Enhanced Enzyme Cascade Reactions Through Coencapsulation in Biocompatible Silica Nanoconfinement

Abstract

Enzyme cascade reactions, where the product of one enzyme serves as the substrate for another, are fundamental to cellular metabolism and essential for constructing efficient multi‐enzyme systems. Spatial confinement of sequential enzymes can enhance cascade efficiency through proximity effects and substrate channeling, mimicking the organization of natural metabolic pathways. Here, we coencapsulated glucose oxidase (GOX) and horseradish peroxidase (HRP) within silica nanocapsules (SiNCs) using an enzyme‐friendly synthesis method. The encapsulated enzymes retained high catalytic activity, and Michaelis‐Menten kinetics analysis revealed decreased Km for GOX, indicating enhanced substrate affinity under confinement. The coencapsulated system demonstrated faster cascade kinetics compared to spatially separated enzyme configurations, providing direct evidence for the beneficial effects of enzyme proximity on intermediate transfer. Enhanced thermal stability of confined enzymes was also observed, presumably due to preserved hydration shells. This platform enables quantitative studies on enzyme behavior under nanoscale confinement, offering insights into the biochemical principles governing multi‐enzyme cascade systems in both natural and artificial environments.

We coencapsulated glucose oxidase (GOX) and horseradish peroxidase (HRP) within silica nanoconfinement using an enzyme‐friendly synthesis method. The coencapsulated system demonstrated faster cascade kinetics than spatially separated configurations, providing direct evidence for beneficial proximity effects on intermediate transfer. This platform enables quantitative studies on enzyme behavior under nanoscale confinement.

1. Introduction

Enzyme cascade reactions are sequential processes where the product of one enzymatic reaction serves as the substrate for a subsequent reaction [1, 2, 3, 4]. These reactions are fundamental to biological systems and play a crucial role in constructing efficient multi‐enzyme systems. In living cells, metabolic pathways consist of sequential enzymatic reactions that are spatially organized within compartments such as organelles or multi‐enzyme complexes, enabling high metabolic efficiency [5, 6, 7, 8]. Mimicking this natural strategy has attracted significant attention in biotechnology, with applications ranging from biosensing and biocatalysis to prodrug activation and synthetic biology [9, 10, 11, 12, 13].

To achieve efficient cascade reactions in artificial systems, the spatial organization of enzymes is critical. Specifically, enzyme confinement, restricting enzymes within a defined space, has emerged as a key strategy to enhance cascade efficiency by reducing the diffusion distance of intermediates and increasing local substrate concentrations [14, 15, 16]. Two major approaches for enzyme confinement have been developed, which are surface confinement and volume confinement [17, 18]. Surface confinement involves immobilizing enzymes on the surface of solid supports such as nanoparticles, membranes, or scaffolds such as DNA structures. While this approach offers easy accessibility of substrates to enzymes, the exposed enzymes are susceptible to environmental stresses and may suffer from reduced stability. In contrast, volume confinement encapsulates enzymes within a defined interior space, such as liposomes, polymersomes, or porous nanoparticles [19, 20, 21]. This approach provides a protective environment for enzymes while maintaining the proximity between coencapsulated enzymes, making it advantageous for multi‐enzyme cascade systems.

Among volume confinement strategies, encapsulating multiple enzymes within a single nanoscale compartment offers unique benefits for cascade reactions. The close proximity between enzymes enables efficient substrate channeling, where intermediates are directly transferred from one enzyme to the next without equilibrating with the bulk solution [22]. However, a significant challenge remains the configuration of multienzyme cascade systems with volume confinement. Many encapsulation methods cause substantial loss of enzymatic activity due to harsh synthesis conditions, such as exposure to organic solvents, extreme pH, or mechanical stress. This activity loss has limited the practical application of encapsulated enzyme systems and, more importantly, has hindered reliable quantitative studies on the effects of confinement and proximity on cascade reaction kinetics. For instance, ZIF‐8‐based approaches encapsulate enzymes during in situ framework formation in aqueous phase; however, enzyme entrapment occurs stochastically rather than completely, and ZIF‐8 is unstable under acidic conditions [23, 24]. Covalent Organic Framework‐based platforms utilizing covalent immobilization enhance enzyme stability; however, covalent conjugation chemically modifies the enzyme structure, which may limit the ability to study the intrinsic behavior of enzymes [25]. In contrast, our silica nanocapsule platform employs inverse miniemulsion to completely encapsulate the aqueous enzyme solution with high efficiency. The chemically stable silica shell and enzyme‐friendly synthesis conditions preserve substantial enzymatic activity, making this approach particularly suitable for quantitative studies on multi‐enzyme cascade reactions. As a strong candidate for volume‐confinement, silica‐based nanomaterials have been widely explored as enzyme encapsulation platforms due to their biocompatibility, chemical stability, and tunable porosity [26, 27]. However, common synthesis methods often employ organic solvents that can compromise enzyme integrity during the encapsulation process.

