Construction of Microreactors for Cascade Reaction and Their

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Biological and Medical Applications of Materials and Interfaces

Construction of Microreactors for Cascade Reaction and their Potential Application as Antibacterial Agents Tong Li, Jiawei Li, Qian Pang, Lie Ma, Weijun Tong, and Changyou Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20069 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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ACS Applied Materials & Interfaces

Construction of Microreactors for Cascade Reaction and their Potential Application as Antibacterial Agents Tong Li, Jiawei Li, Qian Pang, Lie Ma, Weijun Tong*, Changyou Gao MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China.

Abstract: Enzymatic cascade reactions in confined microenvironments play important roles in cellular chemical transformation. They also have important biotechnological and therapeutic applications. Here, enzymatic cascade microreactors (MRs) coupling glucose oxidase (GOx) and hemoglobin (Hb) (GOx-Hb MRs) were successfully fabricated by co-precipitation of GOx and Hb into MnCO3 template, followed by assembly of a multilayer film on template surface, slight crosslinking and final removal of MnCO3. In the presence of glucose with blood-relevant concentration, the GOx-Hb MRs exhibited higher cascade reaction activity under mild acidic condition than under neutral condition at physiological temperature. The GOx-Hb MRs effectively consumed glucose to generate HO˙ at pH=5, which significantly inhibited bacteria growth and biofilm formation. This kind of enzymatic cascade microreactors might be useful for applications in biomedical fields.

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Keywords:

Glucose

oxidase;

Hemoglobin;

Microreactor;

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Cascade

reaction;

Antibacterial

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1. Introduction Biocatalytic chemical transformations occur efficiently in live cells, which are attributed to enzymatic cascade reactions proceeding in spatially confined microenvironments. These reactions play essential roles in various cellular functions including signal transduction, metabolism, propagation, and interactions with outer environments.1,

2

Substantial efforts have been made to mimic natural systems by

constructing enzymatic cascades in well confined environments at micro and nano scales. It has been a general principle to take spatial confinement into consideration to achieve more efficient enzymatic cascades. The confined microenvironment containing enzymatic cascades prevents leakage of intermediate products and benefits their channeling, which is known as proximity effect.3,

4, 5

Recent evidences

demonstrated that the improved efficiency of confined enzymatic cascades results not only from proximity effect, but also the increased local enzyme concentrations and chance of substrates reaching enzyme active sites .6, 7 The enzymatic cascade reactions in confined microenvironments can find important biotechnological and therapeutic applications. A variety of materials including protein or DNA scaffolds,8,

9

hydrogel microparticles,10 liposomes,11 polymer vesicles,12

metal–organic framework (MOF) nanoparticles13, etc. have been adopted to construct spatially confined enzymatic systems. A facile and efficient method to encapsulate proteins is co-precipitation using MnCO3 as templates. Protein particles can be obtained after crosslinking encapsulated payload and dissolution of templates.14 Compared to other methods, mixing two salt solutions and proteins for 3



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co-precipitation is straightforward, and an excellent protein entrapment efficiency of nearly 100% can be achieved under mild reaction conditions. Single component submicron hemoglobin particles as oxygen carriers have been successfully fabricated using this method,14 so have the hybrid biopolymer particles containing proteins and hyaluronic acid (HA).15 These colloidal carrier systems are promising for pharmaceutical and biomedical applications. Although the potential of encapsulating multi-biomacromolecules

through

the

MnCO3-template

method

has

been

demonstrated, few studies have reported the construction of enzymatic cascade systems using the same strategy. In this work, we effectively co-encapsulated glucose oxidase (GOx) and hemoglobin (Hb) into MnCO3 microparticles through a co-precipitation process. A multilayer film was assembled on particle surface and slightly crosslinked to improve particle dispersity in water and prevent leakage of loaded proteins. MnCO3 was subsequently removed using ethylenediaminetetraacetic acid disodium salt (EDTA), generating enzymatic cascade microreactors (GOx-Hb MRs) (Scheme 1). The hypothesis is that GOx first catalyzes glucose oxidation into gluconic acid and hydrogen peroxide (H2O2), and the latter can be subsequently decomposed by Hb in the same microreactor to generate hydroxyl radicals (OH˙), which is highly toxic to bacteria. The GOx-Hb MRs can potentially be used as antibacterial agents to combat drug-resistant bacteria, e.g. methicillin-resistant Staphylococcus aureus (MRSA) in the presence of glucose at physiologically relevant concentrations. (Scheme 1).

