Fabrication of Activity-Reporting Glucose Oxidase Nanocapsules with

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Biological and Medical Applications of Materials and Interfaces

Fabrication of Activity-Reporting Glucose Oxidase Nanocapsules with Oxygen-Independent Fluorescence Variation Dong Chen, Yu Huang, Huangyong Jiang, Wumaier Yasen, Dongbo Guo, Yue Su, Bai Xue, Xin Jin, and Xinyuan Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06348 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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ACS Applied Materials & Interfaces

Fabrication of Activity-Reporting Glucose Oxidase Nanocapsules with Oxygen-Independent Fluorescence Variation Dong Chen, Yu Huang, Huangyong Jiang, Wumaier Yasen, Dongbo Guo, Yue Su, Bai Xue, Xin Jin,* and Xinyuan Zhu School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China KEYWORDS: glucose oxidase, flavin derivatives, mediated electron transfer, fluorescence variation, enzymatic activity reporting

ABSTRACT: Glucose oxidase (GOx) has seen large-scale technologically applications and the determinations of its activity that is directly related to the enzymatic functions are extremely important. However, conventional methods to analyze the enzymatic activity involving high oxygen dependency and indirect redox reactions are usually tedious and restricted in complicated environments. For analyzing enzymatic activity by direct detection of the electron signals from the active centers, mediators are often used for facilitating the electron transfer. Differing from common methods of preparing electron mediators-contained GOx composites, a strategy aiming at remolding of the enzyme itself has been proposed in this work. Cofactor-like molecule DAA-

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Flavin derived from riboflavin is synthesized and incorporated as cross linker into the polyacrylamide (PAAm) network around GOx surface by in situ polymerization to obtain enzyme nanocapsules termed as GOx@Fla-c-PAAm. The peripheral polymer shell confines the orientation of GOx and prevents it from denaturing, while incorporated DAA-Flavin can replace the oxygen as alternative electron acceptor to interact with the active centers of GOx in presence of the substrate, thus gives the nanocapsules oxygen-independent characteristics. The introduced unlimited cofactor-like molecules endow the nanocapsules redox-related fluorescence and the intensity variation is closely correlated with the enzymatic activity. There is a high goodness of fitting (R2 ~ 0.990) between the slope of linear fluorescence-time plots and enzymatic activity, thereby, makes the nanocapsules a reliable activity-reporting enzymatic nanosystem with oxygen-independent fluorescence variation for further extended potential application in biofuel cells and biosensors.

INTRODUCTION As an oxidoreductase of fungal origin (e.g. Aspergillus niger), GOx has seen large-scale technological applications in biosensors1-3 and biofuel cells4-6 since 1950’s due to its high catalytic activity towards glucose and stability under physiological conditions.7-8 Accordingly, kinds of enzyme composites have been fabricated to meet diverse requirements.9-17 Due to the performance of GOx-based materials is directly related to the functions of the enzyme, facile analysis of enzymatic activity has become extremely important especially under some harsh conditions, which may lead to easy denaturation of the enzyme. However, the determination of enzymatic activity of GOx is generally achieved via electrochemical or colorimetric detection of enzymatically-produced hydrogen peroxide (H2O2) in the presence of oxygen. This is due to that


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oxygen plays an important role as electron acceptor from the reductive cofactor FADH2 to form H2O2 (reactions (1) and (2)).18 GOx − FAD + glucose → GOx − FADH + gluconic acid (1) GOx − FADH + O → GOx − FAD + H O (2) GOx − FADH + M(ox) → GOx − FAD + M(re) (3) Consequently, high oxygen dependency of the redox process limits the enzyme’s applications under the anaerobic or oxygen level fluctuant conditions.19 In addition, the activity analysis of GOx is also highly correlated with the oxygen and usually relies on the presence of a second enzyme horseradish peroxidase (HRP) that catalyze produced H2O2 releasing detectable electrons or oxidizing the indicators to colored, resulting in a tedious determination of the enzymatic activity and difficulties to be operated in the complex environments containing various interference factors.20-22 Since the by-product H2O2 is formed via the oxidization of reduced cofactor FADH2,23 enzymatic activity of GOx is expected to be analyzed by straight detection of the electrons trapped in the active centers.24-25 However, given that cofactor FAD is deeply buried within the prosthetic shells, the redox active centers of GOx are usually shielded from external matrix by the glycoprotein coat that prevents the efficient electron tunneling,26-27 thus makes enzymatic activity can hardly be determined by receiving the electron signals directly. To minimize the oxygen dependency and facilitate electron transfer for the straightforward activity determination, currently, some redox active molecules (e.g. ferrocenemethanol) are used to serve the mediated electron transfer (MET), as illustrated in reaction (3).28-30 Nevertheless, current researches are mostly focused on the modification of the matrix with separated decorations with GOx and the


