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Boosting Gas Involved Reactions at Nanochannel Reactor with Joint Gas−Solid−Liquid Interfaces and Controlled Wettability Li Mi,† Jiachao Yu,† Fei He,† Ling Jiang,† Yafeng Wu,† Lijun Yang,⊥ Xiaofeng Han,† Ying Li,† Anran Liu,† Wei Wei,† Yuanjian Zhang,† Ye Tian,*,‡ Songqin Liu,*,† and Lei Jiang⊥
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†
Key Laboratory of Environmental Medicine Engineering, Ministry of Education, Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China ‡ Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Green Printing, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China ⊥ Key Laboratory of Bioinspired Smart Interface Sciences, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, China S Supporting Information *
ABSTRACT: The low solubility of gases in aqueous solution is the major kinetic limitation of reactions that involve gases. To address this challenge, we report a nanochannel reactor with joint gas−solid−liquid interfaces and controlled wettability. As a proof of concept, a porous anodic alumina (PAA) nanochannel membrane with different wettability is used for glucose oxidase (GOx) immobilization, which contacts with glucose aqueous solution on one side, while the other side gets in touch with the gas phase directly. Interestingly, it is observed that the O2 could participate in the enzymatic reaction directly from gas phase through the proposed nanochannels, and a hydrophobic interface is more favorable for the enzymatic reaction due to the rearrangement of GOx structure as well as the high gas adhesion. As a result, the catalytic efficiency of GOx in the proposed interface is increased up to 80-fold compared with that of the free state in traditional aqueous air-saturated electrolyte. This triphase interface with controlled wettability can be generally applied to immobilize enzymes or catalysts with gas substrates for high efficiency.
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INTRODUCTION Oxygen and other gas molecules have an important role in a variety of reactions such as laboratory synthesis, energy utilization, and biochemical processes.1−4,5,6 Due to the extremely low solubility of gases in aqueous solution, for example, O2, CO2, and CH4, the efficiency of these reactions is usually significantly suppressed.7−10 To address this limitation, numerous approaches have been taken to adapt; Pickering emulsions,11 microfluidic devices,12 or “tube-in-tube” techniques13 have been developed for increasing the interfacial areas or gas adsorption.14,15 Those methods require extra additives or complicated design techniques, and the low concentration of gas molecules in solution is still a major limitation. In these regards, a novel method for solving the gas-deficit problem and adjusting the concentration of gases for the specific systems is highly anticipated. In this work, we report a nanochannel reactor with joint gas− solid−liquid interfaces and controlled wettability for boosting gas involving reactions. A porous anodic alumina (PAA) nanochannel membrane is used for immobilizing an enzyme, which contacts the aqueous solution on one side, while the other side contacts the gas phase directly. Glucose oxidase (GOx), a type of aerobic oxidase, is selected as a model © 2017 American Chemical Society
enzyme. Oxygen molecules from the gas phase can transport through the modified PAA nanochannels and reach the active sites of the GOx directly, significantly improving the concentration of oxygen for the reaction. Moreover, it is observed that the reaction efficiency of the enzyme at the hydrophobic interface is much greater than that at the hydrophilic interface in our system. Further mechanistic investigation reveals that at the hydrophobic interface the protein exhibits a favorable conformation and orientation,16−19 as well as high adhesive force for oxygen. As a result, in the nanochannel reactor with joint gas−solid−liquid interfaces, the reaction efficiency of enzyme with oxygen diffusing directly from air is enhanced up to 80 times compared with the free state in traditional aqueous air-saturated electrolyte. Such proposed strategy is expected not only to access the biocatalytic reaction but also to be of use in industrial catalysis and synthesis. Received: May 22, 2017 Published: June 30, 2017 10441
