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Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX-XXX

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High Activity and Convenient Ratio Control: DNA-Directed Coimmobilization of Multiple Enzymes on Multifunctionalized Magnetic Nanoparticles Ye Yang, Ruiqi Zhang, Bingnan Zhou, Jiayi Song, Ping Su,* and Yi Yang* Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, College of Science, Beijing University of Chemical Technology, Beijing 100029, P. R. China S Supporting Information *

ABSTRACT: The development of new methods for fabricating artificial multienzyme systems has attracted much interest because of the potential applications and the urgent need for multienzyme catalysts. Controlling the enzyme ratio is critical for improving the cooperative enzymatic activity in multienzyme systems. Herein, we introduce a versatile strategy for fabricating a multienzyme system by coimmobilizing horseradish peroxidase (HRP) and glucose oxidase (GOx) on magnetic nanoparticles multifunctionalized with dopamine derivatives through DNA-directed immobilization. This multienzyme system exhibited precise enzyme ratio control, high catalytic efficiency, magnetic retrievability, and enhanced stability. The enzyme ratio was conveniently adjusted, as required, by regulating the quantity of functional groups on the multifunctionalized nanoparticles. The optimal mole ratio of GOx/HRP was 2:1. The Michaelis constant Km and specificity constant (kcat/Km, where kcat is the catalytic rate constant) of the multienzyme system were 1.41 mM and 5.02 s−1 mM−1, respectively, which were approximately twice the corresponding values of free GOx&HRP. The increased bioactivity of the multienzyme system was ascribed to the colocalization of the involved enzymes and the promotion of DNA-directed immobilization. Given the wide variety of possible enzyme associations and the high efficiency of this strategy, we believe that this work provides a new route for the fabrication of artificial multienzyme systems and can be extended for a wide range of applications in diagnosis, biomedical devices, and biotechnology. KEYWORDS: multienzyme system, ratio control, enzyme coimmobilization, multifunctionalized magnetic nanoparticles, DNA-directed immobilization

1. INTRODUCTION Artificial multienzyme systems that catalyze a variety of multistep cascade or coupling reactions through a combination of enzymes have been used for the development of numerous applications in biodiagnostics,1,2 bioanalysis,3−5 and biosynthesis.6 To establish a multienzyme system that contains two or more enzymes working together in a cascade or coupling reaction, an optimized stoichiometric ratio of the enzymes is needed to ensure the best overall activity.7,8 Coimmobilization is a relatively straightforward protocol that also allows convenient recovery and promoted stability of immobilized enzyme.9,10 However, it is difficult to control the ratio of the enzymes used during the coimmobilization process11−13 because it is difficult to distinguish one enzyme from others that have a similar structure. Other methods can be used to control the coimmobilization process of enzymes, but these are elaborate and require much more time and effort to design the multienzyme systems.14−16 Mazur and co-workers17 reported a versatile one-pot method for fabricating multifunctionalized magnetic nanoparticles (MPs) using dopamine (DA) derivatives. The multiple functional groups allowed postfunctionaliza© XXXX American Chemical Society

tion of nanoparticles with different molecules. This encouraged us to develop a method for fabricating a multienzyme system that allows control of the stoichiometric ratio of the enzymes. This ratio should be adjustable, as required, by regulating the proportion of the different functional groups on the multifunctionalized nanoparticles. However, the direct anchoring of enzymes on nanoparticles to control the enzyme ratio is still difficult if the enzymes have similar structural characteristics. The direct anchoring of enzymes also reduces the overall activity of the multienzyme systems owing to partial denaturation and covering of the active sites of the enzymes.10,18−20 DNA-directed immobilization (DDI) is a simple and versatile chemical technique for specific immobilization of biological molecules based on the specific Watson−Crick base pairing (A−T, G−C) of DNA molecules.21 The selectivity and specificity of DNA hybridization are beneficial for improving Received: June 14, 2017 Accepted: October 4, 2017