In this study, we developed an enzyme‐friendly silica nanocapsule synthesis method that significantly preserves enzymatic activity after encapsulation. We aimed to coencapsulate two model enzymes, glucose oxidase (GOX) and horseradish peroxidase (HRP), within hollow silica nanocapsules (SiNCs) featuring an aqueous core. These two enzymes were selected as a model cascade system because they can perform a sequential reaction. GOX catalyzes the oxidation of glucose to produce hydrogen peroxide (H₂O₂), which subsequently serves as a substrate for HRP to oxidize chromogenic substrates. By confining both enzymes within the same aqueous ‘nanocompartment’ of SiNCs, we hypothesized that the close proximity between GOX and HRP inside the SiNCs would facilitate efficient substrate channeling of the intermediate H₂O₂, thereby enhancing the overall cascade reaction efficiency (Figure 1). The high encapsulation efficiency and retained enzymatic activity enabled us to quantitatively investigate the cascade reaction behavior and directly demonstrate the beneficial effects of enzyme colocalization on cascade efficiency. Our platform provides not only a practical tool for biotechnology but also a reliable system for fundamental studies on multi‐enzyme reactions under nanoscale confinement.

FIGURE 1.

Coencapsulated GOX and HRP into silica‐based nanoconfinement (SiNCs), and cascade reactions. GOX catalyzes the oxidation of glucose to produce hydrogen peroxide (H₂O₂), which subsequently serves as a substrate for HRP to oxidize chromogenic substrates.

2. Results and Discussion

2.1. Synthesis and Characterization of SiNCs

SiNCs were synthesized based on the sol–gel reaction using tetraethyl orthosilicate (TEOS) and (3‐aminopropyl)‐trimethoxysilane (APTMS) as precursors, combined with an inverse miniemulsion technique. The aqueous phase consisted of 20 mM phosphate buffer (pH 7.4), while olive oil was employed as the organic phase. Polyglycerol polyricinoleate served as the emulsifier, and potassium fluoride was used as a catalyst for the sol–gel reaction (Figure 2a). The use of olive oil as the organic phase represents a key methodological innovation in this study. In our previous attempts using common organic solvents such as toluene or cyclohexane, significant loss of enzyme activity was observed. Through control experiments, we found that these solvents themselves do not substantially contribute to enzyme denaturation. However, when the aqueous enzyme solution was subjected to ultrasonication in the presence of these solvents during emulsion preparation, nearly complete loss of enzymatic activity occurred, particularly for HRP. This synergistic detrimental effect is presumably attributed to protein unfolding at the oil–water interface. During ultrasonication, enzymes are repeatedly exposed to the interface where the hydrophobic core of proteins tends to interact with the nonpolar phase, leading to conformational changes and activity loss, potentially facilitated by cavitation effects. Olive oil, being a biocompatible oil with milder interfacial properties, likely reduces such interfacial denaturation, thereby preserving enzyme activity during emulsification. This enzyme‐friendly approach is one of the core contributions of this work.

FIGURE 2.

Synthesis and characterization of enzyme@SiNCs. (a) Inverse miniemulsion‐based synthesis of SiNCs with encapsulated GOX, HRP, or both. (b) TEM micrograph of GOX/HRP@SiNCs. (c) Size distribution of GOX/HRP@SiNCs analyzed by image analysis.

Using this method, we successfully synthesized three types of nanocapsules, GOX‐encapsulated (GOX@SiNCs), HRP‐encapsulated (HRP@SiNCs), and GOX/HRP coencapsulated silica nanocapsules (GOX/HRP@SiNCs). Transmission electron microscopy (TEM) analysis revealed that all nanocapsules exhibited a spherical morphology with a distinct core–shell structure (Figure 2b, and Figure S1). For the GOX/HRP@SiNCs, size distribution was determined by analyzing 200 number of TEM images using ImageJ software, as dynamic light scattering (DLS) measurements were not feasible due to poor dispersibility in water. The size distribution ranged primarily from 100 to 360 nm, with a mean diameter of 228 ± 59 nm (Figure 2c).

The dispersion stability of nanocapsules in aqueous suspension was evaluated by monitoring turbidity over time. When the suspension was under agitation, the nanocapsules did not readily settle down. Under static conditions, significant changes in turbidity did not occur within the typical assay timeframe. Turbidity was measured at 600 nm under static conditions using a TECAN plate reader with top‐to‐bottom measurement configuration. The optical density (OD) values gradually increased from 0.4534 at the starting point to 0.4751 (5 min), 0.4905 (10 min), 0.5366 (15 min), and 0.5685 (20 min). This gradual increase in OD values is attributed to partial settling of nanocapsules toward the bottom of the well, which increases the OD reading in the top‐to‐bottom measurement configuration rather than indicating aggregation or significant sedimentation. These results confirm that the nanocapsules remain sufficiently dispersed during the typical enzymatic assay duration.