4


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2. Experimental Section 2.1 Materials Bovine hemoglobin (Hb) was purchased from Yuan Ju Co., Ltd. Poly (allylamine hydrochloride) (PAH, Mw~17.5 kDa), poly (sodium 4-styrenesulfonate) (PSS, Mw~70 kDa) and dimethyl pyridine N-oxide (DMPO) were purchased from Sigma-Aldrich. Glucose oxidase (GOx, ≥180 U), MnCl2, 3,3′,5,5′-tetramethylbenzidine (TMB, 99%) and terephthalic acid (99%) (TA) were obtained from Aladdin. Na2CO3, D-glucose monohydrate, crystal violet (CV) and glutaraldehyde (GA) were purchased from Sinopharm Chemical Reagent Co., Ltd. Tryptone soybean broth medium (TSB) and tryptone soybean agar medium (TSA) were purchased from Bai Si Co, Ltd. Methicillin-resistant Staphylococcus aureus (ATCC 43300) was obtained from Guangdong culture collection center. 2.2 Instruments The size and zeta potential of particles were measured using a Zetasizer Nano ZS (Malvern Instruments) at ambient temperature. A UV-vis spectrometer (UV-1800, Shimadzu) was used to study the cascade reaction activity of microreactors. Inductively coupled plasma mass spectrometry (ICP-MS) was performed on an Agilent7700x using calibration curves obtained from standard solutions (5-500 ppb). Scanning

electron

microscopy

(SEM,

S-4800,

HITACHI)

equipped

with

energy-dispersive X-ray spectroscopy (EDX) was used to image the particles at an acceleration voltage of 3 keV and analyze their elements. The fluorescence spectra were characterized by a Shimadzu RF-5301PC spectrofluoro-photometer. A 5



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microplate reader (Tecan Sunrise) was used to measure the absorbance value at particular wavelength. 2.3 Fabrication of GOx-Hb MRs GOx-Hb MRs were fabricated using MnCO3 as template through a co-precipitation process. Typically, 12.5 mg GOx and 62.5 mg Hb were dissolved in 10 mL 0.25 M MnCl2 solution with a final protein concentration of 7.5 mg/mL. Then an equal volume of 0.22 M Na2CO3 solution was added under magnetic agitation (1000 rpm) for 1 min at room temperature. The feeding ratio of GOx to Hb (w/w) during co-precipitation process was changed to investigate their influence on the cascade reaction activity of final microreactors, while the final protein concentration in MnCl2 was fixed to 7.5 mg/mL. MnCO3 particles were washed three times with water, and a PSS/PAH/PSS three-layer film was assembled on the particles through layer-by-layer method.16 PSS and PAH were dissolved in water at 1.5 mg/mL. The assembly process was performed by dispersing ~1 wt% MnCO3 particles into PSS solution first and shaking for 15 minutes. After washing by water three times through centrifugation, the PAH layer was assembled similarly. After the final PSS layer was assembled, the particles were then dispersed into 0.025% GA water solution for 1 h and then treated with 2 mg/mL glycine water solution both with a final volume of 30 mL. GOx-Hb MRs were obtained after MnCO3 template removal by 0.1 M EDTA solution and then washed twice with EDTA solution and three times with water, and finally dispersed in water at 4 °C. As for denatured GOx (dGOx)-Hb microparticles (MPs), GOx was first dissolved in MnCl2 solution and heated at 80 °C for 10 min, and the remaining 6


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procedures were same to that of GOx-Hb MRs. 2.4 Cascade Reaction Activity Assays To investigate the influence of pH on cascade reaction activity of GOx-Hb MRs, the reaction was carried out in buffer solution with pH range of 3-8 (3-6 AcOH buffer was prepared from acetic acid and sodium acetate, 6-8 phosphate buffer was prepared from sodium salts of phosphoric acid, the concentration of both buffers is 0.2 M) containing GOx-Hb MRs (100 µg/mL), glucose (5 mM), TMB (0.75 mM) at 25 °C for 10 min. Absorbance at 652 nm was measured using a UV−vis spectrophotometer. To investigate the influence of temperature on cascade reaction activity of GOx-Hb MRs, AcOH buffer solution (pH=5, 25 ° C) was utilized while the temperature was changed from 25 to 65 °C. Other conditions were kept the same as in the above pH-dependence experiment. 2.5 Kinetics Studies The reaction kinetics measurements were carried out in time course mode by monitoring the absorbance change at 652 nm using an UV−vis spectrophotometer. The experiments were performed using 100 µg/mL GOx-Hb MRs and 0.75 mM TMB at pH=5.0, 25 °C with addition of glucose of different concentrations (1, 2, 5, 10, 22.5 mM). Michaelis-Menten Fitting and Lineweaver-Burk fitting were performed for the kinetics measurements. The Michaelis-Menten constant (Km) and the maximal reaction velocity (Vmax) were then calculated according to the methods reported in reference.17 2.6 Terephthalic Acid Probing Technique 7