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mediators via blending or connecting methods but few works are related with remolding of the enzyme itself. By consideration of the special features of GOx, herein, we have synthesized a riboflavinderived diene molecule (DAA-Flavin) and incorporated it into polyacrylamide (PAAm) network around GOx surface formed via in situ polymerization method to prepare fluorescent GOx nanocapsules with oxygen-independent characteristics (GOx@Fla-c-PAAm). As illustrated in Scheme 1, DAA-Flavin not only acts as an electron-mediator but also a fluorescent probe in the nanocapsules since the isoalloxazine group is preserved in the molecular structure. Due to the similarity and high proximity with the active centers, the surface-fixed DAA-Flavin is capable of replacing oxygen to interact with cofactor FAD in the presence of substrate β-D-glucose, thus makes the nanocapsules oxygen-independent and can still work under anaerobic conditions. Despite of weak autofluorescence of the native GOx, the apparent difference of fluorescence intensity between oxidized and reductive DAA-Flavin endows the nanocapsules distinct fluorescent characteristics under external irradiation in the presence of substrate. According to the interactions between DAA-Flavin and FAD mainly proceeding via electron transfer, the rate of fluorescence intensity change should be closely related to the enzymatic activity, thereby, makes it a reliable activity-reporting enzyme nanosystem with highly retained activity by simply monitoring the fluorescence variation. The potential applications of this nanocapsules can be extended to the modification of anode of high efficient biofuel cells, durable biosensor for glucose detection under severe conditions and so on.


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Scheme 1. The different catalyzed mechanism of GOx and GOx@Fla-c-PAAm nanocapsules.

1. EXPERIMENTAL SECTION 2.1. Reagents and Materials. Riboflavin (RF, >98%), acrylamide (AAm, 99%), diallylamine (DAA, 98%), N, N, N′, N′-tetramethylethylene diamine (TEMED, 99%), sodium glycolate (C2H3NaO3, 98%), sodium cyanoborohydride (NaBH3CN, ≥98%), o-dianisidine dihydrochloride (o-DADH, ≥98.5%), β-D-(+)-glucose (D-glucose, AR) and peroxidase from horseradish (HRP, >160 U/mg) were purchased from Aladdin Chemical Reagent Co., Ltd (China). Glucose oxidase (GOx, ~100U/mg, molecular weight: 160 kDa) from Aspergillus niger, N, N′-methylene bis-acrylamide (BIS, 98%), ammonium persulfate (APs, ≥98%) and periodic acid (PA, for electrophoresis, ≥99%) were purchased from Sigma-Aldrich Chemical Reagent Co., Ltd. Agarose and bicinchoninic acid (BCA) protein assay knit were purchased from Beyotime Biotechnology. All other reagents and solvents were provided by domestic suppliers. All of the chemical reagents were used as received without further purification. 2.2. Measurements. Nuclear magnetic resonance (NMR) analyses were registered on a Varian Mercury Plus 400MHz spectrometer with deuterated DMSO (d6-DMSO) as solvent at 298 K.