The shell thickness was also determined through TEM image analysis, yielding an average thickness of 5.42 ± 1.43 nm (Figure S2). This thin shell structure is advantageous for cascade reactions, as it allows facile diffusion of small substrate molecules such as glucose and chromogenic substrates across the shell while retaining the encapsulated enzymes within the aqueous core. Furthermore, the thin shell minimizes diffusion for the intermediate hydrogen peroxide, potentially enhancing the efficiency of the cascade reaction between GOX and HRP.

To determine the encapsulation efficiency of enzymes within the SiNCs, we dissolved the silica shell using ammonium bifluoride and quantified the released protein content. The fluoride ions react with silica to form soluble silicon hexafluoride complexes, thereby releasing the entrapped enzymes into solution without compromising protein integrity. Following silica dissolution, the solution was neutralized and the protein concentration was determined using Bradford method. The encapsulation efficiency was calculated by comparing the amount of protein recovered from the nanocapsules to the initial amount of enzyme added during synthesis. The results showed encapsulation efficiencies of 88 ± 3.2% for GOX@SiNCs, 83 ± 4.5% for HRP@SiNCs, and 82 ± 3.8% for GOX/HRP@SiNCs (Table 1). These high encapsulation efficiencies (>80%) indicate that the majority of enzymes introduced during the synthesis process were successfully entrapped within the nanocapsules, with minimal loss to the surrounding medium. Such high encapsulation efficiency is advantageous for several reasons: it ensures cost‐effective use of enzymes, enables precise control over the enzyme loading amount, and allows for accurate normalization when comparing catalytic activities between encapsulated and native enzymes. For all subsequent activity comparisons between encapsulated and native enzymes, the enzyme amounts were adjusted based on the encapsulation efficiency to ensure accurate and fair comparison.

TABLE 1.

Encapsulation efficiency of enzymes in SiNCs.

Nanocapsules	Encapsulation efficiency, %
GOX@SiNCs	88 ± 3.2
HRP@SiNCs	83 ± 4.5
GOX/HRP@SiNCs	82 ± 3.8

2.2. Enzymatic Activity Analysis of Encapsulated Enzymes

The enzymatic activity of encapsulated enzymes was evaluated using an Amplex Red‐based fluorescence assay. In this assay, GOX catalyzes the oxidation of glucose to produce H₂O₂, which is subsequently utilized by HRP to convert non‐fluorescent Amplex Red into fluorescent resorufin. The resulting fluorescence intensity was measured to quantify enzymatic activity (Figure 3a).

FIGURE 3.

Enzymatic reaction of SiNCs. (a) Amplex‐based enzyme assay principle for GOX. (b) Michaelis‐Menten kinetics of native GOX, GOX@SiNCs, native HRP, and HRP@SiNCs. (c) Enzyme retention and leakage of GOX/HRP@SiNCs (n = 3). (d) Thermal stability of encapsulated enzymes in GOX/HRP@SiNCs (n = 3). The enzymes were exposed in 50°C. For c and d, error bars represent standard deviation.

Michaelis‐Menten kinetics analysis was performed to characterize the catalytic properties of encapsulated enzymes. Since the simultaneous encapsulation of both enzymes in GOX/HRP@SiNCs would result in mutual interference between the two enzymatic reactions, single enzyme‐encapsulated nanocapsules (GOX@SiNCs and HRP@SiNCs) were used for kinetic analysis instead (Figure 3b and Figure S3). For GOX@SiNCs, the Michaelis‐Menten kinetics analysis revealed that the Km value of encapsulated GOX decreased compared to native GOX, indicating enhanced substrate affinity. The kcat value also decreased, suggesting a reduced turnover rate. The overall catalytic efficiency (kcat/Km) of GOX@SiNCs was 84.2% relative to native GOX. The effectiveness of olive oil as the organic phase was demonstrated by comparing the retained enzymatic activity with our previous study. In our earlier, GOX‐encapsulated nanocapsules fabricated using the same protocol with toluene as the organic phase retained approximately 66.7% catalytic efficiency (kcat/Km) relative to native GOX [26]. This significant improvement in activity preservation confirms that olive oil provides a milder interfacial environment during the emulsification process, reducing enzyme denaturation at the oil–water interface.

