI/PSS + (PEI/silica)2 + (PEI/GOx)2; the diameters of silica nanoparticles used were 9 nm (b), 20 nm (c), and 45 nm (d).
efficiency of the nanoreactors depending on the number of nanoparticle layers deposited before glucose oxidase adsorption. Assuming close-packed silica particle layers, the increment of the surface area of the latex sphere in dependence on the number of layers (n) will be
4π(Rlatex + nRsilica)2 π(nRsilica)2
2π(nRsilica)2 ) 8π(Rlatex + nRsilica)2 (1)
where Rlatex is the radius of the latex particles and Rsilica is the radius of the silica particle. In this work, for Rlatex ) 210 nm (diameter is 420 nm), Rsilica ) 4.5 nm (the diameter of the silica particle used is 9 nm), and n ) 0,
Figure 4. Activity (A500/min) of enzyme multilayers prepared above different silica nanoparticle coated latexes: (a) templates were coated with different numbers of silica layers {PEI/PSS + (PEI/9-nm silica)0-4 + (PEI/GOx)1}; (b) silica nanoparticles of varied diameter were precoated on latex (circle, latex without silica; triangle, 9 nm; square, 20 nm; diamond, 45 nm). The shell layer sequence was {PEI/PSS) + (PEI/silica)2 + (PEI/ GOx)2; (c) activity (A500/min) of enzyme for latex coated with one to three PEI/GOx layers. The concentrations of GOx and latex in (c) were about 15-20% of those in (a). The concentrations of latex in different preparation batches was roughly estimated by weight (drying 0.03 mL of solution on a QCM electrode).
1, 2, 3, and 4, the increments of surface area of each silica layer were estimated. The increment 1 corresponds to “bare” latex, 2 to one layer, 2.15 to two layers, 2.31 to three layers, and 2.46 to four layers of silica. Activity of these reactors as a function of the relative increase in surface area (calculated using eq 1) is shown in Figure 4a. It is clear that with the increase in surface area, the enzymatic activity was also increased. The last point deviated from linear activity increase; this result may potentially be explained by incomplete washing out of nonadsorbed GOx.
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Figure 5. SEM image of 420-nm latex covered with two monolayers of magnetite particles; less dense GOx layers are not visible.
Next, we studied the influence of the radius of underlayer nanoparticles on bioactivity. While the silica nanoparticle diameters were varied from 9 to 45 nm, the activity of GOx multilayers was increased 3-5 times as is evident from Figure 4b (in comparison with GOx multilayers directly immobilized on latex spheres) (Figure 4b). Calculated from formula 1, the increments of surface area of three different radiuses of underlayer nanoparticles were 2.15, 2.19, and 2.45. Thus the influence of one layer of the 9-nm and 20-nm silica nanoparticles was almost the same. Due to experimental error, Figure 4b showed that the bioactivity of GOx on the 20-nm silica underlayer was less than the one on the 9-nm silica underlayer, but their absorbance increments characterizing enzymatic activity were close, which agreed with the estimation. Another possibility to increase the enzymatic activity is increasing the enzyme layer numbers on latex. Therefore, additional experiments were performed to check the activity of the latex with different numbers (one to three) of GOx layers. Figure 4c shows that the activity was also increased using this approach, and this agrees with previous work.11 However, the key result of these experiments is this: three layers of GOx are less effective than an assembly of three underlying nanoparticle layers with a single GOx layer, as is evident from a comparison of increments of the dependences in parts a and c of Figure 4. Magnetic Functions of Nanoreactors. One 14-nm magnetite nanoparticle bilayer was deposited in alternation with PEI in the shell to provide the nanoreactors with both increased bioactivity (via geometric properties, as noted above for silica) and magnetic momentum (via material properties) to allow their manipulation with an external magnetic field. The shell sequence employed was {(PEI/PSS) + (PEI/12-nm magnetite)2 + (PEI/GOx)2}, as was confirmed by surface potential measurements (Figure 2). Figure 5 demonstrates such a coverage. One can see that magnetite particles cover the latex surface, and their diameter is about 14 ( 1 nm; GOx cannot be resolved because of low protein density. In a standard measurement, reaction solutions are mechanically stirred when enzymatic activity is tested. In our experiment, the enzymatic activity of magnetic biocolloids was analyzed using self-stirring magnetic nanoparticles (no stirring bar was used in the cuvette). Therefore, we studied activity to compare two different situations: application of a rotating magnetic field directly to magnetic nanoparticles (no stir bar) and without magnetic field. The results of these tests are shown in Figure 6, and these indicate that the catalytic activity was 2 times higher when a rotating external magnetic field was applied to the solution as compared to the one without any stirring. In a separate experiment, we did
Figure 6. Time trace of absorbance at 500 nm for the enzymatic activity assay of {(PEI/PSS) + (PEI/12-nm magnetite)2 + (PEI/ GOx)2} biocolloids in two different conditions: without magnetic field (open circles) and within magnetic field (filled circles).