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Different concentrations of GOx-Hb MRs (0, 10, 20, 50, 100, 200 µg/mL) were incubated with TA (4 mM), glucose (1 mM) at pH=5.0 or pH=7.4 at 37 °C for 40 min. The fluorescence spectra were observed with excitation at 315 nm using a fluorescence spectrophotometer. 2.7 Electron Spin Resonance (ESR) Spectroscopy Measurements GOx-Hb MRs (24 µg/mL) were mixed with 30 mM DMPO at pH=5.0 (0.2 M AcOH buffer) or 7.4 (0.2 M phosphate buffer) and the reaction was triggered by the addition of 30 mM glucose. The reaction solution was transferred into a glass capillary and monitored by ESR after incubation at 25 °C for 6 min, and finally ESR spectra of DMPO-OH was obtained. Measurements were performed at room temperature with 20 mW microwave power, 1 G modulation amplitude and 100 G sweep width. 2.8 MRSA Culturing and Antibacterial Assays Typically, 100 µL MRSA suspension (106 CFU/mL) mixed with 100 µL GOx-Hb MRs or dGOx-Hb MPs suspension of different concentrations in TSB medium with 12.5 mM glucose were placed into 96-well plates to culture for 24 h at 37 °C. Then the MRSA solution after co-incubation with particles was diluted to several concentrations and 100 µL was taken for spread plate cultivation on TSA plates for another 24 h at 37 °C, the MRSA colonies formed on the TSA plates were counted. 2.9 MRSA Biofilm Inhibition Assays MRSA biofilm inhibition was performed by extending co-incubation time from 24 h to 48 h, and the particle concentration was set as 2.4 µg/mL. Then the biofilms 8


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formed after co-incubation with particles were washed once with PBS carefully, 1% CV was used for the biofilm staining for 15 min and after that washed 3 times by PBS gently. As for biomass analysis, acetic acid solution (33%) was used to dissolve the attached CV and the absorbance at 590 nm was detected by a microplate reader. 3. RESULTS AND DISCUSION 3.1 Fabrication and Characterizations of GOx-Hb MRs The GOx-Hb MRs were fabricated by a co-precipitation method using MnCO3 as template,14, 15 the process was illustrated in Scheme 1. Firstly, the template MnCO3 containing Hb and GOx was fabricated by adding equal volume of Na2CO3 solution to MnCl2 solution in which Hb and GOx were dissolved. As shown in Figure 1a, these particles showed peanut-like shape, the size was 837 ± 68 nm measured from SEM images. Their surface was rough, and was built from smaller nanoparticles (Figure 1a, inset). Then PAH and PSS were assembled onto the surface of MnCO3 particles to improve their dispersity in water, after that a relatively low concentration of GA solution was used to crosslink the particles. Finally, the particles were treated by glycine to react with residual aldehyde groups. After removal of MnCO3 particles by EDTA, GOx-Hb MRs were obtained, as shown in Figure 1b, the shape of GOx-Hb MPs was similar to that of MnCO3 templates, their surface was smoother than that of MnCO3 templates. EDX results showed that after MnCO3 removal via EDTA treatment, the manganese in final GOx-Hb MRs was not detected, confirming the complete removal of MnCO3 (Figure S1). The cascade reaction activity could be easily regulated by the feeding ratio of GOx 9



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to Hb (w/w) during co-precipitation process. The reaction activity of GOx-Hb MRs with different feeding ratios of GOx to Hb was investigated using TMB as substrate in the presence of glucose. The cascade reaction can result in the formation of oxidized TMB (oxTMB), which has a blue color and can be monitored by a UV-vis spectrometer.18 As shown in Figure S2, the cascade reaction activity of GOx-Hb MRs increased with the decrease of GOx to Hb ratio. The absolute protein compositions of MRs fabricated with three feeding ratios of GOx to Hb (5:1, 1:1, 1:5) were checked by ICP-MS. Each Hb molecule has four iron atoms,19 thus the mass of Hb in the particle can be calculated according to the detected amount of iron. The mass of assembled thin PSS/PAH/PSS multilayer can be neglected since the film thickness is only around 5 nm,