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DOI: 10.1021/acsami.7b08553 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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115.6, 115.3 (CH, Ar); 134.1 (CHCH); 40.8 (CH2−N); 37.6 (CH2−NH); 36.4 (CH2−CO−NH); 34.9 (CH2−Ar); 28.2 (N− CH2−CH2); 26.1 (CH2−CH2−CH2); 25.0 (CH2−CH2−CO−NH). 2.4. Synthesis of Dopamine-Functionalized MPs. 2.4.1. Functionalization with One Dopamine Derivative. Briefly, 25 mg of the prepared MPs was dispersed in anhydrous methanol (10 mL) along with dopamine (DA, 5 mg) or a maleimide-terminated dopamine derivative (MA, 9 mg), and the suspension was sonicated for 60 min at room temperature. To remove the unreacted chemicals, the resulting functionalized MPs were washed/centrifuged three times with water and acetone and then stored in water at 4 °C. 2.4.2. Simultaneous Functionalization with Two Dopamine Derivatives. The MPs functionalized simultaneously with DA and MA (DM@MP) were synthesized according to the following procedure. The MPs (25 mg) were dispersed in an anhydrous MeOH solution containing DA (5.3 mM) and MA (5.3 mM), and the dispersion was sonicated for 60 min at 25 °C. The resulting DM@MP were washed three times with water and then stored in water at 4 °C. 2.5. Conjugation of DM@MP with Probe DNA. Two different probe DNA strands were anchored on multifunctionalized DM@MP through a condensation reaction and Michael addition. First, 5′carboxylic single-stranded DNA probe (P1) was connected to the surface of DM@MP through the condensation reaction of the NH2 groups of DA with the COOH groups of P1. To activate the carboxyl, 0.5 optical density (OD) 5′-carboxylic single-stranded DNA probe (P1) was diluted in 2-(N-morpholino)ethanesulfonic acid (MES) buffer and subsequently introduced into 500 μL of 25 mM MES buffer solution (pH 6.0) containing 20 mg mL−1 NHS and 40 mg mL−1 EDC. The mixture was incubated at room temperature for 20 min and then added to a 5 mL suspension of MPs (5 mg mL−1) in MES buffer. The resulting dispersion was incubated at 29 °C under continuous stirring for 6 h, and the prepared P1-anchored MPs (P1@DM@MP) were rinsed with phosphate-buffered saline (PBS) buffer solution (10 mM, pH 7.4, 0.1 M NaCl). Then, a 5′-thiol single-stranded DNA probe (P2) was attached to P1@DM@MP through Michael addition of the maleimide groups with the thiol groups of P2. A 2 mL solution of PBS containing P2 was added to P1@DM@MP and incubated at 29 °C for 6 h. After the reaction, the multiprobe DNA-conjugated DM@ MP (P1−P2@DM@MP) was separated and washed with PBS containing 0.1% Tween-20 (PBST) to remove any excess probe DNA. P1−P2@DM@MP was then immersed in a solution of 0.5% BSA in PBST for 30 min at 29 °C to reduce any nonspecific adsorption from P1−P2@DM@MP. The resulting P1−P2@DM@MP was washed with PBST, immersed in PBS, and stored at 4 °C for further use. 2.6. Conjugation of Enzymes with Complementary DNA (cDNA). The procedure for the preparation of single-stranded complementary DNA (cDNA)-conjugated enzymes was adapted from the previous research.26 To cleave the disulfide bonds of the oligonucleotide, a solution of C1 (0.5 OD) in PBS (200 μL) was mixed with a 30 mM aqueous solution of TCEP (30 μL), and the mixture was incubated at 37 °C for 2 h with gentle shaking. For enzyme conjugations, GOx (2 mg) and sulfo-SMCC (1 mg) were dissolved in PBS (400 μL) and transferred to a shaker for 2 h at 37 °C. The resulting mixtures of C1 and GOx were purified six times with Amicon-3K and Amicon-10K, respectively, using PBS as an eluent. The purified solutions of C1 and sulfo-SMCC-activated GOx were mixed and subsequently reacted at 29 °C for 24 h with gentle shaking. The resulting GOx−C1 conjugates were purified six times with Amicon-10K using PBS to remove excess C1. The HRP−C2 conjugates were prepared using the same approach. 2.7. Preparation of Fluorescence-Labeled Enzymes. The synthetic approach for the preparation of FITC-labeled GOx and rhodamine B-labeled HRP was adapted from a previously reported procedure.27 A solution of FITC (5 mg mL−1) in dimethyl sulfoxide (0.2 mL) was added dropwise to a 2 mL solution of GOx (5 mg mL−1) in a carbonate buffer (50 mM, pH 9.0), and the mixture was stirred for 4 h at 25 °C. A 50 mM NH4Cl aqueous solution (2 mL) was added to the resulting mixture to quench the reaction and then the solution was dialyzed against PBS (50 mM, pH 7.0) at 4 °C for 48 h to remove the