The decrease in Km observed for GOX within SiNCs is consistent with our previous findings [10, 26]. Although the exact mechanism remains to be elucidated, several hypotheses may explain this phenomenon: (1) increased local substrate concentration in the vicinity of the enzyme, leading to easier saturation of the active site, (3) a slightly lower local pH within the silica interior, which may enhance substrate binding affinity (notably, GOX is known to exhibit lower Km values under mildly acidic conditions), and (4) hydrogen bonding interactions between glucose and the silica shell, which may increase substrate retention within the nanocapsule. These hypotheses warrant further investigation in future studies. The decrease in kcat may be attributed to diffusion limitations imposed by the silica shell. Specifically, the product H₂O₂ generated by GOX may accumulate within the nanocapsule interior due to restricted diffusion through the silica shell. This slower product release would delay the regeneration of free enzyme from the enzyme–substrate complex, thereby reducing the overall turnover rate. Nevertheless, the high retained activity after encapsulation demonstrates the advantage of our enzyme‐friendly synthesis approach.

For HRP@SiNCs, the catalytic efficiency (kcat/Km) was 52.8% relative to native HRP. The kinetic analysis revealed that the Km value increased while the kcat value decreased compared to native HRP. Several factors may contribute to these kinetic changes. First, Amplex Red, which serves as the cosubstrate and chromogen in this assay, has relatively low hydrophilicity, which may result in slower diffusion through the hydrophilic silica matrix. This restricted access of Amplex Red to the encapsulated HRP would lead to an apparent increase in Km. Second, the fluorescent product resorufin may become partially trapped within the silica interior, potentially causing an underestimation of the actual enzymatic activity due to reduced fluorescence detection. Despite these limitations, the retained activity of HRP@SiNCs represents a remarkable achievement compared to the toluene‐based synthesis method, where HRP activity was almost completely lost due to ultrasonication‐induced denaturation. The successful preservation of substantial HRP activity further validates the effectiveness of using olive oil as a biocompatible organic phase in our synthesis protocol [26].

2.3. Enzyme Retention and Reusability of Silica Nanocapsules

To verify that encapsulated enzymes remain confined within the SiNCs without leakage, we conducted enzyme retention experiments. Freshly synthesized nanocapsules were dispersed in water for 24 and 48 h, followed by centrifugation to separate the nanocapsules from the supernatant. The enzymatic activity of the redispersed nanocapsules and the supernatant was then compared to that of the freshly synthesized nanocapsules.

The results demonstrated that the redispersed nanocapsules retained nearly equivalent enzymatic activity compared to the freshly synthesized samples for all three types of nanocapsules: GOX@SiNCs, HRP@SiNCs, and GOX/HRP@SiNCs. In contrast, the supernatant (the 1st supernatant for 24 h, and the 2nd supernatant for 48 h) exhibited negligible or no detectable enzymatic activity (Figure 3c and Figure S4). These findings indicate that the encapsulated enzymes remain stably entrapped within the nanocapsules and do not leach out into the surrounding aqueous medium. To complement the activity‐based assessment, we performed direct protein quantification of the supernatants using the Bradford assay. The protein concentration in the supernatant was below the detection limit of the assay after 24 and 48 h incubation. Negligible amounts of proteins were determined from both supernatant. These results confirm that the enzymes are stably retained within the silica nanocapsules with negligible leakage.

This selective permeability of the silica shell is a critical feature of our nanocapsule system. The ultrathin silica shell (approximately 5.42 nm in thickness) allows the free diffusion of small molecules such as glucose, oxygen, hydrogen peroxide, and Amplex Red, which are essential substrates and products for the cascade reaction. However, macromolecules such as GOX (approximately 160 kDa (80 kDa x 2)) and HRP (approximately 44 kDa) are successfully retained within the aqueous core. This size‐selective permeability ensures that the enzymatic cascade reaction can proceed efficiently while maintaining the structural integrity of the multi‐enzyme system within the nanocapsules.

To further confirm that enzymes are encapsulated within the nanocapsule interior rather than adsorbed on the surface, we performed a protease digestion assay using proteinase K, a broad‐spectrum protease capable of digesting exposed proteins. The rationale of this assay is that proteinase K cannot penetrate the silica shell to access internally encapsulated enzymes, while surface‐bound enzymes would be readily digested. GOX/HRP@SiNCs were incubated with proteinase K for 24 and 72 h, followed by thorough washing to remove the protease. The remaining enzymatic activity was then measured and compared to untreated samples. To determine the activity of two enzymes simultaneously, Amplex red assay was employed. The results demonstrated that encapsulated enzymes retained more than 95% of their initial activity even after 24 h as well as 72 h of protease treatment, indicating that the enzymes are well‐protected within the nanocapsule interior and inaccessible to external protease attack (Figure S5). In contrast, native (bare) enzymes subjected to the same protease treatment lost more than 80% of their activity. These findings provide evidence that the enzymes in our system are predominantly encapsulated within the core of the silica nanocapsules rather than adsorbed on the outer surface.

