Nanoparticle Mediated Remote Control of Enzymatic Activity

Abstract

Nanomaterials have found numerous applications as tunable, remotely controlled platforms for drug delivery, hyperthermia cancer treatment, and various other biomedical applications. The basis for the interest lies in their unique properties achieved at the nanoscale that can be accessed via remote stimuli. These properties could then be exploited to simultaneously activate secondary systems that are not remotely actuatable. In this work, iron oxide nanoparticles are encapsulated in a bisacrylamide-crosslinked polyacrylamide hydrogel network along with a model dehalogenase enzyme, L-2-HAD_ST. This thermophilic enzyme is activated at elevated temperatures and has been shown to have optimal activity at 70 °C. By exposing the Fe₃O₄ nanoparticles to a remote stimulus, an alternating magnetic field (AMF), enhanced system heating can be achieved, thus remotely activating the enzyme. The internal heating of the nanocomposite hydrogel network in the AMF results in a 2-fold increase in enzymatic activity as compared to the same hydrogel heated externally in a water bath, suggesting that the internal heating of the nanoparticles is more efficient than the diffusion limited heating of the water bath. This system may prove useful for remote actuation of biomedical and environmentally relevant enzymes and find applications in a variety of fields.

Nanomaterials with unique energy absorbing (e.g., radiofrequency, optical, etc.) and physical properties have built the foundation for a new field of remote controlled (RC) materials.^1-3 Due to such unique features, these materials have found many uses in medicine and biotechnology.^4-7 One example is the use of gold nanoshells for photothermal cancer therapy.⁸ These materials absorb energy in the form of near-infrared light (NIR) and dissipate it as heat, which can be applied to induce photothermal ablation of cancer cells. Additionally, Wijaya et al. demonstrated that laser-induced melting of nanorods was shown to selectively release oligonucleotides from the nanorods, which may prove useful in drug delivery applications.⁹ Carbon nanotubes (CNTs) have also been used for their unique heating abilities in the presence of NIR and other wavelengths of energy. Miyako et al. were able to control the activity of two thermophilic enzymes, Taq DNA polymerase and cyclomaltodextrin glucanotransferase, via heating CNTs with NIR laser irradiation.¹⁰ This was one of the first demonstrations of remotely controlled enzymatic reactions using the heating properties of CNTs. Although these materials have been shown to be heated remotely, their uses in vivo are limited due to the minimal penetration depth of NIR light.¹¹ To overcome this limitation, nanoparticles have been explored as an alternative source for the production of localized heat when exposed to an alternating magnetic field (AMF).^12-14 Iron oxide nanoparticles are of particular interest because they not only function as heating sites and as vehicles for drug delivery, but they can also be visualized using conventional imaging techniques.^15-18 For instance, Thomas et al. developed a drug delivery system based on zinc-doped iron oxide nanocrystals encapsulated within mesoporous silica nanoparticles.¹⁹ When heated in the presence of an AMF, temperature responsive valves opened to release the encapsulated drug. Importantly, it was demonstrated that no compound was released in the absence of the field. Recently, Stanley et al. demonstrated that by coating iron oxide nanoparticles with antibodies, they could be targeted to a temperature-sensitive Ca²⁺ ion channel.²⁰ When the nanoparticles are heated with an AMF, the channels open allowing for an influx of Ca²⁺ ions. In turn, a bioengineered Ca²⁺ driven promoter is activated, thus intitiating the transcription of insulin. This is one of the first examples of using the heating properties of iron oxide nanoparticles to remotely control gene expression.

Nanocomposites containing magnetic nanoparticles have also been used to enhance or control the properties of stimuli-responsive hydrogels.^21-24 There are numerous classes of stimuli-responsive hydrogels that have been developed and are able to, depending on their composition, respond reversibly to external stimuli such as temperature, pH, electric potential, and analyte concentration.^25-27 By combining the remote heating capabilities of the nanoparticles with the stimuli-responsive properties of hydrogels, multifunctional materials can be designed for targeted applications such as drug delivery, hyperthermia cancer treatment, and as stimuli-responsive microfluidic valves.^{13, 28-30} Zadrazil et al. encapsulated iron oxide nanoparticles in a thermo-responsive polymer poly(N-isopropylacrylamide), (PNIPAM), to form a temperature responsive hydrogel sponge that could release an oil, kerosene, in the presence of an alternating magnetic field.³¹ It was demonstrated that by combining the hydrophilic responsive polymer with the iron oxide nanoparticles, a drug delivery system for hydrophobic compounds could be developed with on-demand release by exposure to an AMF.

