Coupled Enzyme Reactions in Multicompartment Microparticles

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Biomacromolecules 2010, 11, 1480–1487

Coupled Enzyme Reactions in Multicompartment Microparticles Hans Baümler* and Radostina Georgieva Berlin-Brandenburg Center of Regenerative Therapies, Charite´-Universita¨tsmedizin Berlin, Charite´platz 1, 10117 Berlin, Germany Received January 29, 2010; Revised Manuscript Received May 7, 2010

Spherical biopolymer particles have been fabricated, applying coprecipitation with calcium carbonate, followed by cross-linking of the macromolecules and dissolution of the inorganic support. Particles made of roughly 80% horseradish peroxidase (HRP) as well as glucose oxidase (GOX) were prepared and enzyme activities were confirmed, applying the Amplex Red assay. The enzyme particles were reusable for at least six times, with a remaining activity of 30-50% from the initial one. When multiple coprecipitation steps and one or several crosslinking procedures were applied, multicompartment particles were obtained. Each of the resulting concentric compartments could be independently loaded with biomolecules. Three coupled enzymes, β-glucosidase (β-Glu), GOX, and HRP have been incorporated stepwise in such particles. Each of these enzymes was located in a separate compartment, in a desired sequence, and at a defined position. The distance between the enzyme containing compartments was also varied, including spacing compartments consisting of bovine serum albumin (BSA). When fluorogenic substrates for β-Glu and HPR were used, the start and the end of the coupled enzyme reaction were visualized and recorded inside of individual particles, applying confocal laser scanning microscopy. A strong influence of the spacing on the reaction kinetics of the last enzyme was observed, suggesting an impaired diffusion of the intermediate products of the chain reaction through the spacing compartments made of BSA. The influence of the spacing between compartments containing different enzymes on the reaction kinetics was demonstrated on the microscopic scale within one microparticle, which to the best of our knowledge was not achieved until now.

1. Introduction The fabrication of biocompatible micro- and nanoparticles has attracted widespread interest due to their potential application in biotechnology as tools for catalysis, sensing, and separation and in medicine as systems for drug delivery,1 diagnostics, and in vivo imaging.2,3 In particular, protein particles and especially enzyme containing particles are gaining more and more attention due to their unique properties and bioactivity. Research on enzyme immobilization and encapsulation has largely been driven by the benefits of achieving higher pH and temperature stability, facile separation from reaction mixtures, and reusability.4-6 A number of methods based on spray drying,7 liposome encapsulation,8,9 immobilization in porous particles,10,11 microemulsions,12,13 polyelectrolyte microcapsules,14-18 and so on have been developed and are currently applied for entrapment and encapsulation of proteins (e.g., enzymes). An increasing number of studies deal with multicompartment systems motivated by the need of multifunctional, controllable, and triggered carriers that are desired, for example, in drug delivery or as bioreactors and biosensors. Multicompartmentalization is envisioned to be the next step of development19 in the area of drug carriers due to possibilities of simultaneous delivery of various substances in one vehicle but separated spatially. In some biomedical applications multicompartmentalization can be indispensable. This is the case, for example, if a triggered control of reaction is desired, modifying a prodrug into an active drug. Examples of multicompartment systems include hybrid polymer microspheres,20 shell-in shell polyelectrolyte capsules hierarchically templated on melamine formaldehyde, silica21 or * To whom correspondence should be addressed. E-mail: hans. [email protected].

CaCO3,22 polymeric micelles,23,24 and two-compartment vesicles.25,26 However, most of these structures have different limitations, such as nonbiofriendly processing techniques, low loading capacity, low stability and leakage of macromolecules, lack of permeability toward small compounds, or complicated and expensive synthesis. Recently, two-enzyme-coupled reactions have been introduced with respect to compartmentalization by Kreft et al.22 in polyelectrolyte capsules and by Kuiper et al.24 in polymersomes. Van Doven et al.27 presented polymersomes as three-compartment systems, placing three coupled enzymes in three separated compartments: on the outer interface, in the polymer shell, and in the inner volume. However, the influence of the spatial separation on the coupled enzymes on the reaction kinetics was not investigated in these studies. Compartmentalization plays an indispensible role in the biological cell where an enormous number of reactions and transport processes are executed in parallel. Besides this, the processes are directed and coordinated, allowing storage of material and free energy in gradients of material and potential. The present study was motivated by the need of a deeper understanding of the influence of compartmentalization on the multiple functionalities and coupled reactions inside of one particle. We present here a simple and inexpensive concept for the fabrication of single and multicompartment particles fully made of biomacromolecules. The technique is based on the process of coprecipitation of biopolymers with an inorganic salt such as calcium carbonate28-31 followed by one or several crosslinking procedures and dissolution of the inorganic support. We prepared particles with several concentrically arranged compartments, placing up to three coupled enzymes in their own compartments with and without spacing compartments between them. Visualizing the first and the last enzyme in a three-enzyme