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Fourier transform infrared spectrometer (FTIR) analyses were recorded on a Perkin Elmer Paragon 1000 spectrophotometer and measured as KBr pellets within the range of 4000～400 cm-1. Ultraviolet-visible (UV-Vis) absorption spectra were carried out on a Perkin Elmer Lambda 20 UV-Vis spectrometer within the range of 200~600 nm. Fluorescence emission spectra (FL) were performed on a Perkin Elmer LS 50B fluorescence spectrometer within the range of 400~700 nm. The samples of UV-Vis and FL were dissolved in water and placed into a 1 cm quartz cuvette. Dynamic light scattering (DLS) measurements were recorded on a Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at λ=633 nm in aqueous solution. The samples were measured at room temperature and at a scattering angle of 173° Thermogravimetric analysis (TGA) was performed on a Perkin-Elmer Pyris analyzer under nitrogen atmosphere from 50 °C to 700 °C. Transmission electron microscopy (TEM) studies were carried out on a JEOL 2010 instrument at 120 kV. The samples were dispersed in aqueous solution and dropped onto carbon-coated copper grids whereafter 1% (w/w) phosphotungstic acid aqueous solution was dropped to stain the sample. Circular dichroism (CD) spectroscopy was performed on a Jasco J-815 spectrometer in 10 mM PBS at room temperature, and the cell length was 1 mm. The surface properties of the samples were analyzed by X-ray photoelectron spectroscopy (XPS, AXIS Ultra DLD, Shimadzu). All the binding energy values were calibrated by using C1S = 284.6 eV as a reference. Cyclic voltammetry (CV) spectrum was determined by CHI 660C electrochemical work-station (Shanghai Chenhua Instrument, CHI, China) with a three-electrodes system (WE: 3mm glassy carbon electrode, CE: platinum wire electrode, RE: Ag/AgCl electrode). The samples were frozen by liquid nitrogen rapidly and lyophilized by a freeze dryer (Martin Christ, α-1-4) at -55 °C before measurements.


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2.3. Synthesis of 2′-oxoethyl flavin. The intermediate 2′-oxoethyl flavin was synthesized using the method reported by Svoboda with slight modification.31 Briefly, riboflavin (5.60 g, 14.9 mmoL, 1 eq.) was suspended in 140 mL 1: 35 (v/v) dilute sulfuric acid cooled by ice bath. 90 mL aqueous solution of periodic acid (12.50 g, 54.8 mmoL, 3.7 eq.) was added dropwise into the suspension using a dropping funnel to control the rate as constant. After addition, ice bath was withdrawn to make the reaction solution warmed back to room temperature naturally. Activated carbon (2 g) was added to the mixture and stirred gently for another 30 min. The solids were removed by filtration while the filtrate was collected and pH was adjusted to 3.9 with 40 % conc. NaOH aqueous solution. The obtained yellowish precipitates were separated by centrifugation and washed with ice-cold water and dried in vacuum to afford crude product. For further purification, crude product was suspended in toluene, the mixture was heated to reflux and small portions of the distillate were removed using Dean-Stark apparatus. The residual solvent was removed by rotary evaporation with subsequent dryness in vacuum to afford earthy yellow powder with a yield of 49 %. 1H NMR (d6-DMSO, ppm): δ 2.37 (s, 3H, 1-CH3), 2.46 (s, 3H, 2-CH3), 5.61 (s, 2H, 3-CH2), 7.67 (s, 1H, 4-ArH), 7.90 (s, 1H, 5-ArH), 9.71 (s, 1H, 6-CHO), 11.31 (br, 1H, 7-NH).

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C NMR (d6-DMSO, ppm): δ 195.7, 159.0, 154.4, 150.5, 146.0, 136.7,

135.2, 132.6, 130.3, 115.3, 86.1, 53.1, 20.4, 18.0. FT-IR (KBr cm-1): 3387 (br, νO-H), 3152, 3070 (aromatic νC-H), 2926, 2826 (νO=C-H), 2320, 1694, 1649 (νC=O), 1555, 1522 (νC-N), 1432 (aromatic νC=C), 1369, 1341, 1243 (CH3). Melting point: 248.2 °C. 2.4. Synthesis of 2′-diallyamino-ethyl flavin (DAA-Flavin). The fluorescent cross linker 2′diallyamino-ethyl flavin (DAA-Flavin) was synthesized by reductive amination starting from intermediate 2′-oxoethyl flavin. In brief, 2′-oxoethyl flavin (400 mg) was placed into 60 mL methanol to form good dispersion and heated up to 50 °C. Diallyamine (0.9 mL) was then added