the precision and operability of the ratio control of immobilized biomolecules. Moreover, enzymes that are immobilized through this strategy provide enhanced activity, improved stability, and assay reproducibility compared to conventional directly immobilized enzymes, which is attributed to the great mechanical rigidity and physicochemical stability of DNA and the conformational freedom of the immobilized enzymes.22−24 In this study, we report the first example of colocalizing multiple enzymes on MPs multifunctionalized with dopamine derivatives through DDI. MPs are a convenient support for immobilizing biomolecules owing to their magnetic properties, which allow them to be rapidly separated from reaction mixtures.25 Glucose oxidase (GOx) and horseradish peroxidase (HRP) were employed as model enzymes to prove the high catalytic performance of the multienzyme system. A precise and flexible control of the enzyme ratio can be achieved by using this strategy.

2. EXPERIMENTAL SECTION 2.1. Materials. GOx from Aspergillus niger and HRP were purchased from Sigma-Aldrich (St. Louis, MO). All DNA molecules used in this study were synthesized and purified by Shanghai Sangon Biological Science & Technology Co., Ltd. (Shanghai, China). The details of the DNA sequences used in this study are listed in Table S-1. Dopamine hydrochloride and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Acros Organics (Geel, Belgium). 6-Maleimidohexanoic acid was obtained from Ark Pharm, Inc. (Ventura, CA). Tris(2-carboxyethyl)phosphine (TCEP), sulfosuccinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC), 2-(Nmorpholino)ethanesulfonic acid (MES), bovine serum albumin (BSA), fluorescein isothiocyanate (FITC), and rhodamine B isothiocyanate (RhoB) were purchased from Aladdin (Beijing, China). N-Hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) were purchased from J&K Scientific (Beijing, China). All of the other reagents used in this study were of analytical grade. 2.2. Synthesis of MPs. Fe3O4 MPs used in the current study were fabricated using a precipitation method. FeCl3·6H2O (16.0 g) and FeCl2·4H2O (8.0 g) were dissolved in water (100 mL) under sonication and subsequently transferred to a nitrogen-protected threeneck flask. The mixture was heated at 75 °C for 30 min and then ammonium hydroxide (50 mL) was slowly added into the mixture under vigorous stirring and kept at a constant temperature (75 °C) for 90 min. The mixture was cooled to room temperature and filtered to give a black product, which was then washed several times with water and ethanol and dried under vacuum at 50 °C for 3 h. 2.3. Preparation of Maleimide-Terminated Dopamine. The procedure for the preparation of maleimide-terminated dopamine derivative (MA) was adapted from a previously reported procedure.17 Dopamine hydrochloride (1.5 g, 0.008 mol) and 2 mL of triethylamine were dissolved in 20 mL of anhydrous MeOH. The mixture was added dropwise into a solution of 6-maleimidohexanoic acid N-hydroxysuccinimide ester (2 g, 0.007 mol) in 40 mL of anhydrous CH2Cl2. The reaction was vigorously stirred for 48 h under a nitrogen atmosphere. Then, the solvent of the reaction mixture was evaporated under reduced pressure and the residue was dissolved in CH2Cl2 (20 mL). The organic phase was washed three times with HCl (0.1 M, 20 mL) and dried with Na2SO4. After filtration, the solvent was evaporated under reduced pressure and the crude product was purified by column chromatography over SiO2 (CH2Cl2/MeOH 10:1). The prepared product was a light yellow solid (Figure S-1). 1H NMR (400 MHz, CDCl3): 1.25 (m, 2H, CH2−CH2−CH2), 1.60 (m, 4H, N−CH2−CH2, CH2−CH2−CO−NH), 2.14 (t, J = 7.2 Hz, 2H, CH2− CO−NH), 2.68 (t, J = 6.8 Hz, 2H, CH2−Ar), 3.44−3.52 (m, 4H, N− CH2−CH2, CH2−NH−CO), 5.71 (t, J = 5.2 Hz, 1H, NH−CH2), 6.58 (dd, J = 8, 1.6 Hz, 1H, ArH), 6.68 (s, 2H, CHCH), 6.80 (d, J = 8 Hz, 1H, ArH), 6.82 (d, 1H, ArH); 13C NMR (100 MHz, CDCl3): 173.6 (CO−NH); 171.1 (CO−N−CO); 144.1, 143.0, 130.7, 120.5, B