10.1021/bm1001125  2010 American Chemical Society Published on Web 05/20/2010

Enzyme Reactions in Multicompartment Microparticles

reaction by fluorogenic substrates and confocal laser scanning microscopy, we studied the time shift between start and end of the chain. In such a way, the influence of the spacing between compartments containing different enzymes on the reaction kinetics was demonstrated on the microscopic scale within one microparticle, which to the best of our knowledge was not achieved until now.

2. Materials and Methods 2.1. Materials. The materials were purchased as follows: glucose oxidase (GOX), 208 U/mg protein, horseradish peroxidase (HRP), 970 U/mg protein, β-glucosidase (β-GLU), 24.8 U/mg protein, and fluorescein-diglucopyranoside (FDGlu) from Fluka; bovine serum albumine (BSA), dextran (Dx), MW 64-76 kD, fluorescein isothiocyanate-BSA (FITC-BSA), divinyl sulfon (DVS), and 25% glutaraldehyde (GA) from Sigma-Aldrich; BSA-Alexa Fluor 680 (BSA-AF680) and Amplex Red Assay from Molecular Probes, Invitrogen; diethylaminoethyl-dextran (DEAE-Dx), MW 500 kD, from pK Chemicals (Copenhagen, Denmark); and citrate-coated magnetite particles (diameter )10 nm), 1% (w/w) suspension in water, was provided by Magnetic Fluids (Berlin, Germany). All chemicals were used without further purification. Stock solutions with a concentration of 20 mg/mL were prepared from each biosubstance with sterile distilled water (Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany) and kept at 4 °C for a maximum of 2 weeks. 2.2. Enzyme Chain Reaction. The enzymes β-GLU, GOX, and HRP were chosen for the preparation of particles. These three enzymes can be coupled in a chain reaction. Additionally, the starting enzyme in the chain, β-GLU, and the final one, HRP, can be detected and visualized by the commercially available fluorogenic substrates, FDGlu and Amplex Red, respectively (Figure 1a). 2.3. Fabrication of Biopolymer/Enzyme Particles. For the preparation of the biopolymer/enzyme particles, we applied the coprecipitation method.30,31 Briefly, 0.5 mL of 0.3 M CaCl2 solution was mixed with 0.5 mL of a biopolymer/enzyme stock solution and incubated under gentle stirring at room temperature for 15 min. The biopolymer/enzyme stock solutions used were BSA/HRP, BSA/GOX, and Dx/DEAE-Dx/ β-GLU. The final concentration of all enzymes during the fabrication of the particles was 4 mg/mL. The supporting biopolymers BSA, Dx, and DEAE-Dx were applied with final concentrations of 1, 1, and 5 mg/mL, respectively. Then, 1 mL of 0.15 M Na2CO3 was added and rapidly agitated on a magnetic stirrer for 20 s at room temperature. After the precipitation was completed, the obtained particles were separated by centrifugation (300 × g, 1 min) and washed three times with water. The particles were then subjected to a cross-linking agent, depending on the biomaterial coprecipitated. In the case of BSA, HRP, and GOX, GA at a final concentration of 2% (w/v) was used. For Dx and DEAE-Dx, the cross-linking was performed with 20 µL of DVS in 0.1 M NaOH. In both cross-linking procedures, the samples were incubated at room temperature for 1 h, followed by five washing steps with water. The pellet was then resuspended in water to a total volume of 1 mL and the CaCO3 support was dissolved by adding 4 mL of 0.2 M EDTA solution (pH 7.4). Finally, the obtained biopolymer particles were centrifuged at 1000 × g for 5 min, washed 4 times with water, and resuspended for further use in sterile water to a final particle concentration of 5% (v/v). To obtain multicompartment biopolymer particles, 10 µL of a magnetite nanosuspension were added to the Na2CO3 solution during the first precipitation step.22 In such a way we obtained magnetic biopolymer/CaCO3 cores that were subjected to multiple further coprecipitation steps (Figure 1b), including different compounds and the enzymes of the cascade. The initial particles with the growing multicompartments could be collected using a strong permanent magnet separating them from the new simple ones after every new precipitation step. This procedure was repeated up to five times to obtain microparticles with five separated microcompartments. The cross-linking steps and the decomposition of the CaCO3 cores were performed as described