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in dropwise under constant stirring and allowed to react for 1 h. To the end, a brown-color Schiff base semi-synthetic product was obtained. The mixture was cooled down to room temperature naturally and the pH was adjusted to ~5.5 with glacial acetic acid. After that, NaBH3CN (220 mg, 3.5 mmoL) was added into the mixture under stirring to react for another 16 h at room temperature. pH value of the final obtained golden solution was adjusted back to neutral state using saturated NaHCO3 aqueous solution with a subsequent filtration to remove insoluble components. The filtrate was evaporated to dryness and the residue was re-suspended in a small quantity of ice-cold ultrapure water followed by re-filtration. The filter was washed with ice-cold water several times and dried in vacuum to afford orange-red powder with a yield of 50 %. 1H NMR (d6-DMSO, ppm): δ 2.38 (s, 3H, 1-CH3), 2.46 (s, 3H, 2-CH3), 2.77 (tri, 2H, 3-CH2), 3.14 (tetra, 4H, 4-CH2), 4.68 (tri, 2H, 5-CH2), 5.03 (tetra, 2H, 6-CH=CH2), 5.13 (tetra, 2H, 7CH=CH2), 5.61 (tetra, 4H, 8-CH=CH2), 7.70 (s, 1H, 9-ArH), 7.87 (s, 1H, 10-ArH), 11.31 (br, 1H, 7-NH).

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C NMR (d6-DMSO, ppm): δ 159.2, 155.0, 149.7, 145.6, 136.2, 135.2, 134.9, 132.9,

116.7, 115.4, 86.2, 55.3, 48.3, 41.5, 20.0, 18.0. FT-IR (KBr cm-1): 3417 (br, νO-H), 3136, 3068 (aromatic νC-H), 3023 (νC=C-H), 2802 (νC-C-H), 2306, 1690, 1638 (νC=O), 1604 (alkene νC=C), 1551, 1518 (νC-N), 1426 (aromatic νC=C), 1367, 1332, 1243 (CH3). Melting point: 137.9 °C. 2.5. Fabrication of Enzyme Nanocapsules. Enzyme nanocapsules (GOx@Fla-c-PAAm, GOx@cPAAm) were fabricated by surface in situ polymerization method. Native proteins were first conjugated with N-acryloxysuccinimide (NAS) to attach acryloyl groups onto their surfaces. A typical procedure is described as followed. GOx (10 mg·mL-1) was firstly dialyzed against phosphate buffer (20 mM, pH 8.0) to remove any ammonium sulfate that usually exists in the stock protein powder. After the dialysis, protein solution was diluted to 5 mg·mL-1 with phosphate buffer (20 mM, pH 8.0), followed by adding NAS solution (10% in DMSO, w/v) to


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perform the conjugation with a molar ratio of NAS: GOx =20:1. The conjugation is achieved by keeping the reaction at 4 °C for 1 h. The solution was then thoroughly dialyzed against sodium acetate buffer (20 mM, pH 5.1) with a dialysis tubing membrane (MW 10 kDa) to remove any unreacted NAS. Acryloylated enzyme solutions were store at 4 °C for further uses. The obtained acryloylated proteins were encapsulated with PAAm by in situ polymerization method. AAm was firstly prepared as stock solution (100 mg·mL-1) in ultra-pure water while cross-linker DAAflavin was dissolved in a small quantity of anhydrous DMSO, respectively. Then AAm and DAA-flavin were added orderly into the dilute solution of acryloylated GOx (1 mg·mL-1) being encapsulated with a specific molar ratio (listed in Table 1). Polymerization was initiated by the addition of APS and TEMED and kept at 4 °C under constant stirring for 2 h. After the polymerization, the samples were dialyzed against sodium acetate buffer (20 mM, pH 5.1) to remove unreacted monomers and by-products. Finally, the solution was lyophilized by a freeze dryer to afford golden fiberized powder. GOx@cPAAm nanocapsules were prepared under the same conditions. Table 1. Synthesis parameters of different enzyme nanocapsules Sample

GOx

AAm

DAA-Flavin

BIS

APS

TEMED

GOx@Fla-c-PAAm

1

8000

200

200

400

800

GOx@cPAAm

1

8000

\

400

400

800

*All numbers indicate the molar ratios. 2.6. Protein Quantitative Assay. The protein content in the form of nanocapsules was determined by bicinchoninic acid (BCA) colorimetric method. In a typical procedure, a tertrate buffer (pH 11.25) containing 25 mM BCA, 3.2 nM CuSO4, and appropriately diluted protein/nanocapsules were incubated at 60 °C for 1 h. After that the solution was cooled down to