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ACS Applied Materials & Interfaces Scheme 1. Fabrication of the Multienzyme System

in the figures were determined from the standard deviation of three measurements. 2.11. Characterization. Magnetic characterization was performed on a Lake Shore 7410 vibrating sample magnetometer (Westerville, OH). A Mettler Toledo 1100SF thermogravimetric analyzer (Columbus, OH) was used for the thermogravimetric analysis (TGA) of the samples. The TGA experiments were conducted at temperatures from 25 to 1000 °C at a heating rate of 10 °C min−1 under nitrogen atmosphere. 1H NMR spectra were recorded on a Bruker 400 MHz spectrometer. 13C NMR spectra were recorded at 100 MHz. The immobilization of FITC-labeled GOx and RhoBlabeled HRP on the multifunctionalized DM@MP was confirmed by confocal laser scanning microscopy (CLSM) on a Leica TCS SP5II CLSM system (Wetzlar, Germany). Fourier transform infrared (FTIR) spectra (4000−400 cm−1) were collected on a Nicolet spectrometer (Thermo Fisher Scientific, Waltham, MA) from KBr pellet samples. All of the enzymatic assays were performed on a UV−vis spectrophotometer (U3010; Hitachi, Tokyo, Japan) at room temperature.

excess FITC. The rhodamine B-labeled HRP was prepared using the same approach. 2.8. Synthesis of Multienzyme Catalysts through DDI. GOx and HRP were immobilized on the multifunctionalized DM@MP through the DDI technique. The GOx−C1 conjugates and HRP−C2 conjugates were diluted in PBS (3 mL) and subsequently incubated with P1−P2@DM@MP (25 mg) at 37 °C for 3 h. The resulting multienzyme catalyst (GOx−HRP@DM@MP) was rinsed thoroughly with PBST to remove unbonded enzyme−cDNA conjugates and then stored at 4 °C for further study. Mild basic conditions were used for dissociation of the hybridized oligonucleotide complex, thus enabling fast surface regeneration. For example, an aqueous solution of NaOH (1 mL, 0.05 M) was mixed with GOx−HRP@DM@MP and incubated at 25 °C for 5 min to unwind double-stranded DNA (dsDNA). 2.9. Preparation of Immobilized GOx−HRP through a CrossLinking Method. The traditional procedure for the preparation of GOx−HRP-modified MPs was adapted from our previous paper.28 Aminated-silica-coated MPs were prepared sequentially through a solvothermal reaction, sol−gel coating strategy, and silanization process. The common silanization reagent 3-aminopropyltriethoxysilane (APTES) was used to functionalize the MPs during the silanization process to obtain the aminated MPs (APTES@MP). Then, GOx and HRP were coimmobilized on the surface of APTES@ MP using glutaraldehyde as the cross-linking reagent. The resulting GOx- and HRP-anchored MP (GOx−HRP@APTES@MP) was stored at 4 °C for further study. 2.10. Enzymatic Assays. Glucose was dissolved in the enzymolysis buffer (50 mM PBS containing 1 mM TMB) and diluted to varied concentrations, followed by incubation at 37 °C with the GOx−HRP@DM@MP composite. Then, the multienzyme catalyst was separated with magnetic field and H2SO4 aqueous solution (20 μL, 2 M) was added into the remaining supernatant. The absorbance of the resulting yellow solution was recorded at 450 nm. The error bars