Herein, we demonstrate that by combining enzyme-modified responsive hydrogels with Fe₃O₄ nanomaterials, RC nanocomposites can be developed that could find applications not only in the remote decontamination of environmental samples, but also as ‘on/off’ switches for heating and activating proteins in other biomedical, industrial, and pharmaceutical applications. Specifically, the incorporated Fe₃O₄ nanoparticles controllably heat the hydrogel matrix in the presence of an AMF, thus allowing for the remote control of the temperature within the system.¹³ By exploiting this selective heating capability, enhancement of catalytic activity was achieved by turning on the co-encapsulated thermophilic enzyme. The reactivity of the biomolecules is enhanced at higher temperatures (Figure 1a), where combining the divergent materials results in a remotely controlled system. Specifically, we demonstrated the ability to remotely activate a model enzyme, L-2-HAD_ST, a thermophilic dehalogenase isolated and characterized from the thermophile archaea Sulfolobus tokodaii. This enzyme catalyzes the stereospecific dehalogenation of L-2-haloalkanoates to L-2-hydroxyalkanoates (Figure 1b). By encapsulating the enzyme into the hydrogel, it allows for the use of the biocatalyst through many enzymatic cycles with different substrates without the need for complicated separation and/or regeneration steps.

Figure 1.

a) L-2-HAD_ST immobilized in a hydrogel network containing Fe₃O₄ nanoparticles. At room temperature, the enzyme activity is minimal. In the presence of the AMF, the nanoparticles heat, thus heating the environment around the L-2-HAD_ST and increasing the activity of the enzyme. b) L-2-HAD_ST functions by replacing the halogen (X) on an L-2-haloalkanoate with a hydroxyl group. The result is the formation of an L-2-hydroxyalkanoate.

Results and Discussion

L-2-HAD_ST catalyzes the dehalogenation of organic compounds, such as the model substrate S-2-chloropropionic acid (CPA). The reaction results in the stereospecific dehalogenation of CPA and formation free chloride, which can be measured by a chloride ion-selective electrode (ISE). L-2-HAD_ST has optimal activity at pH 9.5 and 70 °C, and maintains its activity when immobilized on a solid matrix.³² The demonstration of the long-term activity of the enzyme in solution, as well as its ability to work under extreme conditions and retain activity upon immobilization, allowed us to formulate the hypothesis that L-2-HAD_ST can maintain its enzymatic activity after chemical conjugation to acrylic acid monomers and polymerization into a hydrogel network. Purification of the enzyme used for all the studies was performed using a previously established protocol.³² After purification, L-2-HAD_ST was incorporated into a hydrogel network by chemical conjugation to acrylic acid functional groups via the lysines on the protein. An acrylamide hydrogel containing the L-2-HAD_ST was then polymerized with 1.5 wt% bisacrylamide crosslinking in the presence of 10% (w/v) Fe₃O₄ nanoparticles. Previous modeling studies indicated that such a nanoparticle concentration would be needed to reach and maintain the desired temperatures.³³ Additionally, a control hydrogel was polymerized by replacing the enzyme solution with the same volume of 20 mM glycine buffer, pH 9.5. The hydrogel was used as a mechanism by which encapsulation of both the enzyme and the nanoparticles was possible, thereby allowing for remote heating of the system in the presence of an AMF. This is envisioned to allow for activation of the system for bioremediation capabilities with facile recollection of the materials when the process is finished. Although this technique was implemented to immobilize the enzyme in the hydrogel, other strategies may be employed to directly immobilize enzymes to the nanoparticles.³⁴ The immobilization can lead to enhanced stability of the enzyme, as previously demonstrated.³⁵