To evaluate the reusability of the encapsulated enzymes, GOX/HRP@SiNCs were subjected to repeated cascade reaction cycles. In each cycle, the nanocapsules were incubated with 10 mM glucose solution for 30 min, and the cascade reaction activity was measured. The nanocapsules were then recovered by centrifugation, washed with buffer, and reused for the subsequent cycle. This process was repeated for a total of 5 cycles. The results demonstrated that GOX/HRP@SiNCs retained approximately 80% of their initial cascade activity after 5 consecutive cycles (Figure S6). The gradual decrease in activity (∼30%) is presumably attributed to the mechanical loss of nanocapsules during the centrifugation and recovery process rather than enzyme inactivation or leakage, as our enzyme retention experiments demonstrated negligible enzyme release from the nanocapsules. These findings indicate that our silica nanocapsule system offers reasonable recyclability, supporting its potential for practical applications in repeated cascade reactions.

2.4. Permeability of SiNCs

To verify the selective permeability of the silica shell, we performed a dye diffusion experiment using Rhodamine B as a model small molecule. Rhodamine B was incorporated into the aqueous phase during nanocapsule synthesis, and all purification steps were conducted in the organic phase. The nanocapsules were then dispersed in water, incubated for 10 min, and centrifuged to collect the supernatant. This process was repeated for 5 cycles. Intense fluorescence was observed in the first supernatant, indicating rapid diffusion of the encapsulated Rhodamine B (MW. 479.1 Da) through the silica shell upon exposure to the aqueous environment (Figure S7). This result, combined with the observation that enzymatic reactions proceed efficiently without enzyme leakage, confirms that the ultrathin silica shell (∼5.4 nm) functions as a semi‐permeable membrane that allows free diffusion of small molecules while retaining macromolecular enzymes within the nanocapsule interior.

2.5. Thermal Stability of Encapsulated Enzymes

To examine the thermal stability of each enzyme individually, we conducted separate experiments using GOX@SiNCs and HRP@SiNCs along with their respective native enzymes. For GOX, native GOX lost approximately 90% of its initial activity after 30 min of exposure at 50°C, whereas GOX@SiNCs retained 50% of its activity under the same conditions (Figure 3d and Figure S8). This enhanced thermal stability of encapsulated GOX is consistent with our previous findings. For HRP, which inherently possesses higher thermal stability than GOX, a longer exposure time was employed to evaluate the protective effect of encapsulation. After 6 h of incubation at 50°C, native HRP retained 60% of its initial activity, while HRP@SiNCs preserved 80% of its activity. This result indicates that the silica nanocapsule provides significant thermal protection for HRP as well. The enhanced thermal stability of encapsulated enzymes is presumably attributed to the preservation of the hydration shell within the silica interior. The confined aqueous environment within the nanocapsule may maintain a stable hydration layer surrounding the enzyme molecules, which plays a critical role in preserving protein conformation and preventing thermal denaturation. Additionally, the silica shell itself may provide a physical barrier that limits the conformational fluctuations of enzymes at elevated temperatures, thereby contributing to the improved thermal stability.

2.6. Cascade Reaction of Coencapsulated Enzymes

To investigate the cascade reaction behavior of coencapsulated enzymes, we conducted enzymatic assays using GOX/HRP@SiNCs with varying concentrations of glucose (0 ‐ 352 mM). In this cascade system, only glucose, the substrate for the first enzyme (GOX), was supplied along with Amplex Red. GOX catalyzes the oxidation of glucose using dissolved oxygen in the aqueous medium, generating H₂O₂ as a product. This H₂O₂ then serves as a substrate for the second enzyme (HRP), which catalyzes the oxidation of Amplex Red to produce the fluorescent product resorufin (excitation 550 nm, emission maximum 585 nm). The key feature of this assay is that by supplying only the substrate for the first enzyme, the sequential reaction through to the second enzyme can be monitored, thereby demonstrating the cascade reaction (Figure 4a). The results confirmed the successful cascade reactions within GOX/HRP@SiNCs, and the reaction rate was dependent on the glucose concentration.

FIGURE 4.

Cascade reactions of GOX to HRP in SiNCs. (a) Cascade reaction of GOX/HRP@SiNCs in various glucose concentraton. (b) Cascade reaction inside GOX/HRP@SiNCs (•), free GOX to HRP@SiNCs (▾), GOX@SiNCs to free HRP (○), GOX@SiNCs to HRP@SiNCs (△), and without any enzymes (▪) (n = 3). For b, error bars represent standard deviation.