3. RESULTS AND DISCUSSION 3.1. Functionalization and Characterization of MPs with Dopamine Derivatives. As illustrated in Scheme 1, the multienzyme system was fabricated by coimmobilizing enzymes on multifunctionalized MPs through DNA-directed immobilization. The size and morphology of the MPs were observed by transmission electron microscopy (TEM) (Figure S-1), and the diameter of the MPs was approximately 14 nm. The presence of DA and MA on the MPs was confirmed by FTIR spectroscopy. DA and MA were grafted on the MPs through the chelation of the catechol ligand of dopamine with the C


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primary amine of DA, which was located at 1627 cm−1 and was also superposed with the OH vibration of adsorbed water molecules on the MP surface. The peaks at 1484 and 1263 cm−1 were assigned to the bending vibration of C−H and the C−O stretching of the phenolic hydroxyl group, respectively. The strong band at 1705 cm−1 was attributed to the CO stretching vibration of MA. These results were clear evidence of the successful multifunctionalization of the particles with DA and MA. In addition, the reaction solvent (including water, acetonitrile, and methyl alcohol) and time (30−120 min) for the synthesis of the DA-functionalized MPs were optimized. The intensities of the C−H bending and C−O stretching vibrations were the strongest when MeOH was used as a solvent (Figure S-2a), and 60 min was used as the reaction time (Figure S-2b). Thus, we chose MeOH and 60 min as the optimal reaction conditions for subsequent experiments. 3.2. Preparation and Characterization of a Multienzyme System Using the DDI Strategy. Simultaneous immobilization of GOx and HRP on MPs through DDI was proven by CLSM. GOx and HRP were labeled with FITC and RhoB, respectively. CLSM clearly indicated that both GOx− FITC (green) and HRP−RhoB (red) were anchored on the MPs with the construction of spatial colocalization (Figure 2a). The TGA validated the anchoring of dopamine derivatives and enzymes (Figure S-3a). The magnetic properties of these nanomaterials were investigated using a vibrating sample magnetometer (Figure S-3b). The magnetization saturation

surface iron atoms of the MPs. As shown in Figure 1, the C−N stretching overlapped with the N−H bending vibration of the

Figure 1. Fourier transform infrared spectra of magnetic nanoparticles (MPs), and MPs functionalized with dopamine (DA@MP), maleimide-terminated dopamine derivative (MA@MP), or DA and MA simultaneously (DM@MP).

Figure 2. (a) Confocal laser scanning microscopy images of magnetic nanoparticles functionalized simultaneously with dopamine (DA) and maleimide-terminated dopamine derivative (MA), on which glucose oxidase (GOx) and horseradish peroxidase (HRP) were simultaneously immobilized (GOx−HRP@DM@MP). GOx and HRP were labeled with fluorescein isothiocyanate (green) and rhodamine B (red), respectively. (b) Catalytic efficiency of GOx−HRP@DM@MP using different reaction buffers and at different times. (c) Overall activity of GOx−HRP@DM@ MP, HRP−GOx@DM@MP, and [GOx−HRP]@DM@MP. D