With the enzyme incorporated into the hydrogel network, a time study was conducted to determine the optimal incubation time for the substrate. The hydrogel-encapsulated enzyme was incubated with 0.5 mM CPA for various periods of time ranging from 10 min to 1 h at 70 °C. The incubation time used for subsequent studies was determined to be 30 min (Figure 2a). Although the time study showed a constant increase in activity over the hour incubation, the 30 min time point is long enough to detect activity significantly above the blank, yet short enough to optimize assay time. After determining the time of incubation for the hydrogel-encapsulated enzyme with the substrate, it was important to demonstrate the activity trend when exposed to varying temperatures. To demonstrate temperature effects on activity, we evaluated the enzyme turnover at three different temperatures (~21, 50, and 70 °C). The highest temperature chosen was 70 °C, the optimal temperature for enzyme activity. Higher temperatures were not used because the aim of the study was to evaluate if the enzyme could be remotely heated using an AMF, not to determine stability of the hydrogel-encapsulated enzyme. First, the activity of the hydrogel-encapsulated enzyme at room temperature (RT, ~21 °C) was evaluated. The hydrogels were placed in a 20 mM glycine buffer, pH 9.5, containing various substrate concentrations (Figure 2b). After incubating for 30 min, the hydrogels were removed and the chloride concentration was measured. For each substrate concentration, triplicate measurements were performed using hydrogels containing the enzyme and control hydrogels prepared in the absence of the enzyme. The latter was used to determine the contribution of substrate autohydrolysis during the incubation period, which was taken into account by subtracting the chloride concentration in the controls from the total chloride concentration in the test samples. The protein in the hydrogel was found to have a k_cat of 4.02 ± 0.18 × 10⁻⁵ s⁻¹ and a K_m of 1.03 ± 0.27 mM. Furthermore, the enzymatic efficiency at room temperature was found to be 0.39 ± 0.10 × 10⁻⁴ mM⁻¹ s⁻¹.

Figure 2.

a) The protein was encapsulated in the hydrogel and a time study was performed by allowing the nanocomposite hydrogel pieces to incubate with 0.5 mM L-2-CPA for various periods of time. Data are blank subtracted and shown as averages ± one standard deviation (n=3). The time point chosen for subsequent experiments was 30 min. b) To test the activity of the immobilized protein, the hydrogel-encapsulated L-2-HAD_ST was incubated for 30 min at 21 °C (circles), 50 °C (squares), and 70 °C (triangles) with various concentrations of CPA. Data are blank substracted and shown as averages ± one standard deviation (n=3).

Next, the activity of the hydrogel-encapsulated enzyme was evaluated at 50 °C, which is still lower than the optimum temperature for reactivity (70 °C). The experiments were performed as described above by incubating for 30 min at 50 °C in a water bath. After the incubation period, the hydrogels were removed and the samples allowed to cool to room temperature before measuring the chloride concentration. The enzyme was found to have a k_cat of 17.9 ± 2.6 × 10⁻⁵ s⁻¹ and a K_m of 0.41 ± 0.01 mM. The enzymatic efficiency was 4.39 ± 0.65 × 10⁻⁴ mM⁻¹ s⁻¹, representing a >7 fold increase in functionality as compared to the system at room temperature, which was expected as L-2-HAD_ST is a thermophilic enzyme with optimal reactivity observed at higher temperatures. Additionally, the K_m also decreased at 50 °C compared to the hydrogel at room temperature. This demonstrates that the enzyme is still able to more favorably bind the substrate for enzymatic activity at the higher temperature when encapsulated in the hydrogel.

Finally, the experiments were repeated as above by incubating the hydrogels for 30 min in a water bath at 70 °C, the optimum temperature for enzymatic activity. The k_cat was determined to be 27.6 ± 0.6 × 10⁻⁵ s⁻¹, the K_m was 0.52 ± 0.15 mM, while the enzymatic efficiency was 5.33 ± 1.54 × 10⁻⁴ mM⁻¹ s⁻¹. The efficiency increased by almost 1.5 times when the hydrogel encapsulated enzyme was heated at 70 °C as compared to 50 °C and by ~15 fold as compared to the enzyme at room temperature. This remarkable increase in activity as a function of temperature is based upon the thermophilic nature of the system. To that end, higher temperatures are required for maximal activity. The K_m value did not vary significantly for the enzyme at 70 °C as compared to 50 °C. Previously published studies also suggest that the K_m values at these temperatures are comparable.^32,36 Unfortunately, global heating of the system, which includes the hydrogel and surrounding environment, is highly energy inefficient and can result in unwanted side effects. This is especially true when applying the materials for selective functionality in a complex matrix.