mechanical stirring and the activity curve coincided with the self-stirring activity curve. By this, we prove the ability of self-stirring and believe that it is reached by rotation of the nanoreactors with the same angular speed as the applied magnetic field (2-10 Hz and ca. 200 Oe were used). This is a promising approach demonstrating the ability to manipulate with magnetic nanoreactors. In the future, we plan to apply this for focusing and targeting of drugs encapsulated in magnetic shells. Let us consider the motion of a nanoreactor in aqueous solution under the action of a magnetic field. The energy equation in this case can be written as
Em ) Ek + Ev
(2)
Here Ev ) AηSv is the energy of viscous interaction of the nanoreactor with the environment, where η is the viscosity of the environment, S ) πD2 is the cross-sectional area of the nanoreactor (D is the diameter), v is the velocity of the motion, and A ∼ 1 is a coefficient that characterizes shape and surface of the reactor. Ek ) mv2/2 is a kinetic energy of the nanoreactor, where m is the mass. In our case, the kinetic energy is at least an order of magnitude smaller than Ev and can be neglected. Em is the energy of magnetic interaction of magnetic moment M of the nanoreactor with magnetic field H. This term should include the energies of magnetic anisotropy and dipole-dipole interactions, but for the sake of simplicity these contributions are not considered here.21 Omission of these terms does not lead to considerable errors in the evaluations. Thus, Em ) MH and M ) nIpVp, where n is the number of ferromagnetic particles in the nanoreactor, Ip is their magnetization, and volume Vp is equal to πd3/6 (d is the diameter of the magnetite nanoparticle). Small magnetic particles become superparamagnetic at room temperature; therefore, Ip can be evaluated from the equation
( )
Ip ) IsL
IsHVp kBT
(3)
where Is is the saturation magnetization of the bulk magnetic material, T is the temperature, kB is the Boltzmann constant, and L(x) ) coth(x) - 1/x is the Langevin function.22 (21) Golub, V.; Kakazei, N.; Kravets, A.; Lesnik, N.; Pogorelov, Y.; Sousa, J.; Vovk, A. Mater. Sci. Forum 2001, 373-376, 197. (22) Chazelle, B.; Lvov, A. Discr. Comput. Geometry 2001, 25, 519524.
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Figure 7. Squares: Fluorescence peak intensity ratio response with 5 mM glucose additions added every minute. Triangles: Peak intensity without glucose additions. Scheme 2. Illustration of the Layer-by-Layer Assembly of Polyelectrolyte, Dye Complex, and Enzyme (Glucose Oxidase) on Latex Particles
Thus we can calculate the velocity of the reactor under the action of applied magnetic field:
v)
nd3IsH 6AD2η
( )
L
πd3IsH 6kBT
(4)
intensity increases as local oxygen levels decrease. For the sensors, oxygen is consumed locally by the reaction between GOx molecules and glucose (Scheme 2). Additional fluorescence layers are employed as an internal intensity reference, allowing ratiometric monitoring. The glucose sensing assay was assembled on latex particles with the following organization: (PDDA/FITCdextran complex/PSS)2 + (Ru-bpy/PSS complex/PDDA)4 + (PDDA/GOx)5. Monitoring the ratio of the Ru-bpy peak intensity to the FITC peak intensity tested the sensitivity of nanoparticle suspensions to additions of glucose, which occurred at 1-min intervals. The preliminary data presented in (Figure 7) compare the baseline peak intensity ratio of the particles with ratios for increasing glucose concentration. The two sets of data exhibit different apparent slopes, suggesting two separate sources of change. The increasing slope when no glucose is added is believed to be caused by photobleaching of FITC at a slightly faster rate than Ru-bpy. Glucose additions cause an increase in Ru-bpy fluorescence, while the FITC peak decreases slightly due to photobleaching, thus yielding an increasing peak ratio with a greater rate of change than that explained only by photodestruction. The variations in peak ratio intensity during each minute interval are similar for both experiments, representing noise from the experimental setup, and can be decreased with a new protocol. The results demonstrate the feasibility of combining the GOx nanoreactors with sensing chemistry to monitor reactions in progress, as well as build devices with other functions such as sensors.