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above. After four or five washing steps, the resulting biopolymer particles were resuspended in sterile water at a volume concentration of 10% (v/v) and stored at 4 °C. 2.4. Determination of Enzyme Activity. Enzymatic activities of GOX and HRP particles were monitored applying a commercial standardized Amplex Red glucose/glucose oxidase assay from Molecular Probes, Invitrogen. The measurements were performed in 96-well microplates. The total sample volume was 100 µL per well. Each sample contained 50 µL of glucose solution, with concentrations from 0 to 50 µM and 50 µL working solution containing the enzymes and the enzyme particles, as well as 10 µM Amplex Red reagent. For quantitative analysis, absorbance measurements at 560 nm were performed using a plate reader Bio-Tek Power Wave 340 (BioTek Instruments Inc., Winooski, Vermont). The enzyme activity was monitored depending on the substrate concentration, and the reaction rate was followed for up to 3 h. The enzyme activity of the particle suspensions was calculated in proportion to the activity of the control using the corresponding absorbance values. The control contained 0.1 U GOX and 0.01 U HRP. The number of particles per well was in the order of 7-8 × 106. 2.5. Confocal Laser Scanning Microscopy (CLSM). The enzyme cascade was followed in situ using a confocal microscope LSM 510 Meta equipped with a 100× oil-immersion objective (Carl Zeiss MicroImaging GMBH, Jena Germany). Time series were recorded and analyzed applying the LSM software. The reactions were performed in phosphate buffer (0.01 M sodium phosphate, 0.1 M NaCl) in 18well microslides (ibiTreat µ-Slides, Ibidi GmbH, Martinsried, Germany). The total volume per well was 20 µL. Single enzyme particles HRP and GOX particles (approximately 1.5 × 106 per well) were measured with the corresponding enzyme in the buffer (0.02 U GOX and 0.002 U HRP, respectively), 50 µM D-glucose, and 10 µM Amplex Red reagent. The substrates were 10 µM Amplex Red reagent and 50 µM D-glucose for the two-enzyme particles and 20 µM FDGlu and 10 µM Amplex Red for the three-enzyme particles.

3. Results 3.1. Single Enzyme Particles. Single-enzyme particles were fabricated using HRP or GOX in combination with 20% (w/w) BSA. The size of the resulting particles reproduced the size of the CaCO3 cores after precipitation and ranged from 2 to 5 µm. It was slightly dependent on the duration and intensity of agitation during the precipitation, shifting the mean particle diameter from 2.5 µm for high velocity of the stirrer to 4 µm for low velocity. The protein content of the particles was calculated from the difference between the protein concentration in the supernatant after coprecipitation and the initial concentration in the stock solutions taking into account the dilution during the fabrication process. After determination of the number of particles in the samples, a protein content of roughly 1 pg per particle (related to particles with a mean diameter of 3 µm) was estimated. Finally, subtracting 20% BSA content, the enzyme content of a 3 µm particle is assumed as 0.8 pg. An example of such microparticles made of a cross-linked single enzyme and BSA with incorporated magnetite nanoparticles is shown in Figure 2a. The particles are spherical with a mean diameter of 3 µm. The magnetite nanoparticles are visible inside the microparticles as dark spots. The substrate sensitivity of each type of enzyme particles was investigated separately with the corresponding second enzyme and the substrates glucose and Amplex Red in the suspending medium. In Figure 2b the absorption of the fluorescent reaction product resorufin is plotted in dependence of the glucose concentration. For the recommended incubation time of 30 min the absorbance measured with the particles was roughly 30% of that for the enzyme solutions. Thus, the corresponding total enzyme activities of the particle samples

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Figure 1. (a) Chain reaction of the three coupled enzymes with fluorogenic substrates for the first and third enzyme. (b) Fabrication scheme of multicompartment particles and coupled enzyme reaction.