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room temperature, absorbance reading at 562 nm was determined with a UV-Vis spectrometer. GOx solutions with known concentration were used as standards. 2.7. Enzymatic Activity and Stability Assay. The activity of native GOx and GOx nanocapsules were determined by monitoring absorbance change at 500 nm for 1 min in the mixture of protein samples and assay solution (50mM sodium acetate buffer (pH 5.1) with 95.5 mM β-D-glucose, 0.5 mM o-DADH and 0.01 mg·mL-1 HRP) with the same content of proteins. One unit is defined as oxidation of 1.0 µmole of β-D-glucose and D-gluconolactone per minute. For thermo-stability assay, native GOx and GOx nanocapsules were both incubated at the same concentration at 50 °C and 60 °C in sodium acetate buffer (20 mM, pH 5.1) for 10, 30 and 60 min. Samples were taken out at varying time intervals during the incubation and placed immediately on ice bath with following activity determination. For pH-dependent stability assay, enzyme activities were recorded in varying buffer solutions with pH ranging from 3.0~8.0 that is the activated region of GOx. 2.8. Enzyme Kinetics Assay. The kinetic parameters of GOx and GOx nanocapsules were obtained by measuring initial oxidative rate of β-D-glucose with different concentrations of substrate while enzyme content was set as constant. The kinetic parameters were determined by fitting to Lineweaver-Burk reciprocal plot. All assays were performed in triplicate and the average and standard deviation were recorded. 2.9. Cyclic Voltammetry Measurements. Cyclic voltammetry measurements were performed using the method reported by previous publication.44 It was using a BAS Electrochemical Analyzer Model 660A at room temperature in PBS (0.1 M, pH 7.4) in a standard threecomponent sealable cell under N2 atmosphere equipped with a 3 mm-O.D. glassy carbon disk working electrode, platinum wire counter electrode as Ag/AgCl reference electrode which was in


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saturated aqueous KCl solution. All potential values were given versus Ag/AgCl electrode. All sample solutions were deoxygenated by N2 bubbling for 30 min just before the experiments, covered with N2 atmosphere and maintained above the liquid during the course of measurement at room temperature. 2.10. Fluorescence Measurements. FL spectrum was monitored by fluorescence spectrophotometer. The emission spectrum was recorded under a fixed excitation wavelength of 450 nm at a scan rate of 20 nm·s-1. GOx@Fla-c-PAAm powder was dissolved into phosphate buffer (20 mM. pH 7.4) to prepare 1 mg·mL-1 sample solution. For the study of the effect of substrate oxidation on GOx@Fla-c-PAAm, 100 µL sample solution was mixed with 2.9 mL phosphate buffer (20 mM, pH 7.4) with β-D-glucose (55.5 mM) immediately by inversion and then was placed into spectrophotometer to record the fluorescence spectrum within the range of 475~600 nm. For the investigation of systematic relative fluorescence intensity (λex=450 nm, λem=520 nm) during the redox process, 200 µL of 1 mg·mL-1 (protein concentration) GOx@Flac-PAAm nanocapsules was placed into 3792 Costar 96 well plate and the fluorescence was recorded by BioTek Synergy H4 plate-reader with background correction. To obtain re-oxidized GOx@Fla-c-PAAm, the solution of nanocapsules was dialyzed against phosphate buffer solution (20 mM pH 7.4) after the fluorescence remained unchanged. Afterwards, H2O2 (1.0 %, wt%) was added into the nanocapsules solution and the pH was adjusted to about 5.0 using 2.4 M hydrochloric acid. For the activity-fluorescence correlation experiment, 10 µL 1 mg·mL-1 GOx@Fla-c-PAAm nanocapsules (protein concentration) in phosphate buffer solution (20 mM pH 7.4) incubated at 60 °C for 0 min, 10 min, 30 min and 60 min were mixed with 100 µL 55.5 mM β-D-glucose phosphate buffer solution (20 mM pH 7.4) immediately by vortex mix with