Studies were also carried out to characterize the free enzyme in order to compare its activity to that of the enzyme immobilized in the hydrogel network. A time study was performed using 2.0 mM of CPA (Figure 3a), which demonstrated an optimal incubation time at 70 °C of 10 min. Next, L-2-HAD_ST was incubated with various concentrations of substrate in 20 mM glycine buffer, pH 9.5 for 10 min and its activity was measured by determining the concentration of chloride with an ISE. Characterization of the Michaelis-Menten kinetics (Figure 3b) found a k_cat of 213.4 ± 8.7 × 10⁻⁵ s⁻¹, K_m of 1.25 ± 0.16 mM, and an enzymatic efficiency (k_cat/K_m) of 17.02 ± 2.28 × 10⁻⁴ mM⁻¹ s⁻¹. The activity of the free enzyme in the water bath at 70 °C was compared to the hydrogel-encapsulated enzyme at varying temperatures are summarized in Table 1. From this study, it was determined that the enzymatic efficiency of the immobilized enzyme was about 30% of the efficiency of the free enzyme at the same temperature; however, activity was maintained even after protein modification and hydrogel encapsulation. A decrease in enzymatic efficiency was seen previously when the enzyme was immobilized on a sepharose resin.³⁶ The decreased enzymatic efficiency observed in our study is likely due to the immobilization of the enzyme in the hydrogel network and the diffusion limitation of the substrate into the hydrogel.^37-38 By conjugating the enzyme to the hydrogel through non-specific lysines, some protein may become denaturated, or the enzymatic pocket may become inaccessible to the substrate as a result of immobilization. Even so, the enzymatic efficiency increased with increasing the temperature from room temperature to the optimum temperature for activity (70 °C) within the hydrogel. This suggests that although there may be enzyme molecules where the binding pocket is inaccessible to the substrate, there is a significant fraction of immobilized enzyme that maintains function.

Figure 3.

a) Time study of the free L-2-HAD_ST . The protein was incubated at 70 °C with 2 mM S-2-CPA for various periods of time. Data are blank subtracted and shown as averages ± one standard deviation (n=3). The optimal incubation time chosen was 10 min. b) To test the activity of the free protein, 0.003 mg L-2-HAD_ST was incubated at 70 °C in the presence of various concentrations of L-2-CPA for 10 minutes. Data are blank subtracted and shown as averages ± one standard deviation (n=3).

Table 1.

Summary of kinetic data for L-2-HAD_ST under various conditions. Data are shown as averages ± one standard deviation (n=3).

	FP, 70 °C*	HE, 21 °C	HE, 50 °C*	HE, 70 °C*	HE, AMF
kcat (x 10−5 s−1)	213.4 ± 8.7	4.02 ± 0.18	17.9 ± 2.6	27.6 ± 0.6	31.3 ± 8.1
Km (mM)	1.25 ± 0.16	1.03 ± 0.27	0.41 ± 0.01	0.52 ± 0.15	0.30 ± 0.07
kcat/Km (x 10−4 mM−1s−1)	17.02 ± 2.28	0.39 ± 0.10	4.39 ± 0.65	5.33 ± 1.54	10.11 ± 3.52