20

so the rest of the mass is that of GOx. With feeding ratio

changing from 5:1 to 1:5, the absolute weight percentage of Hb in MRs increased from 12.5% to 61.3% while GOx decreased from 87.5% to 38.7%, (Figure S3). In the range of feeding ratio we investigated, the MRs with Hb content of 61.3% showed highest reaction activity (feeding ratios of GOx to Hb 1:5). This result indicates that the amount of Hb plays a more important role in the cascade reaction. In the range of feeding ratio we investigated, the GOx could always produce enough H2O2, while more Hb could supply more reactive center for subsequent reaction using H2O2 as substrate. Further decrease in GOx:Hb ratio resulted in unstability of obtained particles, thus ratio of GOx to Hb was fixed to 1:5 for following experiments. The ratio of GOx to Hb was much higher than the feeding ratio. This result is possibly due to greater binding ability of GOx to Mn2+ compared with that of Hb. Thus, more GOx 10


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can participate in the formation of nuclei or primary nanoparticles during co-precipitation with MnCO3.15 We compared the cascade reaction activity of GOx-Hb MRs with equivalent free GOx and Hb. As shown in Figure S4, the initial reaction rate of GOx-Hb MRs was lower than that of free enzymes. This is mainly because that co-precipitation and crosslinking processes decreased the absolute activities of enzymes, and the multilayers restrained glucose transportation to some extent at the beginning.

21, 22

However, the crosslinked GOx-Hb MRs can simultaneously transport two enzymes to desired locations and are more stable than free enzymes.

21, 22

The control sample

dGOx-Hb MPs were fabricated through the same procedure except that GOx dissolved in MnCl2 solution was firstly deactivated by 10 min heating at 80 °C. As shown in Figure S5a, the shape of dGOx-Hb MPs was same to that of GOx-Hb MRs. As measured by DLS, the size of GOx-Hb MRs was 957.1±17.1 nm and zeta-potential was -24.0±0.1 mV. The dGOx-Hb MPs had similar size (1023.5±34.2 nm) and zeta-potential (-33.4±0.7 mV). These two kinds of particles could be both well dispersed in water. Kinetic study results showed that dGOx-Hb MPs had a significantly lower cascade reaction activity compared to GOx-Hb MRs, (Figure S5b). 3.2 Cascade Reaction Activity Studies TMB is a widely used substrate for peroxidase which could be oxidized into oxTMB with a blue color and easily monitored by UV-vis spectrometer.18 In order to investigate the influence of pH on cascade reaction activity of GOx-Hb MRs, pH 11



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values were changed from 3.0 to 8.0. As shown in Figure 2a, GOx-Hb MRs exhibited a higher activity at lower pH in the presence of glucose, rather than under neutral condition. It is known that GOx has higher activity in mild acid condition than in neutral condition.23 Besides, Hb releases Fe3+ in the presence of excess H2O2, inducing Fenton reaction, a reaction which also has a higher rate at lower pH.24 As a result, rate of the cascade reaction based on GOx and Hb was higher at lower pH. The effect of temperature on cascade reaction activity was studied employing temperatures from ambient temperature to 65 °C at pH=5.0. As illustrated in Figure 2b, the reactivity first increased with the increase of temperature until 37°C and then decreased with further temperature increase. Below 37 °C the protein structures are stable and increase of temperature can accelerate the reactions, while with further temperature increase, the protein structures of GOx and Hb would change and lose enzymatic activity.25, 26 It is worth mentioning that although pH 5 is not optimal, the cascade reaction activity is still about 80% of the highest activity. Thus, this system can work well at mild acidic pathological environments, such as infection and tumor, at physiological temperature. The reaction efficiency of the cascade reaction was also controlled by the concentration of GOx-Hb MRs. As the concentration increased, the efficiency increased, reaching a plateau around 100 g/mL (Figure S6). In order to acquire kinetic parameters, the cascade reaction activity of GOx-Hb MRs was analyzed with enzyme kinetics theory and methods. TMB and glucose of different concentrations were added to reaction at ambient temperature and pH=5.0. UV-vis spectrometer was utilized to monitor the absorbance changes of oxTMB at 12


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652 nm. The time-course absorbance at different glucose concentrations was plotted as shown in Figure S7a. The initial reaction rates were calculated by five periods from start according to Beer-Lambert law. The initial rates were then plotted against glucose concentration and fitted using Michaelis-Menten curves (Figure S7b). The Michaelis-Menten constant (Km) and maximal rate (Vmax) were obtained by Lineweaver-Burk fitting the linear double-reciprocal plot (Figure S7c).