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followed determination of fluorescence change at 520 nm in 8 min by BioTek Synergy H4 platereader. The slope of the linear region of each sample was recorded. 2.11. Statistical Analysis. Statistical analyses were performed using one-way ANOVA in SPSS Statistics; p < 0.05 was considered significant. Data were expressed as mean ± SD. Scheme 2. The design of GOx@Fla-c-PAAm nanocapsules. a) Synthetic routes of DAA-Flavin and its redox mechanism; b) Fabrication of GOx@Fla-c-PAAm nanocapsules.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of DAA-Flavin. The intermediate 2′-oxoethyl flavin was synthesized by the oxidation of riboflavin polyhydric side chain on the 2′-C site to aldehyde group using periodic acid and sulfuric acid as oxidants. As shown in Scheme 2a, after purification, the aldehyde group on 2′-C site of 2′-oxoethyl flavin was then reacted with the secondary amine group of DAA to form Schiff base with followed reductive amination using mild reductant NaBH3CN to obtain the final product 2′-diallyamino-ethyl flavin (DAA-Flavin). It is well known that the functional unit of riboflavin derivatives including FAD and FMN are


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isoalloxazine-type condensed rings. And during the whole reactions, there are no changes taking place on its ring structure but introducing diene monomer onto the 2′-C site to make it polymerizable so that this FAD-like artificial cofactor can be incorporated into the polymer network around GOx surface and take part in the enzyme’s physiological processes. The chemical structures of 2′-oxoethyl flavin were confirmed by 1H NMR measurement (Figure 1a), which was highly consistent with the results of existing publications.31-32 Compared with the spectrum of 2′-oxoethyl flavin, the signal at 9.71 ppm assigned to the aldehyde disappeared in the spectrum of DAA-Flavin. Moreover, a set of multi-peaks of diallyamine in the range of 3.0 to 6.0 ppm are generated, indicating that the reductive amination was completed and diallyamine was successfully conjugated onto the 2′-C site of flavin. The results of

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C NMR

spectrum (Figure S1) were also consistent with the object molecular structure. The FTIR spectra of riboflavin, 2′-oxoethyl flavin and DAA-Flavin are shown in Figure 1b. It was found that the peak observed at 3380 cm-1 (ν, O-H) in riboflavin apparently weakened in 2′-oxoethyl flavin, instead double peaks at 2926 cm-1 and 2826 cm-1 (ν, O=C-H) was generated. The peaks assigned to aldehyde group disappeared while the peak located at 3023 cm-1 (ν, =C-H) strengthened significantly after that 2′-oxoethyl flavin transformed into DAA-Flavin, indicating the success of reductive amination and the total conversion of aldehyde to tertiary-amine. In addition, DSC measurements were also performed to determine the melting points of these two compounds (Figure S2).


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Figure 1. 1H NMR spectrum of a) 2′-oxoethyl flavin and DAA-Flavin in d6-DMSO; b) FTIR spectrum of riboflavin, 2′-oxoethyl flavin and DAA-Flavin. The fluorescence of riboflavin originates from the isoalloxazine chromophore. Accordingly, UV-Vis absorption and fluorescence spectrum were utilized to investigate if there is any structure change of isoalloxazine ring during the synthesis. It was found that there were no obvious peak shifts in UV-Vis absorption (Figure S3a) between reactant riboflavin, intermediate 2′-oxoethyl flavin and product DAA-Flavin, suggesting the reservation of isoalloxazine structure. The functional group linked on 2′-C position has a pivotal effect on the fluorescent characteristics of isoalloxazine by electronic effect and stacking interactions. It was further revealed by the fluorescence spectrum (Figure S3b) that the fluorescence intensity of DAAFlavin decreased distinctly compared with riboflavin and 2′-oxoethyl flavin under the same condition (0.05 M methanol solution). It may be attributed to the decreased electron-donating effect of substituted diallylamine part in DAA-Flavin. 3.2. Fabrication and Characterization of GOx@Fla-c-PAAm. The in situ polymerization method was employed to prepare enzyme nanocapsules. DAA-Flavin was used as cross linker to facilitate the formation of PAAm network around GOx surface due to its diene-containing structure (Scheme 2b). A common used cross linker BIS was used to prepare nanocapsules