^*incubation in a water bath at the specified temperature

FP- Free protein, HE- hydrogel-encapsulated protein

Once the above parameters were established, we hypothesized that the hydrogel with encapsulated Fe₃O₄ nanoparticles could be heated remotely via an AMF, thus increasing the activity of the incorporated enzyme. Successful remote heating would allow for use of the materials in self-contained remote sites, making them ideal for environmental field applications. Initially, studies were performed with the hydrogel-encapsulated enzyme containing Fe₃O₄ nanoparticles to ensure that it could be remotely heated to 70 °C, which is required for optimum L-HAD_ST activity. The hydrogels were exposed to a magnetic field amplitude of 19.9 kA/m for 15 min on an induction power supply equipped with a solenoid (1.5 cm diameter, 5 turns). Using this approach, the temperature of the hydrogel was measured every 30 s with an infrared thermal imaging camera. Approximately 2.5 min into the exposure, the hydrogel heated to 70 °C and maintained that temperature for at least 15 min (Figure 4a). The images taken using the IR camera demonstrate that when the hydrogel was not in the presence of the AMF (time=0), there is no heating. After exposure to the AMF after 60 seconds, the hydrogel begins to heat up and continues to do so until about 150 seconds. At this time, the hydrogel is at the maximum temperature for that field amplitude and it maintains that temperature until the field is removed (Figure 4b). Although this study was important to demonstrate that the temperate could be controlled in a narrow range by heating with the AMF, the temperatures were measured with an IR camera and not in solution. Due to the need for the hydrogel-air interface, the parameters used in this study may not directly correlate to the system when immersed in buffer. Therefore, further optimization for the hydrogel heating had to be conducted for the hydrogel in solution. This was assessed by using 0.5 mM substrate and measuring L-2-HAD_ST activity after a 10 min exposure time to various magnetic field amplitudes (14.2, 21.7, 26.2, and 30.6 kA/m). As a control, the hydrogels were incubated with 0.5 mM substrate for 10 min at room temperature in the absence of the AMF. The control chloride concentration was then subtracted from the values found in the samples exposed to the field to ensure that the enzyme activity was due to the field and not to other environmental factors. As expected, as the field amplitude increased, the activity of the enzyme increased; however, as the amplitude passed 26.2 kA/m, the activity decreased (Figure 5a). This is consistent with previous data where the activity of free L-HAD_ST decreased after the temperature increased over 70 °C, possibly due to protein denaturation.³² Although this study does not allow for direct correlation of field amplitude to temperature, it demonstrated that the activity of the enzyme followed a similar trend in the presence of the AMF as it did when heated in the water bath. Therefore, it was determined that the optimum activity was obtained at a field amplitude of 26.2 kA/m, which was used for all subsequent activity experiments. Characterization of the remotely heated hydrogels was performed by determining enzymatic activity after a 10 min incubation time at 26.2 kA/m in the presence of various substrate concentrations (Figure 5b). From this study, the enzyme was determined to have a k_cat of 31.3 ± 8.1 × 10⁻⁵ s⁻¹ and a K_m of 0.30 ± 0.07 mM. The catalytic efficiency of L-2-HAD_ST was found to be 10.11 ± 3.52 × 10⁻⁴ mM⁻¹ s⁻¹ (Table 1). The k_cat increased when the hydrogel was heated with an AMF and the K_m decreased. This suggests that the affinity of the substrate to the enzyme increased when heated with the AMF, which indicates the remote heating is a more efficient heating method for optimal activity as compared to external approaches. Furthermore, the increase in k_cat demonstrates that the product turnover is higher when heated in the AMF as compared to the water bath. This is interesting because the time of exposure, and thus the heating, in the AMF (10 min) is three times less than in the water bath (30 min), which indicates that the enzyme reaches and maintains the optimal temperature more rapidly in the AMF. Interestingly, compared to the free protein, the k_cat of the immobilized enzyme does decrease. Such results were previously observed when the enzyme was immobilized via the lysine residues on sepharose beads.³⁶ The kcat of the free enzyme is roughly 10 times greater than the encapsulated materials, which, as discussed previously, likely arises from the diffusion limitation of the substrate through the porous hydrogel and the modification of the enzyme for hydrogel encapsulation. It is important to note, however, that the purpose of this study was not to compare the activity of the free and hydrogel-encapsulated protein, but to evaluate the efficiency of remotely controlling the heating of the enzyme in a system that is easily recoverable. Therefore, the more significant value in this work is the comparison of the k_cat of the hydrogel-encapsulated enzyme heated in the water bath versus heated in the AMF.

Figure 4.

Demonstration of heating capabilities of the nanocomposite hydrogels when exposed to the AMF. They hydrogels were exposed to a magnetic field amplitude of 19.9 kA/m for 15 min on an induction power supply equipped with a solenoid (1.5 cm diameter, 5 turns). a) A picture was taken with an IR camera every 30 sec. The IR images of three time points (0 sec, 60 sec, and 600 sec) are shown b) and the temperature was plotted at each time point.

Figure 5.

a) To find an optimal power setting on the alternating magnetic field, the hydrogels were incubated in the specified field strength for 10 minutes in the presence of 0.5 mM CPA. The optimum field amplitude was determined to be 26.2 kA/m. b) The hydrogels were then placed at this field strength in the presence of various substrate concentrations. After 10 min incubation, the chloride concentrations were measured to calculate the specific activity of the enzyme. Data are blank subtracted and shown as averages ± one standard deviation (n=3).

To study the long-term stability of the enzyme-encapsulated hydrogel, the same nanocomposite systems were used over a three month period. The specific activity was calculated and plotted every month for three months. Enzymatic activity was maintained even after storage at 4 °C in glycine buffer for the three month period (Figure 6). As evident, no decrease in activity was observed over this time period indicating potential long-term stability.

Figure 6.

Demonstration of long-term stability of hydrogel encapsulated enzyme over a three month period. Each month, the hydrogels were incubated with varying substrate concentrations and heated via a water bath at 70 °C. Data are shown as averages ± one standard deviation (n=3).