17

Km and

Vmax were calculated to be 2.60 mM and 2.94×10-8 Ms-1. The Km value demonstrates that GOx-Hb MRs has a relatively high cascade reaction activity in the presence of glucose with normal blood-relevant concentration from about 4 mM to 7 mM.27 Compared to other cascade systems containing GOx28, 29, Km value in our study is much lower, demonstrating their higher affinity to glucose. This supports GOx-Hb MRs to react effectively with high concentration glucose (e.g. in diabetic wounds) and manage drug-resistant bacteria infections.30, 31 3.3 Detection of Hydroxyl Radicals In order to detect HO˙ production, a classic reagent TA was used to react with HO˙ to generate fluorescent TA-OH, which was monitored by a fluorescence spectrometer with excitation at 315 nm (Figure 3a).32, 33 The experiments were conducted at pH 5.0 and 7.4. As shown in Figure 3b, c, fluorescence intensity increased with increased GOx-Hb MRs concentration. At same GOx-Hb MRs concentrations, fluorescence intensity at pH=5.0 was significantly higher than that at pH=7.4, which demonstrated that GOx-Hb MRs produced more HO˙ at pH=5.0 than at pH=7.4 in the presence of glucose. 13



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To further confirm such effect, DMPO, a classic electron paramagnetic resonance (EPR) reagent was employed to identify HO˙, as the produced DMPO-OH has a characteristic 1:2:2:1 hydroxyl radical signal with a hyperfine coupling of aN=aH ≈15 G.34, 35 As shown in Figure 4, with DMPO and glucose, GOx-Hb MRs produced a typical hydroxyl radical signal at pH=5.0, while no signal was generated at pH=7.4. No obvious hydroxyl radical signal was detected under either pH without glucose. 3.4 Antibacterial Experiments and Inhibition of MRSA Biofilm Formation Bacterial infection is a serious threat to humankind. Drug-resistant bacteria species including MRSA is a result of abuse of antibiotics. It is urgent to adopt more effective methods to combat bacteria. Toxic reactive oxygen species (ROS) including HO˙ have been reported to effectively kill drug-resistant bacteria.36 Inorganic particles displaying peroxidase-like activity have been employed to produce antibacteria HO˙, but usually external H2O2 was needed.37, 38 Glucose in human body supplies energy for bacteria growth in bacterial infection. Meanwhile, bacteria produces a mild acidic environment due to carbonic acid, lactic acid, etc. generated in respiration and fermentation.39 GOx-Hb MRs have been shown to produce HO˙ through a cascade reaction in a mild acidic environment using local glucose. The antibacterial ability against MRSA of this material was further tested, with the hypothesis that GOx-Hb MRs can simultaneously cut off the energy supply to bacteria and kill them by consuming glucose to produce HO˙. MRSA was incubated with GOx-Hb MRs with different concentrations in the presence of glucose, dGOx-Hb MPs served as control. Standard plate spread method was used to count the living bacteria using TSA plates. 14


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As shown in Figure 5, dGOx-Hb MPs exhibited no antibacterial effectiveness. In sharp contrast, with increased GOx-Hb MRs concentration, the colony-forming units of remaining bacteria decreased significantly: 1.2 µg/mL GOx-Hb MRs significantly limited colony formation, and no colony was formed after being treated by 2.4 µg/mL GOx-Hb MRs (Figure S8). This result demonstrated that GOx-Hb MRs have a significant effect on killing MRSA, with the proposed mechanism. Bacteria have the ability to form biofilms on medical device surfaces, protecting bacteria from being eradicated by host immune system and antibiotics.40, 41 Therefore, it is necessary to inhibit biofilm formation, especially for managing drug-resistant bacteria. To investigate the ability of GOx-Hb MRs to inhibit MRSA biofilm formation, MRSA was incubated with GOx-Hb MPs (2.4 µg/mL) at 37 °C for 48 h with TSB medium containing 12.5 mM glucose. After 48 h, the formed MRSA biofilms were washed once by PBS slightly and stained by CV42,