can be estimated as 0.03 U for GOX and 0.003 U for HRP. Taking a mean number of 7.5 × 106 particles in the test samples one can estimate an activity of 4 × 10-9 U per GOX/BSAparticle and 4 × 10-10 per HRP/BSA-particle. The time dependency of the reactions was then followed at a glucose concentration of 50 µM and 10 µM Amplex Red reagent. Figure 2c shows the reaction rate of a mixture of equal quantities of HRP/BSA and GOX/BSA particles (7.5 × 106 each) for 150 min in comparison to that of enzymes in solution monitored by measuring the absorbance of resorufin. It can be

seen that the substrate is almost completely consumed by the dissolved enzymes within 30 min. In the samples where the enzymes are immobilized as microparticles the reaction rate is lower but the resorufin absorbance steadily increases during the whole period of time reaching almost 80% of the absorbance measured in the control. Thus, the sensitivity of the particle suspension for glucose can be improved with increasing incubation time. The enzyme reaction was also studied on the microscopic scale inside individual HRP/BSA particles using confocal



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Figure 2. Single enzyme particles: (a) Transmission microscopy image of HRP/BSA particles with incorporated magnetite nanoparticles (dark spots); (b) Glucose sensitivity of HRP/BSA-particles with 0.1 U GOX (squares), GOX/BSA-particles with 0.01 U HRP (diamonds), compared with a control of HRP (0.01 U)/GOX (0.1 U)-solution (circles), as measured by a commercial AmplexRed Assay (Molecular Probes, Invitrogen). The absorbance of the coproduct resorufin at 560 nm was a measure for the enzyme activity. The microplates were incubated for 30 min at dark and room temperature. (c) Reaction rates of a mixture of HRP/BSA- and GOX/BSA-particles (filled squares) compared with a control of HRP (0.01 U)/GOX (0.1 U)-solution (open squares) with a glucose concentration of 50 µM and 10 µM AmplexRed reagent; (d) CLSM image of a mixture of HRP/BSA particles with fluorescence intensity profiles of resorufin in five Regions of Interest (ROI) of the image with time: inside of individual HRP/BSA particles (ROI 1, 2, 3, and 4) and in the background (ROI 5); (e) Reproducibility of glucose sensing by a mixture of HRP/ BSA and GOX/BSA particles. Equal volumes of the particles (50 µL each) were mixed and incubated with 50 µM glucose and 10 µM AmplexRed at room temperature for 30 min. After centrifugation, 100 µL of the supernatant were placed in a microplate and the absorbance at 560 nm was immediately measured. The pellet was washed to white color with water, resuspended in a reaction buffer and subjected to a new reaction start with glucose and AmplexRed. After five reaction runs, the particles were washed, stored for 48 h at 4 °C in water, and then subjected to an additional reaction run.

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Figure 3. CLSM images of enzyme particles with four compartments. (a) Multifunctional particle consisting of four concentric compartments: HRP/BSA compartment with magnetite (the dark spots in the transmission image mode) in the center; two fluorescent compartments of FITCBSA with a GOX/BSA compartment between them; (b) Particle (HRP/FITC-BSA/GOX/FITC-BSA) reacting with glucose; glucose as well as AmplexRed were added to the suspension, the strong red color appeared in the central compartment, which contains HRP; (c) Particle (GOX/ FITC-BSA/HRP/FITC-BSA) reacting with glucose in the presence of Amplex Red: the strong red color appeared in the peripheral compartment between the green fluorescing FITC-BSA compartments, which contains HRP in this sample; glucose as well as Amplex Red were added to the bulk solution.