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GOx@cPAAm as the control group. The formation of GOx@Fla-c-PAAm was confirmed by the size distribution monitored by DLS (Figure 2a). The native GOx exhibits a size distribution centered at 8 nm, which is consistent with its molecular dimension. For comparison, GOx@Flac-PAAm shows a size distribution centered at 30 nm, indicating the successful formation of the polymer network around the enzyme surface. Further confirmation of the encapsulation of GOx was also evidenced by agarose gel electrophoresis. As shown in Figure 2b, the negative-charged native GOx migrated to anode while GOx@Fla-c-PAAm nanocapsules can hardly move out of the sample groove due to the charge shielding by the outer PAAm shell. For TEM images (Figure 2c-2d), compared with native GOx, increased sizes of GOx@Fla-c-PAAm are observed. Moreover, it is also found that individual spherical nanocapsules has a clear core-shell structure (Figure 2e). The sizes of GOx and GOx@Fla-c-PAAm obtained from TEM images are in highly accordance with the DLS results. Enzyme composite with core-shell nanostructure was fabricated after the encapsulation of GOx by PAAm polymer network and it was analyzed by TGA measurement. As shown in Figure 2f, by comparison, onset temperature at 300 ℃ assigned to the thermal decomposition of PAAm was observed in sample GOx@Fla-c-PAAm. The mass fraction of PAAm composition of the complex reaches up to ~50.0% (w/w). According to the close relationship between the conformation and activity of enzyme, we utilized CD spectroscopy to quantitatively calculate the secondary conformations contents of the enzyme samples in 10 mM PBS by the CDPro software with selcon3 method and investigate if there was any conformation change of the enzyme during the preparation of nanocapsules (Figure 2g and Table S-1). Referred to the publications,33 it is inferred that the pair of plus-minus peaks respectively centered at 212 nm and 238 nm are assigned to the α-helix secondary structure while the peak located at 272 nm is assigned to the β-


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sheets secondary structure of native GOx. Additionally, the results from calculation showed that there is a decreasing amount of α-helix (9.3% decreased to 6.4%) and β-sheets (45.7% decreased to 39.1%) in the nanocapsules’ secondary structure while a relevant increase of components of turn and unordered structures (44.1% increased to 54.2%). According to the results, the change of secondary structure cannot be considered as significant between GOx and GOx@Fla-c-PAAm, suggesting retained intact structure of the enzyme after encapsulation. That might be attributed to the interactions between the enzyme and polymer shell is mainly weak Van der Waals force, exclusive of a small quantity of vinyl-decorated lysine residues acting as anchored points.10, 34 XPS are showed in Figure 2h, native GOx and GOx@Fla-c-PAAm exhibit different features and chemical microenvironment of active sites on the surface. After encapsulation, relative intensity of N1s peak (Figure S4) increased significantly accompanying with a slight attenuation of C1s peak, indicating an enrichment of AAm upon the enzyme surface. Additionally, for the high resolution carbon spectra (Figure 2i), it was clearly observed that the peak located at 286.5 eV assigned to C-O bond of amine group disappeared and a new peak centered at 288.5 eV assigned to the N-C=O bond of acylamino group appeared after encapsulation, demonstrating that PAAm shell had indeed formed around the enzyme surface. Combining the results, it is reasonable to confirm that the GOx nanocapsules are successfully prepared.


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Figure 2. The formation of GOx@ Fla-c-PAAm Fla-c-PAAm nanocapsules. a) Size distribution of GOx and GOx@Fla-c-PAAm; b) Agarose gel electrophoresis fluorescent images of FITC labeled 1. GOx and 2. GOx@Fla-c-PAAm; TEM images of c) GOx, d) GOx@Fla-c-PAAm and e) enlarged image of GOx@Fla-c-PAAm with negative staining of the outer shell (scale bar represents 100 nm in c and d, 20 nm in e); f) Thermogravimetry and differential curve of native GOx and GOx@Fla-c-PAAm; g) CD spectrum of GOx and GOx@Fla-c-PAAm; h) XPS spectrum and i) high resolution carbon spectrum of native GOx and GOx@Fla-c-PAAm (C1: CC, 285.0 eV; C2: C-O, 286.5 eV; C3: N-C=O, 288.5 eV). In order to optimize the ratio of GOx and DAA-Flavin, a series of mixtures of GOx and DAAFlavin were obtained by rapid mixing with different molar ratio, while native GOx was set as the sample control without the addition of DAA-Flavin. As shown in Figure 3a, the calculated activity of all kinds of mixtures are higher than GOx with a maximum activity ~110% compared


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with native enzyme at the molar ratio GOx/DAA-Flavin 1: 200, suggesting high affinity of DAA-Flavin and GOx and the occurrence of its interaction with cofactor FAD. The fluorescence intensity of GOx@Fla-c-PAAm reached to maximum when the molar ratio GOx/DAA-Flavin was 1: 200 followed by a dramatically decrease with further increasing the proportion of DAAFlavin (Figure 3b). It is indicative of that when the enzyme surface is covered with DAA-Flavin reaching to the saturation, further introduced molecules may tend to pile up with the pre-existing congeners to form a com-pact structure by the π-π stacking interaction, resulting in a decrease of the fluorescence by intermolecular electron transfer. As illustrated in Figure 3c, the results of enzymatic activity and fluorescence were highly consistent. Thereby, the final molar ratio GOx/DAA-Flavin was kept at 1: 200 and used in subsequent experiments.