To further verify that coencapsulation of both enzymes within a single nanocapsule enhances cascade reaction efficiency, we compared the cascade reaction rates of GOX/HRP@SiNCs with systems where only one enzyme was encapsulated while the other remained free: 1) free GOX + HRP@SiNCs and 2) GOX@SiNCs + free HRP. Additionally, to exclude the possibility that the enhanced cascade kinetics in GOX/HRP@SiNCs could be attributed to diffusion or partitioning artifacts rather than enzyme proximity effects, we also performed cascade reactions using a physical mixture of independently encapsulated GOX@SiNCs and HRP@SiNCs at matched total enzyme loading and nanocapsule mass.

The results demonstrated that GOX/HRP@SiNCs exhibited the fastest cascade reaction rate among all tested systems (Figure 4b). Among the other three configurations, the mixture of GOX@SiNCs + HRP@SiNCs showed the slowest cascade kinetics, followed by GOX@SiNCs + free HRP and free GOX + HRP@SiNCs. The comparison between coencapsulated GOX/HRP@SiNCs and the mixture of GOX@SiNCs + HRP@SiNCs is particularly informative, as both systems contain enzymes encapsulated within silica nanocapsules, experiencing identical diffusion barriers and partitioning effects through the silica shell. The critical difference is that in GOX/HRP@SiNCs, both enzymes are colocalized within the same aqueous compartment, whereas in the mixture, they are physically separated in different nanocapsules. Since both systems share equivalent silica shell diffusion barriers, this finding rules out diffusion and partitioning artifacts as the primary cause of enhanced cascade efficiency, and strongly supports the conclusion that enzyme proximity and substrate channeling within the coencapsulated system are responsible for the accelerated cascade reaction.

This enhanced cascade efficiency is attributed to the proximity and confinement effects within the nanocapsule interior. When both enzymes are colocalized within the same aqueous compartment, the intermediate H₂O₂ produced by GOX can be rapidly transferred to nearby HRP molecules without diffusing into the bulk solution. This substrate channeling effect minimizes the loss of intermediate and reduces the diffusion distance, thereby accelerating the overall cascade reaction. In contrast, for the systems combining free enzyme with encapsulated enzyme, the two enzymes are spatially separated, and the intermediate must diffuse across the silica shell to reach the second enzyme. For the mixture of GOX@SiNCs + HRP@SiNCs, the situation is even more challenging: the intermediate H₂O₂ produced inside one nanocapsule must diffuse out through the silica shell, travel through the bulk solution, and then diffuse into another nanocapsule to reach HRP, resulting in the slowest cascade kinetics.

Importantly, these comparative studies were made possible by the unique characteristics of our nanocapsule synthesis platform. The high encapsulation efficiency allows for precise quantification of the encapsulated enzyme amount, and the enzyme‐friendly synthesis approach preserves substantial enzymatic activity after encapsulation. These features enabled accurate and reliable comparison of cascade reaction kinetics across different enzyme configurations. Therefore, our platform can serve not only as a practical tool for various biotechnological applications but also as a valuable research platform for fundamental studies on enzyme behavior, including the effects of confinement and proximity on enzymatic reactions and the kinetic characteristics of multi‐enzyme cascade systems. These findings also highlight the effective mimicking of the confined environment of natural cellular organelles, where sequential enzymatic reactions are spatially organized to maximize metabolic efficiency.

3. Conclusion

In this study, we developed an enzyme‐friendly silica nanocapsule synthesis platform using olive oil as the organic phase, which significantly preserves enzymatic activity compared to common organic solvents such as toluene or cyclohexane. The coencapsulated GOX/HRP@SiNCs exhibited successful cascade reaction behavior, where glucose oxidation by GOX produced H₂O₂ that was subsequently utilized by HRP to generate fluorescent resorufin. Notably, the coencapsulated system demonstrated faster cascade reaction kinetics compared to systems with spatially separated enzymes, providing direct evidence for the proximity and confinement effects on multi‐enzyme cascade reactions.

Our silica nanocapsule platform offers several biochemical advantages: high encapsulation efficiency with precise enzyme quantification, substantial retention of enzymatic activity, enhanced thermal stability, and effective enzyme confinement without leakage. These characteristics make the platform not only suitable for practical applications such as biosensing and biocatalysis, but also valuable for fundamental biochemical studies investigating enzyme behavior under nanoscale confinement. This biomimetic approach of colocalizing sequential enzymes within a single compartment provides insights into the spatial organization strategies employed by natural metabolic systems and opens new avenues for designing efficient artificial multienzyme cascade systems.