microscopy to visualize the fluorescence of resorufin (Figure 2d). It can be seen that the fluorescence increases much earlier and faster inside the particles (ROI 1-4) as in the background (ROI 5). This indicates a higher real activity of the enzyme in the particles as calculated from the macroscopic measurements in their suspensions. One advantage of the immobilization of enzymes in particles is the option of separation and a repeated use for reaction. The particles can be separated by centrifugation, filtration, or even by application of an external magnetic field in the case of simultaneous entrapment of magnetite nanoparticles.22 We tested the potential of our novel enzyme particles for reuse applying a mixture of HRP/BSA and GOX/BSA particles. The reaction was run at a glucose concentration of 0.05 mM for 30 min with equal quantities of both particle types. The supernatant was then separated by centrifugation and the absorption of resorufin was determined as a measure for the enzyme activity. The pellet was washed with distilled water to white color, resuspended in reaction buffer and subjected to a new reaction run under the same conditions. This procedure was repeated five times. The values obtained for the absorbance of the fluorescent product resorufin are displayed in Figure 2e. It can be seen that there is only a little absorbance decrease in the second run followed by a significant reduction of about 50% for the third one and a further decrease to approximately 30% of the initial value for the forth and fifth run. The same particle suspension was then washed and stored in distilled water at 4 °C for 48 h before their remaining activity has been tested again in comparison to a control of particles which have not been used for reaction and stored for the same time under the same conditions. The repeatedly used particles showed again a surprisingly high enzyme activity with even some recovery compared to the activity measured for the fifth run. 3.2. Two-Enzyme Particles. To obtain multicompartment particles we incorporated magnetite nanoparticles during the first coprecipitation and used a strong permanent magnet for particle separation after each further coprecipitation step collecting only the particles with growing compartments (Figure 1a). Applying this approach, we prepared microparticles containing HRP and GOX in separated concentric compartments. For better visualization of the compartmentalization enzyme-free areas were included consisting only of fluorescently labeled albumin (FITCBSA). A CLSM image of such a particle consisting of four

compartments is shown in Figure 3a. It is an overlay of the transmission mode and the fluorescent channel for FITC. The included magnetite nanoparticles are clearly seen as dark spots in the central compartment of the transmission mode. The micrographs presented in Figure 3b and c show particles after the completed reaction in the presence of 50 µM glucose and10 µM Amplex Red (AR) reagent. We placed the reporting enzyme, HRP, either in the central compartment (Figure 3b) or in the second peripheral one (Figure 3c). In both particle types, the enzymes were accessible for their substrates, which could diffuse through the cross-linked proteins due to their small molecular weights. The positions of the enzymes were fixed. The time series corresponding to both samples shown in Figure 3b,c are provided in the Supporting Information, Videos S1 and S2, respectively. 3.3. Three-Enzyme Particles. Finally, we increased the number of enzymes including β-glucosidase (β-GLU), which cleaves glucose monomers from polysaccharides. This enzyme was chosen in particular because it can also be detected using fluorogenic substrates, for example, FDGlu, which is cleaved to glucose and highly fluorescent fluorescein. In such a way one can detect the start of the cascade and measure the time lag between the first and the last enzyme in it. β-GLU appeared to be more sensitive to cross-linking by GA and was incorporated in a compartment supported by a mixture of dextran and DEAE-dextran on top of two enzyme particles. DEAE-dextran is a cationic biopolymer and interacts strongly with the negatively charged enzyme (isoelectric point from 4.4-7.0).32 After the cross-linking of the dextrans with DVS and dissolution of the CaCO3 template the enzyme remained inside the top compartment due to electrostatic interactions. Two types of three-enzyme particles were constructed. In type 1, the enzymes were placed in concentric compartments directly in contact with each other starting with HRP in the central compartment, GOX in the first peripheral compartment and β-GLU on the top. In type 2 particles, alternating layers of BSAAF680 separated the enzyme containing compartments were included, providing roughly micrometer thick barriers for the diffusion of the intermediate products in the reaction chain. The time-dependence of the enzyme cascade was recorded and analyzed for both particle types using CLSM (Figure 4 and Videos S3 and S4, Supporting Information). The two fluorogenic substrates were simultaneously added to the particle suspensions



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Figure 4. Coupled chain reaction of three enzymes placed in separated compartments of single microparticles. The reaction starts with the adding of FDGlu and AmplexRed simultaneously to the bulk solution. (a) CLSM time series of coupled chain reaction in three compartment particles with HRP/BSA and magnetite in the center, GOX/BSA in the first peripheral and ß-GLU/Dx/DEAE-Dx in the outer compartment. The particles do not contain any fluorescent labels. (b) CLSM time series of coupled chain reaction in five compartment particles with three enzymes incorporated in the same manner but additionally separated from each other by BSA-AF680-compartments (blue). (c) Time dependency of resorufin fluorescence in the HRP containing central compartment of a 3-compartment and a 5-compartment particle. (d) A single five-compartment particle: CLSM images before and after performing coupled reaction and time dependencies of fluorescence of fluorescein in the β-GLUcontaining an outer peripheral compartment, and of resorufin in the HRP-containing a central compartment in a 5-compartment particle.