Figure 3. a) Relative activity profiles and b) Fluorescence spectrum of the mixtures of GOx and DAA-Flavin with different GOx/DAA-Flavin molar ratio; c) Respective graphical relationship of enzymatic activity and FL intensity against GOx/DAA-Flavin molar ratio; Relative activity of GOx, GOx@cPAAm and GOx@Fla-c-PAAm d) after incubation at 50 °C and 60 °C for different time and e) determined at varying pH.


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The catalytic performances of GOx composites usually decreased significantly due to the relatively strong enzyme-supporting interaction, mass-transfer problems and harsh con-ditions.3536

As reported by previous researches,37-38 peripheral polymer shell can effectively stabilize the

interior enzymes enabling them robust features with high stability for various applications. In this work, the nanocapsules GOx@Fla-c-PAAm were placed under different conditions (varying temperature and pH) and showed similar significantly improved stability against the denaturation factors in comparison to native GOx. The results (Figure 3d) demonstrated the thermo-stability of native GOx, GOx@cPAAm and GOx@Fla-c-PAAm nanocapsules at 50 °C and 60 °C (denatured temperature of GOx) incubated for varying time. It was found that all the samples showed relative high stability at 50 °C which is an appropriate temperature of GOx (20~60 °C). However, native GOx denatured rapidly at 60 °C that the relative activity decreased to 24.4% and 18.5% after incubation for 10 minutes and 1 h, respectively. In contrast, both GOx nanocapsules exhibited a much slower decrease rate of activity under the same condition. Especially, it is noteworthy that GOx@Fla-c-PAAm shows significant enhanced thermo-stability. The enzymatic activity preserved 91.9% and 67.6% after incubation at 60 °C for 10min and 1 h, respectively, indicating that the multiple covalent attachments between GOx and PAAm shell efficiently prevented the enzyme conformation change upon heating. The stability of enzyme in solution with varying pH was also investigated. As shown in Figure 3e, differing from the observable activity change of native GOx, both GOx nanocapsules show similar stability at pH ranging from 4.0 to 9.0, representing more insensitivity to varying pH that the activity of GOx@cPAAm and GOx@Fla-c-PAAm preserved above 80.0% (activity at pH 6.0 refer to 100%) within the test range. It could be demonstrated by the fixation of GOx core by covalently connecting to the polymer shell and the buffering capability of PAAm’s amide group.


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3.3. Enzyme Kinetics of GOx@Fla-c-PAAm. In enzymatic activity assay, the absorbancetime curves of two kinds of GOx nanocapsules were plotted under the same conditions to investigate the functions of DAA-Flavin. As shown in Figure S5, both nanocapsules have a similar linear range with close slope value at the beginning. The slope declined gradually with time increasing due to the decreased content of dissolved oxygen. Nevertheless, the slope of GOx@Fla-c-PAAm has a much slower downtrend than GOx@cPAAm, demonstrating the function of DAA-Flavin as a substitute of oxygen. To be a comparison, long-term stability of the enzyme nanocapsules stocking in solution at 4 °C after 7 days was also investigated. It was found that the slope of GOx@cPAAm within the linear region declined obviously compared with the original sample. Whereas, there was still 93.5% of enzymatic activity of GOx@Fla-c-PAAm preserved after 7 days. Moreover, with time increasing, GOx@Fla-c-PAAm nanocapsules exhibited more activity reservation, further indicating that the introduced DAA-Flavin has the ability to improve the enzymatic activity and stability by the interaction with the cofactor FAD.

Figure 4. a) Lineweaver-Burk plots of GOx and GOx@Fla-c-PAAm. b) Km and kcat of 1. GOx and 2. GOx@Fla-c-PAAm calculated from Lineweaver-Burk plots. Data are presented as the average ± standard deviation (n=3) (statistical significance level is *p

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