43,

as shown in

Figure 6. The blank group and the dGOx-Hb MPs group both had obvious blue color, indicating the formation of biofilms (Figure 6a, b). Addition of GOx-Hb MRs significantly inhibited MRSA biofilm formation (Figure 6c). Acetic acid solution was utilized to dissolve the attached CV in the biofilms after thorough desiccation. Then the absorbance at 590 nm of the solution was detected by a microplate reader. The quantitative results also confirmed that the GOx-Hb MRs could effectively inhibit the formation of MRSA biofilms (Figure 6d, e). The high antibacterial efficiency may result from the following two aspects: 1) when bacterial infection happens and creates a mild acidic condition, the GOx-Hb MRs can react more effectively and produce 15



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toxic HO˙ to kill bacteria; 2) the cascade reaction can consume glucose to cut the energy supply to bacteria. 4. Conclusion In summary, enzymatic cascade microreactors coupling GOx and Hb (GOx-Hb MRs) were successfully fabricated by co-precipitation of GOx and Hb into MnCO3 template followed by assembly of a multilayer film on the surface, slight crosslinking and final removal of MnCO3. With glucose at physiologically relevant concentrations, the microreactors exhibited higher cascade reaction activity under mild acidic condition than under neutral condition at physiological temperature: GOx-Hb MRs effectively consumed glucose at pH=5.0 and generated significantly more HO˙ compared to same reactions at pH=7.4. Km and Vmax of GOx-Hb MRs were 2.60 mM and 2.94×10-8 Ms-1, respectively, which means the GOx-Hb MRs can perform a relatively high cascade reaction activity in the presence of glucose with normal blood-relevant concentration. The GOx-Hb MRs at a relative low concentration (2.4 µg/mL) effectively limited MRSA growth and inhibited biofilm formation. Therefore, GOx-Hb MRs has a great potential as highly efficient antibacterial agents.

ASSOCIATED CONTENT Supporting Information. EDX results and elemental mappings of MnCO3 and GOx-Hb MRs; The cascade reaction activity of GOx-Hb MRs with different feeding ratios of GOx to Hb during 16


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the fabrication of template; Absolute protein compositions of MRs; Comparison between the activities of GOx-Hb MRs and free enzymes; SEM image of dGOx-Hb MPs and their cascade reaction activity; the dependence of cascade reaction activity on the concentration of GOx-Hb MRs; the steady-state kinetic assays of the GOx-Hb MRs; the photomicrographs of bacterial colony-forming units after co-incubation with different particles. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Email: [email protected] (W. Tong). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest Acknowledgements This study is financially supported by the Natural Science Foundation of China (21374101), the Zhejiang University K. P. Cao’s High Technology Development Foundation, and the Fundamental Research Funds for the Central Universities of 17



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Scheme 1. The construction of microreactors (GOx-Hb MRs) for cascade reaction based on glucose oxidase (GOx) and hemoglobin (Hb). The microreactors can produce hydroxyl radicals (OH˙) using glucose to combat MRSA.

Figure 1. SEM images of MnCO3 with co-precipitated GOx and Hb (a) and GOx-Hb microreactors (MRs) (b). Scale bar=1 µm.

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Figure 2. The pH (a) and temperature (b) dependences of relative cascade reaction activity of GOx-Hb MRs (100 µg/mL) with addition of glucose (5 mM) and TMB (0.75 mM), reaction time=10 min.

Figure 3. Detection of HO˙ generated by GOx-Hb MRs using TA probing method. (a) Reaction of TA and HO˙ results in TA-OH which is fluorescent. Fluorescence spectra of TA buffer solution with addition of 1 mM glucose and GOx-Hb MRs of different concentrations under (b) pH=5.0 and (c) pH=7.4.

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Figure 4. The ESR spectra of GOx-Hb MRs (24 µg/mL) using DMPO (30 mM) as HO˙ trapping agent in the presence or absence of glucose (30 mM) at (a) pH=5.0 and (b) pH=7.4 at ambient temperature.

Figure 5. Quantitative results of standard plate counting assay after MRSA co-incubated with GOx-Hb MRs or dGOx-Hb MPs of different concentrations. The glucose in the culture medium is 12.5 mM.

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Figure 6. (a) Biofilms formed on a glass substrate without addition of any particles, (b) with addition of 2.4 µg/mL dGOx-Hb MPs and (c) with addition of 2.4 µg/mL Gox-Hb MPs were stained with crystal violet (CV). (d) Photomicrographs of CV-stained biofilms dissolved by acetic acid. (e) Absorbance at λ=590 nm of

the

corresponding solution in (d).

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Figure for TOC

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Construction of Microreactors for Cascade Reaction and Their

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