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ACS Applied Materials & Interfaces value of GOx−HRP@DM@MP was 51.92 emu g−1, indicating that it retained excellent magnetic responsiveness after modification with enzymes. To enhance the overall enzymatic activity, we studied the reaction conditions, such as time and type of buffer, for attaching the modified probe DNA on the multifunctionalized DM@MP. The conditions for the Michael addition of the maleimide groups with the thiol groups of P2 are reported in our previous study.26 In this work, we optimized the condensation reaction conditions for aminated DM@MP with the carboxylic group of P1. The influence of the reaction time was evaluated in the range of 1−12 h. As shown in Figure 2b, the efficiency significantly increased from 1 to 6 h, but then declined as the time was increased to 12 h. This indicated that 6 h was the optimum reaction time, which was used for subsequent experiments. The decline in catalytic efficiency might be attributed to the hydrolysis of a few probe DNAs, resulting in longer reaction times. Also, the results indicated that MES buffer (25 mM, pH 6.0) was more effective than PBS (25 mM, pH 7.4) for the immobilization of P1 (Figure 2b). Interestingly, we found that the immobilization order of the probe DNA had a clear influence on the overall activity. GOx− HRP@DM@MP was prepared through the hybridization of GOx−C1 and HRP−C2 with P1−P2@DM@MP, which was prepared by first modifying DM@MP with P1 and subsequently with P2. Similarly to GOx−HRP@DM@MP, HRP−GOx@DM@MP and [GOx−HRP]@DM@MP were prepared by the hybridization of enzyme−DNA conjugates with P2−P1@DM@MP (first modified with P2 and subsequently with P1) and [P1−P2]@DM@MP (simultaneously modified with P1 and P2), respectively. As shown in Figure 2c, GOx−HRP@DM@MP gave the highest overall activity, approximately 110.7 and 124.8% of the activity of HRP− GOx@DM@MP and [GOx−HRP]@DM@MP, respectively. The molecular structures of DA and MA suggest that the chain length of MA is longer than DA, which might enhance the steric effect of P2 on P1 when DM@MP was first modified with P2. The connection between P1 and the NH2 group was hindered by P2, which caused the slightly reduced activity of HRP−GOx@DM@MP. When P1 and P2 were immobilized on DM@MP simultaneously, both the steric effect and competition effect of P1 and P2 probably caused the reduced activity of [GOx−HRP]@DM@MP. Thus, the overall activity of [GOx− HRP]@DM@MP was lower than that of HRP−GOx@DM@ MP and GOx−HRP@DM@MP. Therefore, GOx−HRP@ DM@MP was selected for subsequent studies. 3.3. Optimization of the Enzymatic Activity of the Multienzyme Catalyst. Some key parameters for the enzymolysis process, such as pH and temperature, were optimized to improve the procedure. GOx−HRP@DM@MP sustained higher enzymatic activities over a broad pH range of 5−8 compared to free GOx&HRP (Figure 3a). Although GOx−HRP@DM@MP gave the highest enzymolysis efficiency at pH 5.5, the optimum pH was 6.0 because DNA was easily hydrolyzed in an acidic medium. The optimum temperature for the enzymolysis of both GOx−HRP@DM@MP and free GOx&HRP was 37 °C (Figure 3b). GOx−HRP@DM@MP retained much higher activity and stability in comparison to the free enzymes in a wide temperature range of 50−80 °C and retained 32% activity even at 80 °C. The DNA duplex partially unwound during the incubation at 60−80 °C, which was another reason besides the thermal denaturation of the protein for the continuous decline of activity of GOx−HRP@DM@

Figure 3. Effect of (a) pH and (b) temperature on the performance of GOx−HRP@DM@MP.

MP. In contrast, the activity of free GOx&HRP declined dramatically in the range of 50−80 °C and retained only 9.5% activity at 80 °C. These results demonstrated that GOx− HRP@DM@MP had much higher stability and activity compared to free GOx&HRP under harsh microenvironments. 3.4. Optimization of the Enzyme Ratio for the GOx&HRP System. Significant benefits can be achieved by optimizing the enzyme ratio, such as reaching the maximum overall activity with the minimum amount of enzymes, reducing competitive side reactions, preventing the accumulation of toxic intermediates, and enhancing the rate-limiting reaction step.29 Multienzyme catalysts prepared with different enzyme ratios were observed by CLSM. The fluorescence intensity of GOx (green) was enhanced and that of HRP (red) remained unchanged with increasing GOx/HRP molar ratio (Figure 4a), which indicated that the method reported in this study can be used to control the amount of the enzymes. Figure 4b shows a comparison of the enzymatic activities of multienzyme catalytic systems containing different molar ratios of GOx/HRP; the highest activity was achieved at a molar ratio of 2:1. The above results demonstrated that the enzyme ratio could be precisely controlled and conveniently adjusted to reach the highest enzymolysis efficiency by regulating the quantity of functional groups on the multifunctionalized nanoparticles rather than just regulating the amount of enzymes used, which usually causes waste. 3.5. Enzymatic Assays of the Multienzyme System. The enzymolysis efficiency of free and coimmobilized multiE


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Figure 4. (a) Multichannel CLSM images of the multienzyme system with various enzyme molar ratios. (b) Relative activity of the multienzyme system with various enzyme molar ratios.