at equal concentrations for both samples (50 µM FDGlu and 10 µM AR). It can be seen that the rise of fluorescence inside the individual compartments is time-resolved. The production of resorufin in the central compartment, mediated by HRP, appears within approximately 60 s in type 1 particles, where the enzyme compartments are in direct contact with each other (Figure 4a,c; Video S3 and Figure S1a, Supporting Information). In contrast, the central compartment of type 2 particles, separated by BSA-AF680 compartments, remained dark for 300 to more than 400 s (Figure 4b-d, Video S4 and Figure S1a,b, Supporting

Information). In both sample types, fluorescein, a coproduct of β-Glu, is detectable in the outer compartment within roughly 30 s. The role of separating compartments deterring the diffusion of substrates and products among enzymes located in different compartments appear to be significant. This is evident in one defect particle imaged in Figure 4b (bottom, right). Here the inner compartments are disrupted and the components from the outer compartments penetrate into the central one. The diffusion of substrates and products between the environment and the enzymes in the broken particle is much faster and the rise of

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fluorescence (red) in the central compartment occurs approximately with the same time constant as that of type 1 particles (Video S4 and Figure S1c, Supporting Information).

4. Discussion In summary, we presented a novel and simple approach to fabricate particles made fully of biopolymers. In principle there are no limitations to include charged or uncharged molecules, organic or inorganic nanoparticles. We demonstrated here the preparation of protein/enzyme particles with incorporated magnetite nanoparticles but also the construction of a dextranbased compartment on top of protein particles. The activity of particles containing a single enzyme, HRP or GOX, was detected and estimated applying a commercially available assay. The values obtained in this way were lower in comparison with the starting material. The cross-linking with GA is known to impair the intramolecular mobility of the enzymes and to decrease the accessibility of their active sites.33 However, the interpretation of these estimated values should be carried out with some precaution. It has to be taken into account, that enzyme activity assays are standardized for enzyme solutions. The measured absorbance or fluorescence is an average value over the total sample volume. In the case of particles, additional factors, such as scattering and inhomogeneous distribution of absorbance, can influence the measurements in a way that is difficult to predict. The rate of resorufin release from the particles into the supernatant strongly contributes to the overall absorption of the sample when measured in a batch. In fact, when followed on the microscopic level of individual particles, the reaction rate was significantly higher (Figure 2d) than the rate measured macroscopically (Figure 2c). The microscopically recorded fluorescence intensity is thus more relevant for the enzyme activity of the particles since it represents the product concentration inside the particles and is not influenced by its distribution into the surrounding solution. It can be seen that the distribution of the fluorescence into the supernatant appeared with a certain delay, most probably due to a hindered dissociation of the reaction product resorufin that is known to be less soluble in aqueous milieus takes place. This assumption is supported by the partial recovery of activity after longer storage of repeatedly used particles (Figure 2e). During the storage in water over longer time a part of the undissociated product could be released resulting in more free active sites of the enzymes. It should also be mentioned that the activity of the enzyme particles can be preserved over months in agreement with the finding of others.4-6,34 We demonstrated further the preparation of multicompartment particles with the option to place coupled enzymes in a desired sequence (Figure 3b,c) and at a different distance (Figure 4) from each other. Previously, Kreft et al.22 reported similar systems with two coprecipitation steps. Using polyelectrolyte layer-by-layer coatings only on top of “boll-in-boll” particles they demonstrated the mixing of two initially separated compartments with FITC- and TRITC-BSA during the dissolution of the CaCO3. To achieve sustained separation of two enzymes, “shell-in shell” capsules, they prepared LbL coatings after each coprecipitation step. The microscopically observed “spatially confined enzymatic reaction” of GOX and HRP also presented in22 was very fast due to the high permeability of the polyelectrolyte multilayers separating the enzyme compartments for small molecules. Thus, the substrates and products of reaction distributed and equilibrated between the two compartments within few seconds.