Using parameters of our nanoreactors (the number of magnetite particles with d ) 12 nm assembled on a latex core is n ) 1000; for magnetite Is ) 600 emu/cm3; the used magnetic field is H ) 200 Oe; nanoreactor diameter D ) 420 nm; for dilute aqueous solution η ) 0.01 mPa s, and room temperature T ) 293 K), one can obtain velocity of v ≈ 1 cm/s. If the magnetic field is rotated with the frequency f ) 25 Hz (an optimal steering speed for our experimental conditions), the nanoreactors will move at a circular trajectory with largest radius R ) v/(2πf) ≈ 0.1 mm. A smaller frequency could be effective as well because it provides a larger trajectory radius. Such a rotation provided sufficient mixing of products, and the reaction kinetics appeared to be similar to the case of mechanical bar stirring with the same rate and field. Additionally, when a permanent magnet (∼200 Oe) was placed against the tube containing nanoreactors, all nanoreactors were collected at the wall in about 1 s. This again confirms our estimations of the typical velocity. Function of Nanoreactors in a Sensor Device. To study another function of the bio/nanoreactor, we have designed a so-called “nanostructured fluorescent sensor”23,24 based on a previously studied assay for sensing glucose and oxygen.25,26 The sensors are fabricated using LbL adsorption to create oxygen-quenched fluorescent (ruthenium-based) layers, such that the fluorescent
The preparation, characterization, and multiple applications of nanoparticle/glucose oxidase multilayercoated latex particles with defined shell architectures have been demonstrated. The inclusion of silica layers on latex yields a higher surface area and roughness, adsorbing more GOx and thereby increasing the catalytic activity of the nanoreactors. The introduction of magnetic nanoparticles in these nanobioreactors gives the possibility for self-stirring, which also enhances the effectiveness of the nanoreactor. Therefore, the stable, multifunctional bio-
(23) Grant, P.; Fang, M.; Lvov, Y.; McShane, M. Proc. SPIE-Int. Soc. Opt. Eng. 2002, 4624, 174. (24) Schu¨tz, P.; Caruso, F. Langmuir 2001, 17, 7670.
(25) Moreno-Bondi, M.; Wolfbeis, O.; Leiner, M.; Schaffar, B. Anal. Chem. 1990, 62, 2377. (26) Rosenzweig, Z.; Kopelman, R. Anal. Chem. 1996, 68, 1408.
Conclusion
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colloid nanoreactors prepared combine the benefits of high surface area with magnetic function, making them attractive for use in various applications in the future. The combination of the nanoreactor architecture and fluorescent dyes for use in a glucose microsensor has been demonstrated. Sensor function based on the correlation of increase in Ru-bpy fluorescence with glucose changes was observed in preliminary experiments with GOx layers. The ability to make sensors based on oxygen sensitivity is not limited to glucose oxidase, as any enzyme that consumes oxygen in the reaction is available. Nanoparticle fluorescent biosensors could be used to study basic physiological processes inside cells and used clinically to monitor disease, aiding in diagnosis and treatment.
and Dr. N. Buske (Mediport Kardiotechnik GmbH) for providing magnetic nanoparticles. Dr. I. Ichinose (RIKEN, Japan) is acknowledged for scanning electron microscopy. Acknowledgment is made to the donors of the Petroleum Research Fund (Grant No. 36066), administered by the American Chemical Society, and The Whitaker Foundation for partial support of this work. This material is based upon work supported in part by National Science Foundation Grant No. 0092001, “Micro/Nanodevices and Systems”. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the view of the National Science Foundation.
Acknowledgment. We thank Drs. C. O’Connor, X. Qiao, and Z. Zhong for assistance and useful discussions
LA025731M