DA@MP and free HRP, and free GOx and HRP@MA@MP (Figure 5b), to further validate the improved efficiency of GOx−HRP@DM@MP. The results indicated that GOx− HRP@DM@MP showed approximately 2.0−2.3-fold higher activity than the above three bienzyme mixtures probably because of the closer proximity of GOx and HRP in GOx− HRP@DM@MP. The Michaelis−Menten model was applied to explore the enzymatic kinetic parameters of the free and coimmobilized multienzyme system. Km and kcat were determined on the basis of the Lineweaver−Burk strategy (Figure 5c). As shown in Table S-2, the Km value of GOx−HRP@DM@MP was 1.41 mM, whereas the Km values for free GOx&HRP and GOx− HRP@APTES@MP were 2.63 and 1.92 mM, respectively. This revealed that GOx−HRP@DM@MP showed more affinity toward the substrate than the free and cross-linking coimmobilized bienzymes. In addition, kcat/Km for GOx− HRP@DM@MP was 5.02 s−1 mM−1, which was higher than that for free GOx&HRP and GOx−HRP@APTES@MP, indicating a higher catalytic efficiency. The increased bioactivity of GOx−HRP@DM@MP was ascribed to the colocalization of the involved enzymes at the optimized ratio. The H2O2

enzyme was evaluated using glucose and TMB as model substrates by monitoring the absorbance change using UV−vis spectroscopy. As shown in Scheme 1, glucose was converted into gluconic acid and H2O2 through the catalysis of GOx; then, the resulting H2O2 oxidized TMB in the presence of HRP to blue-colored oxidized TMB. To ensure the termination of the enzymatic reaction, H2SO4 aqueous solution was added to the supernatant of the reaction mixture, thereby converting the oxidized TMB into diamine (yellow), which was detectable at 450 nm. To show the superior catalytic performance of GOx−HRP@ DM@MP, we compared its enzymolysis efficiency to that of GOx−HRP@APTES@MP (which was prepared by a crosslinking method) and free GOx&HRP. Both GOx−HRP@ DM@MP and free GOx&HRP in solution with the same protein amount and corresponding molar ratio as that of GOx− HRP@DM@MP were utilized as controls. As illustrated in Figure 5a, GOx−HRP@DM@MP showed 24.7 and 116.7% increase in activity compared to free GOx&HRP and GOx− HRP@APTES@MP, respectively. Furthermore, the performance of GOx−HRP@DM@MP was compared to that of the mixtures of GOx@DA@MP and HRP@MA@MP, GOx@ F


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Figure 5. (a) Overall and specific activity of GOx−HRP@DM@MP, free GOx&HRP, and GOx−HRP@APTES@MP. (b) Catalytic efficiency of GOx−HRP@DM@MP, GOx@DA@MP with HRP@MA@MP, GOx@DA@MP with free HRP, and free GOx with HRP@MA@MP (in comparison to free GOx&HRP at the same concentration). (c) Lineweaver−Burk plots of GOx−HRP@DM@MP, GOx−HRP@APTES@MP, and free GOx&HRP.

generated by GOx can accumulate inside GOx−HRP@DM@ MP composites, leading to a higher intermediate concentration, which can drive and promote the GOx−HRP cascade. Then, the generated H2O2 was immediately catalyzed by HRP, resulting in the reduction of competitive side reactions. In addition, H2O2 is an inhibitor of GOx; therefore, the quick elimination of H2O2 helped to maintain the enzymatic activity of GOx.30,31 Furthermore, enzymes that are anchored with DNA strands can maintain most of their activity, whereas direct immobilization of enzymes through covalent or noncovalent interactions restricts their conformational freedom and can partially denature their structure, as we reported previously.26,32 3.6. Regeneration of GOx−HRP@DM@MP. Another significant advantage of the DDI strategy for anchoring of enzymes is the capacity for reversible enzyme immobilization and surface regeneration, as presented in Figure 6. GOx and HRP were removed from the surface of P1−P2@DM@MP by denaturing the DNA duplex with 0.05 M NaOH, which was confirmed by the sharp decline in overall activity (less than 3%). After a subsequent cycle of hybridization and dehybridization, the activity of GOx−HRP@DM@MP remained greater than 85% and less than 2% (cycle 2), respectively. These results demonstrated that the DDI strategy exhibited high reversibility

Figure 6. Reversibility of GOx−HRP@DM@MP. Two sequential cycles of hybridization/dehybridization were performed. (A) Enzymolysis efficiency of GOx and HRP coimmobilized onto the DM@MP using the DDI technique. (B) Enzymolysis efficiency of the multienzyme catalyst following the removal of the DNA−enzyme conjugates through a mild dehybridization process.