4. Materials and Methods

4.1. Materials

Glucose oxidase from Aspergillus niger type VII (GOX; EC 1.1.3.4, 149 units/mg), horseradish peroxidase (HRP; EC 1.11.1.7, 200 units/mg), D‐glucose, tetraethyl orthosilicate (TEOS, 98%), and (3‐aminopropyl)trimethoxysilane (APTMS, 97%) were purchased from Merck; potassium fluoride, olive oil, Amplex Red (Ampliflu), and hydrogen peroxide (35%) were all purchased from Merck (Darmstadt, Germany). Polyglycerol polyricinoleate (PGPR) was purchased from Danisco (Copenhagen, Denmark), and Lutensol AT50 was obtained from BASF (Ludwigshafen, Germany). All other chemicals and solvents used in this study were of reagent grade.

4.2. Preparation of SiNCs

SiNCs were prepared using a miniemulsion method [26]. Olive oil (1.5 mL) was used as the continuous phase and contained TEOS (30.2 mg) and PGPR (12 mg). The aqueous phase consisted of sodium phosphate buffer (150 μL, 20 mM, pH 7.4) containing APTMS (5.6 mg), enzymes (0.4 or 0.8 mg), and potassium fluoride (0.1 mM). The two phases were combined and homogenized using a tip‐type ultrasonicator (Branson Ultrasonics, CT, USA) operated at 30% amplitude for a total of 5 min with pulsed mode (20 s on, 30 s off) while maintained on an iced‐bath. After emulsification, the reaction was allowed to react for 24 h at ambient temperature under gentle orbital mixing. The resulting nanocapsules were purified by dispersing the reaction mixture in cyclohexane (10 mL) followed by centrifugation at 11,000 rpm for 20 min. The pellet was then re‐dispersed in sodium phosphate buffer (250 μL, 10 mM, pH 7.4) containing 2 wt% Lutensol AT50.

For nanocapsule synthesis, 0.8 mg of total enzyme (GOX:HRP = 1:1 mass ratio) was used per batch for GOX/HRP@SiNCs, and 0.4 mg of enzyme was used per batch for single‐enzyme nanocapsules. The yield of silica nanocapsules was 7.8–9.5 mg per batch (average 8.2 mg). The enzyme loading was determined by dissolving the silica shell with ammonium bifluoride (2 M) and quantifying the released protein content using the Bradford assay. The calculated enzyme loading values were approximately 83 µg total enzyme per mg SiNCs for GOX/HRP@SiNCs, 43 µg GOX per mg SiNCs for GOX@SiNCs, and 40 µg HRP per mg SiNCs for HRP@SiNCs. For all cascade reaction comparisons, the enzyme amounts were matched based on the encapsulated enzyme content to ensure accurate comparison across different experimental configurations.

4.3. Enzymatic Assay

The enzymatic activity of GOX was evaluated using an Amplex Red‐HRP coupled assay. The reaction cocktail consisted of sodium phosphate buffer (1 mL, 10 mM, pH 7.4), Amplex Red (3.3 μL, 1 mM in DMSO), glucose solution (25 μL, 100 mM), and HRP solution (5 μL, 50 units/mL). For each measurement, 100 μL of the reaction mixture was transferred into a well of a 96‐well plate, followed by the addition of 2.5 μL of either native GOX solution or GOX@SiNCs dispersion containing 0.32 ng of enzymes (ca. 2.0 x 10⁻¹⁵ mole). Fluorescence changes were monitored at an excitation wavelength of 555 nm and an emission wavelength of 585 nm using a TECAN plate reader (Infinite M Plex, Männedorf, Switzerland). The initial reaction velocity measured within the first 3 min with 15 s interval was used to determine the Michaelis‐Menten enzyme kinetics.

The activity of HRP was measured using an Amplex Red‐hydrogen peroxide assay. The reaction cocktail was prepared by mixing sodium phosphate buffer (1 mL, 10 mM, pH 7.4), Amplex Red (3.3 μL, 1 mM in DMSO), and hydrogen peroxide solution (10 μL, 0.35%). A 100 μL aliquot of this reaction mixture was dispensed into each well of a 96‐well plate, after which 2.5 μL of either native HRP solution or GOX/HRP@SiNCs dispersion containing 4.0 ng of enzymes (ca. 9.1 x 10⁻¹⁴ mole) was added. Fluorescence was recorded at excitation 555 nm and emission 585 nm using a TECAN Infinite plate reader. The initial reaction velocity during the first 3 min with 15 s interval was used to determine the Michaelis‐Menten enzyme kinetics.