In contrast to this work, the separation of the proteins in our procedure was maintained by cross-linking and remained stable after the dissolution of the CaCO3 support. Additional coatings for encapsulation and spatial separation of the enzymes were not required, which simplifies the fabrication procedure considerably. Consequently, reducing the steps of adsorption and washing it was possible to produce particles with a higher number of concentric compartments, up to five, without impairment of the quality of the samples. In such a way we were able to include three coupled enzymes in separated compartments in one particle. Moreover, BSA compartments without enzyme activity were incorporated between the enzyme compartments to create additional spacing and barriers. The main benefit was achieved by including a third enzyme (β-GLU) and visualizing the first and the last element of the cascade by corresponding fluorogenic substrates facilitated the observation of the time lag between the spatially separated enzymes. We observed significant changes in the reaction kinetics inside the particles caused by appropriate spacer compartments (Figure 4). Only two separating BSA shells with a thickness of roughly 1 µm each caused the delay between the first and the last enzyme in the chain to increase from seconds to minutes. The matrix of GA-cross-linked BSA in the spacing compartments seems to be poorly permeable for the reaction products. This is probably due to the high density of amino groups in this protein (it contains 60 lysines from totally 585 amino acids per protein molecule35,36). For comparison, GOX contains only 32 lysines from 1250 amino acids in total.37 Consequently, the glucose produced by β-GLU in the outermost compartments diffuses into the surrounding solution more quickly than into the inner compartments causing the glucose concentration in the particles’ environment to increase. In the inner compartments, the glucose is readily consumed by the enzyme GOX. This leads to a concentration gradient accelerating the diffusion of glucose toward the inner compartments of the particle and provides more and more substrate for the next element of the cascade, finally supplying HRP with hydrogen peroxide to close the chain reaction. The overall reaction kinetics in coupled chain reactions in solutions is determined by the slowest element. In our case, the diffusion of the intermediate products through the spacing compartments is clearly the slowest process and controls the final output of the chain. To the best of our knowledge, besides the recent publication of van Dongen et al.,27 this is the second report on a coupled reaction of three enzymes in multicompartment particles and the first one where the influence of the spacing on the reaction kinetics was demonstrated on the microscopic scale.

5. Conclusions We have developed a simple and inexpensive technique for the fabrication of microparticles fully made of biomacromolecules based on coprecipitation with calcium carbonate followed by cross-linking of the macromolecules and dissolution of the inorganic support. Spherical microparticles made of roughly 80% enzyme, HRP or GOX, respectively, could be prepared with confirmed enzyme activity and reusability. Applying multiple coprecipitations and cross-linking procedures, multicompartment particles with concentric compartments were obtained, loaded independently with different biomolecules and magnetic nanoparticles. Two-coupled, GOX and HRP, and three-coupled enzymes, β-Glu, GOX, and HRP, have been incorporated in microreactors, where each of these enzymes was located in a separate compartment. It has to be mentioned that


in principle the number of enzymes is not limited to three and the reaction chain can be extended further. The enzymes were incorporated in different sequences at defined positions. Including two spacing compartments consisting of bovine serum albumin (BSA) between the three-enzyme compartments, particles with five compartments have been prepared exhibiting a prolonged lag period between the first and the last enzyme in the chain. The influence of the spacing between compartments containing different enzymes on the reaction kinetics was demonstrated on the microscopic scale within one microparticle, which to the best of our knowledge was not achieved until now. Our concept certainly opens a way to study coupled reactions in well-defined and structured systems with the possibility to change the environment of the enzymes, the spacing, as well as the properties of the compartments separating them. Acknowledgment. This work was supported by EFREProFIT 10139827 of the EU. Supporting Information Available. CLSM time series of four-compartment particles, HRP/FITC-BSA/GOX/FITC-BSA, with HRP in the central compartment (Video 1) and GOX/FITCBSA/HRP/FITC-BSA with HRP in the second peripheral compartment (Video 2) performing a reaction with glucose in the presence of Amplex Red added simultaneously to the bulk solution. CLSM time series of three-compartment particles, HRP/GOX/β-GLU (Video 3), and five-compartment particles, HRP/BSA-AF680/GOX/BSA-AF680/β-GLU, (Video 4) performing a reaction with FDGlu in the presence of Amplex Red, both added simultaneously to the bulk solution. Time dependency of fluorescence intensity in the outer β-GLU/Dx/DEAEDx compartment (green) and in the central HRP/BSA compartment (red) in a three-compartment particle, an intact fivecompartment particle, and a defective five-compartment particle (Figure S1a, b, and c, respectively). This material is available free of charge via the Internet at http://pubs.acs.org.

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