G


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Figure 7. (a) Recycling performance of GOx−HRP@DM@MP and GOx−HRP@APTES@MP. (b) Retained activity of the coimmobilized and free GOx&HRP after incubating at 60 °C for various times. (c) Long-term stability of GOx−HRP@DM@MP compared to free enzymes and GOx− HRP@APTES@MP incubated in pH 7.4 PBS at 4 °C.

and reproducibility, which could effectively reduce the cost of the synthesis. 3.7. Stability and Reusability. The reusability of the anchored enzyme is a particularly important aspect because of its economic significance. As presented in Figure 7a, GOx− HRP@DM@MP retained a high degree of activity (more than 63%) after 13 cycles, whereas GOx−HRP@APTES@MP retained only 46.1% residual activity. These results indicated that GOx−HRP@DM@MP had satisfactory reusability compared to previous studies,33−35 benefiting from the great mechanical rigidity and high physicochemical stability of double-stranded DNA (dsDNA), which has been well elaborated in our previous studies.26,32 Furthermore, to investigate the thermal stability of GOx−HRP@DM@MP, both coimmobilized and free GOx&HRP were incubated at 60 °C in PBS and their retained activities were measured at different times. As illustrated in Figure 7b, GOx−HRP@DM@ MP retained approximately 89.7% enzymatic activity after 40 min, whereas the enzymatic activity of GOx−HRP@APTES@ MP and free GOx&HRP reduced to 46.0 and 38.5% of the original activity after 40 min, respectively. The DNA duplex partially unwound during the incubation, which was another reason besides the thermal denaturation of the protein for the reduced activity of GOx−HRP@DM@MP. However, GOx− HRP@DM@MP retained approximately 56.6% residual activity after 120 min of incubation (as shown in Figure 7b), which was attributed to the renaturation of uncomplimentary DNA when

GOx−HRP@DM@MP cooled to room temperature. In addition, the long-term storage stability and durability of the enzymes was evaluated by incubating in PBS at 4 °C for 30 days. After incubation for 30 days, GOx−HRP@DM@MP still retained 81.2% of its initial activity and showed 19.4 and 53.7% increase in durability compared to GOx−HRP@DM@MP and free GOx&HRP, respectively (Figure 7c). In general, the immobilized multienzyme system prepared using the method reported in the current study exhibited excellent reusability and high stability. Therefore, it represents a promising platform for biocatalysis.

4. CONCLUSIONS In summary, we developed a strategy for fabricating a multienzyme system with high catalytic efficiency and precise control of the enzyme ratio. Multiple enzymes were coimmobilized on MPs multifunctionalized with DA or MA by using DDI. The resulting multienzyme catalyst exhibited great dispersibility and superparamagnetism. The enzyme ratio was precisely controlled and conveniently adjusted by regulating the quantity of the functional groups on the multifunctionalized nanoparticles rather than just varying the amount of enzymes used, which usually causes waste. In addition, coimmobilizing GOx and HRP through DDI significantly enhanced the overall catalytic efficiency and kinetic properties in comparison to the single-enzyme mixtures and directly immobilized multienzymes. The improved stability, H


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reusability, and reversibility of the multienzyme system in this study is promising for applications in biocatalysis. Given the wide variety of possible enzyme associations and the high efficiency of this strategy, we believe that this work provides a new route for the fabrication of artificial multienzyme systems and can be extended for a wide range of applications in diagnosis, biomedical devices, and biotechnology.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b08553. Additional figures and tables including TEM image of MP; data on NMR analysis of MA; data on FTIR measurement of DA@MP; data on vibrating sample magnetometer and TGA measurements of multienzyme system; enzymatic kinetics of multienzyme system; DNA sequences (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (P.S.). *E-mail: [email protected] (Y.Y.). ORCID

Yi Yang: 0000-0002-4091-8532 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 21675008). REFERENCES

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