4.4. Enzyme Leakage Test

Enzyme leakage from silica nanocapsules was evaluated by incubating GOX/HRP@SiNCs under refrigeration (4°C) and measuring the enzymatic activity in both the nanocapsules and the supernatants. Freshly synthesized GOX/HRP@SiNCs were designated as “as prepared.” The nanocapsules were incubated at 4°C for 24 h under static conditions, followed by centrifugation to separate the supernatant (“1st supernatant”) from the nanocapsules. The recovered nanocapsules were redispersed in fresh buffer and incubated for an additional 24 h under the same conditions, followed by centrifugation to collect the second supernatant (“2nd supernatant"). The remaining nanocapsules after two rounds of supernatant collection were designated as “washed.” Enzymatic activity was measured for all four sample groups (as prepared, 1st supernatant, 2nd supernatant, and washed) using the Amplex Red‐based fluorescence assay. To directly quantify protein leakage, the collected supernatants were concentrated using Amicon Ultra centrifugal filters (10K MWCO) and subjected to Bradford protein assay.

4.5. Thermal Stability Test

The thermal stability of native and encapsulated enzymes was evaluated by incubating samples at 50°C and measuring the residual enzymatic activity. For native enzyme samples, GOX or HRP (0.16 mg/mL) in 10 mM phosphate buffer (pH 7.4) was used. For encapsulated enzyme samples, GOX@SiNCs, HRP@SiNCs, or GOX/HRP@SiNCs (0.5 mg/mL) was dispersed in the same buffer. Each sample (500 µL total volume) was placed in a glass vial and incubated in an oil bath at 50°C without shaking. Independent sample vials were prepared for each time point: 0 (room temperature control), 10, 20, and 30 min for GOX and GOX/HRP samples, and 0 (room temperature control), 2, 4, and 6 h for HRP samples, reflecting the higher intrinsic thermal stability of HRP. After thermal treatment, samples were cooled to room temperature, and the residual enzyme activity was measured using the Amplex Red‐based fluorescence assay. The reaction was monitored for 3 min at 15 s intervals, and the slope from a 2 min linear region was used to calculate the reaction rate. Residual activity was calculated as a percentage relative to the room temperature control sample. Native enzymes and SiNCs‐encapsulated enzymes were compared against their respective controls.

4.6. Reusability Test

To evaluate the reusability of encapsulated enzymes under operating conditions, GOX/HRP@SiNCs were subjected to repeated cascade reaction cycles. In each cycle, the nanocapsules were incubated with 10 mM glucose solution for 30 min, and the cascade reaction activity was measured using the Amplex Red‐based fluorescence assay. After each reaction, the nanocapsules were recovered by centrifugation, washed with buffer, and reused for the subsequent cycle. This process was repeated for a total of 5 cycles, and the enzymatic activity was measured after each cycle.

4.7. Cascade Reaction of Coencapsulated Enzymes

To investigate the cascade reaction behavior, GOX/HRP@SiNCs were incubated with various concentrations of glucose (0–352 mM) in 10 mM phosphate buffer (pH 7.4) containing Amplex Red as the chromogenic substrate. In this cascade system, GOX catalyzes the oxidation of glucose using dissolved oxygen, generating H₂O₂ as a product. The H₂O₂ subsequently serves as a substrate for HRP, which catalyzes the oxidation of Amplex Red to produce the fluorescent product resorufin (excitation 550 nm, emission 575 ‐ 665 nm). The fluorescence intensity was monitored using a TECAN plate reader with continuous orbital shaking to prevent nanocapsule sedimentation.

4.8. Comparison of Cascade Reaction Kinetics Across Different Enzyme Configurations

To evaluate the effect of enzyme colocalization on cascade reaction efficiency, we compared the cascade reaction rates of the following systems: (1) GOX/HRP@SiNCs (coencapsulated), (2) a physical mixture of GOX@SiNCs + HRP@SiNCs, (3) free GOX + HRP@SiNCs, (4) GOX@SiNCs + free HRP, and (5) free GOX + free HRP. For all comparisons, the total enzyme amount was held constant across different experimental groups (2 micrograms for each enzyme in 100 microliters). The amount of encapsulated enzyme in each nanocapsule formulation was determined based on the encapsulation efficiency (see section 4.2), and the amount of free enzyme or other nanocapsule formulations was adjusted to match this value. The cascade reaction was initiated by adding glucose (10 mM final concentration) and Amplex Red, and the fluorescence increase was monitored over time (excitation 550 nm, emission 585 nm). The reaction was performed in triplicate (n = 3), and the reaction rate was determined from the slope of the linear region of the fluorescence time course.

Funding

This study was supported by Pusan National University.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material

Lee Ji‐Soo, Ryu Da‐Hye, Jung Myung Kyung, You Dong‐Hyun, Park So‐Hee, Hwang Dae Youn, An Beum‐Soo, Yang Seung Yun, Seo Sungbaek, Choi Youngwoo, Jo Seong‐Min, Enhanced Enzyme Cascade Reactions Through Coencapsulation in Biocompatible Silica Nanoconfinement, ChemBioChem 2026, 27, e70382. 10.1002/cbic.70382

Data Availability Statement

The data that supports the findings of this study are available in the supplementary material of this article