Based on our results, numerous enhancements over conventional enzymatic applications can be achieved using the nanocomposite system. First, the data show that the catalytic efficiency increased two-fold when the hydrogels were heated via the AMF instead of the water bath at 70 °C. Furthermore, the sample incubation time for the AMF system required heating for only 10 min, as compared to 30 min required by the water bath. The system not only has efficient localized heating, but also requires less time to achieve higher catalytic output as compared to the hydrogel in the water bath. This is because the AMF exposure generates highly localized heating at the reaction location in the system. Due to the encapsulated Fe₃O₄ nanoparticles, the system reaches higher internal temperatures more rapidly than when using traditional heating methods. Unfortunately, the internal temperature of the hydrogel could not be determined, but this could be predicted through modeling/simulations³³ or determined after the development of an appropriate measurement technique. Second, based upon the design of the composite system, recyclability of the functional materials can be readily achieved. By having magnetic materials encapsulated in the hydrogel, magnetic separation can be applied to extract the functional materials from complex mixtures. This is important for the long-term recyclability, while minimizing the need to produce excessive amounts of enzyme. To that end, multiple sets of the composite material were functional for at least a 3 month period.

Conclusions

In conclusion, the formation of a novel composite system for remote controlled enzymatic activity has been demonstrated. This system consists of a hydrogel, a thermophilic enzyme, and magnetic nanoparticles for remote heating in the presence of an AMF. The activity of the system was enhanced via AMF-based heating as compared to conventional heating methods due to the composite nature of the materials. Such materials can be readily dispersed in environmental systems for facile, remote contaminant degradation that is difficult to achieve using current technologies. It is envisioned that our approach can be applied to the creation of other materials that incorporate biological or synthetic elements that can be activated via an ‘on/off’ switch and used in a variety of industrial, environmental and medical applications.

Methods

Chemicals and apparatus

Sodium hydroxide, sodium chloride, ammonium persulfate (APS), sodium phosphate dibasic, sodium phosphate monobasic, acrylamide, and bisacrylamide were purchased from Sigma (St. Louis, MO). L-2-chloropropionic acid, acrylic acid, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), tetramethylethylenediamine (TEMED), and glycine were obtained from Fisher Scientific (Pittsburgh, PA). All chemicals were reagent grade or better. All solutions were prepared using reverse osmosis water (Milli-Q Water Purification System, Millipore, Bedford, MA). Nanoparticles were purchased from Nanostructured & Amorphous Materials Inc. (Houston, TX). The 15-mL centrifuge tubes were purchased from Sarstedt (Newton, NC). Chloride concentrations were determined using a chloride ion selective electrode (VWR, West Chester, PA). Alternating magnetic field studies were performed on an induction power supply (MMF-3-135/400-2, Taylor Winfield, Brookfield, OH) equipped with a solenoid (1.5 cm diameter, 5 turns). All results were analyzed using GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA).

Enzymatic activity determination of free L-2-HAD_ST using the chloride ISE

A stock solution of CPA (10 mM) substrate was prepared in glycine buffer (20 mM, pH 9.5). After addition of the substrate to the buffer, the pH was readjusted with NaOH to 9.5. From this stock solution, dilutions of CPA (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM) were prepared in glycine buffer (20 mM pH 9.5). Triplicate aliquots (2-mL) were obtained from each CPA solution. Then, L-2-HAD_ST (0.003 mg) was added to each sample and incubated for 10 min at 70 °C. Blank samples were prepared by adding an equivalent volume of glycine buffer to the protein without substrate. Upon cooling, a commercial chloride ISE was used to determine chloride concentration.

Hydrogel Fabrication

The first step of the covalent immobilization of the L-2-HAD_ST into a hydrogel network involved the chemical conjugation of the lysine residues of the enzyme to the carboxylic groups of acrylic acid monomers using well-established carbodiimide-mediated coupling reaction protocols. First, acrylic acid (170 μL of a 1 × 10⁻³ M stock solution) was added to a reaction mixture of EDC (0.115 mg) and sulfo-NHS (0.575 mg) and left to react at room temperature for 15 min. To this mixture, enzyme (2.0 mg) was added at RT for 2 h and left to react, without stirring, at 4 °C overnight. The conjugated protein was then centrifuged in an Amicon centrifuge filter with a 3000 MW cutoff to remove any unreacted acrylic acid.

Before polymerization of the hydrogels, the hydrogel cast was weighed, and the mass recorded. For hydrogel fabrication, acrylamide (300 mg) and bisacrylamide (576 μL) were added to 2-(N-morpholino)ethanesulfonic acid (MES) buffer (2.35 mL, pH 5.5). Next, Fe₃O₄ (10% w/v) nanoparticles were added to the precursor solution, which was then sonicated for 20 min to disperse the Fe₃O₄ nanoparticles. After dispersion, either (2 mL,1 mg/mL) of the L-2-HAD_ST-acrylamide functionalized enzyme or buffer (control hydrogels) were added to the solution. Next, APS (60 μL) and TEMED (25 μL) were incorporated into the solution and the hydrogels were allowed to polymerize for 1 h at 4 °C. After allowing it to warm to room temperature, the hydrogel and cast were again weighed. The mass of the cast was subtracted from the mass of the hydrogel plus cast to get an accurate mass of the hydrogel. The polymerized matrix was then removed from the cast and washed overnight in 20 mM glycine buffer, pH 9.5, to remove any of the unreacted monomer from the hydrogel. The hydrogels were then cut into 4-mm diameter round pieces. Eight of these pieces were weighed and their masses averaged. This average was expressed as a percent of the total hydrogel mass. To estimate the average mass of protein in each hydrogel piece, the percentage of mass found for an individual piece was multiplied by the total protein concentration incorporated into the hydrogel matrix.

Determination of hydrogel encapsulated L-2-HAD_ST activity in a water bath

A stock solution CPA (10 mM) substrate was prepared in glycine buffer (20 mM, pH 9.5). After addition of the substrate to the buffer, the pH was readjusted with NaOH to 9.5. From this stock solution, dilutions of CPA (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM) were prepared in glycine buffer (20 mM, pH 9.5). Triplicate aliquots (2-mL) were obtained from each CPA solution. Hydrogel pieces were added to the solutions that either contained the immobilized L-2-HAD_ST or no protein (control hydrogels). Each sample was incubated for 30 min at 21 °C (room temperature), 50 °C (water bath), or 70 °C (water bath). They hydrogel pieces were immediately removed. Upon cooling to room temperature, a commercial chloride ISE was used to determine chloride concentration.

Long-term stability of hydrogel encapsulated L-2-HAD_ST in a water bath

The activity of the enzyme was determined as described above. Specific activity was evaluated at 70 °C each month for three months from the same set of hydrogels.

Alternating magnetic field studies

The hydrogels with the encapsulated enzyme and control hydrogels were incubated at various field amplitudes for 10 min in the presence of CPA (0.5 mM). Simultaneously, hydrogels with the encapsulated enzyme and control hydrogels were also incubated for 10 min in the presence of CPA (0.5 mM) at room temperature. Next, the hydrogel was removed from solution, and the chloride concentration was measured. All measurements were blank subtracted to account for the chloride formation at room temperature. After determining the field amplitude for optimum enzymatic activity, an assay was performed to evaluate the enzymatic efficiency. Stock solutions of the substrate were prepared as described above. The hydrogels were exposed to a field amplitude of 26.2 kA/m for 10 min in the presence of various amounts of substrate as described previously, then the hydrogel was removed and the chloride concentration determined via the chloride ISE.

Time study of free L-2-HAD_ST

A time study was performed to find an optimal incubation time for L-2-HAD_ST activity. The substrate concentration was kept constant at 2.0 mM S-2-chloropropionic acid in 20 mM glycine buffer, pH 9.5. Each sample in triplicate was incubated for the desired time (5, 10, 15, 30, 45, and 60 min) in a water bath at 70 °C. After incubation, the tubes were cooled to room temperature and a commercial chloride ISE was used to determine chloride concentration.

Time study of hydrogel encapsulated L-2-HAD_ST

The hydrogels were incubated in a 20 mM glycine buffer, pH 9.5, with 0.5 mM substrate for different time intervals (5, 10, 15, 20, 30, 45, and 60 min) in a water bath at 70 °C. After incubation, the hydrogels were removed and the samples allowed to cool to room temperature. The chloride concentration was then determined via a commercial chloride ISE.

Heating profile of hydrogel

The wet hydrogel was wrapped in saran wrap and placed on the top of the coil of the AMF apparatus. The field strength was set at 19.9 kA/m and the temperature of the hydrogel was visualized using an AGEMA Thermovision 470 IR camera (FLIR, Boston, MA).