DOI: 10.1021/jacs.7b05249 J. Am. Chem. Soc. 2017, 139, 10441−10446
Article
Journal of the American Chemical Society
Figure 1. Schematic illustration for the construction process of a nanochannel with joint gas−solid−liquid interfaces and controlled wettability. First, the PAA membrane is covered with a gold film via sputtering (Au/PAA). Then, the modified PAA membrane is incubated with NDM solution to obtain a hydrophobic surface (NDM/Au/PAA). Third, Au nanoparticles (AuNPs) are electrodeposited (AuNPs/NDM/Au/PAA). After that, the structure is incubated with either NDM (NDM/AuNPs/NDM/Au/PAA) or MUA (MUA/AuNPs/NDM/Au/PAA) to form a hydrophobic or hydrophilic surface, respectively. Finally, GOx is immobilized at the hydrophobic or hydrophilic interfaces. The electrochemical characteristics of the as-prepared membrane are obtained with a homemade device that has two chambers: one chamber is equipped with a counter Pt wire electrode (CE), an SCE reference electrode (RE), and 0.1 M, pH 7.4, phosphate solution as an electrolyte, and the other chamber is used for gas storage. The GOx modified PAA, which is the working electrode (WE), is fixed between the two chambers, with the GOx immobilization side immersed into the electrolyte and the other side in contact with the gas phase for gas transport through the nanochannels to form a gas−solid−liquid interface.
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PAA) was further modified with NDM or MUA overnight to obtain surface hydrophobic NDM/AuNPs/NDM/Au/PAA or surface hydrophilic MUA/AuNPs/NDM/Au/PAA. Finally, both hydrophobic and hydrophilic PAA membranes were incubated with 750 U mL−1 glucose oxidase solution in 100 mM PBS, pH 7.4, at 4 °C for 4 h, followed by rinsing thoroughly with PBS to obtain enzyme modified three-phase electrodes (GOx/NDM/AuNPs/NDM/Au/PAA or GOx/MUA/ AuNPs/NDM/Au/PAA) and storage at 4 °C for further experiments.
EXPERIMENTAL SECTION
Materials. The porous anodic alumina (PAA) membrane with pore diameter of 90 ± 10 nm and length of 100 ± 20 μm is purchased from Puyuan Nanotechnology (Hefei, China). 11-Mercaptoundecanoic acid (MUA), n-dodecanethiol (NDM), and glucose oxidase (GOx, EC 1.1.3.4, from Aspergillus niger, 196.6 kU g−1) were received from Sigma-Aaldrich (Shanghai, China). HAuCL4·4H2O (Au% = 47.8%) was received from Nanjing Reagent Co. (Nanjing, China). Phosphatebuffer solution (PBS, 100 mM, pH 7.4) was prepared by mixing stock solutions of Na2HPO4 and NaH2PO4. Apparatus. The scanning electron micrograph (SEM) images were recorded with a field emission scanning electron microscope (ULTRA PLUS, ZEISS, Germany), coupled to an energy dispersive spectrometer (EDS). Measurement of the contact angle (CA) of water or oil droplets (dichloroethane) in water was performed with a video-based optical contact angle measuring instrument (Dataphysics, German). The adhesive force of a bubble in water on the PAA membrane surface was measured by a high sensitivity microelectromechanical balance system (Data-Physics DCAT 11, Germany). The electrochemical measurements were performed with a CHI 750 electrochemical workstation (Shanghai Chenhua, China). All experiments were conducted with this three-electrode system, composed of a saturated calomel electrode (SCE), a Pt electrode, and the PAA membrane as reference, counter, and working electrodes, respectively. The secondary structure of GOx was analyzed with a sum frequency generation spectrometer (SFG, EKSPLA, Lithuania). The right-angle CaF2 prisms were distributed in n-trimethoxyoctadecylsilane and succine anhydride to obtain the hydrophobic and hydrophilic surfaces, respectively. GOx was then dropped onto the modified prism and incubated for 4 h to reach equilibrium. SFG spectra in the amide I frequency region were collected from the immobilized enzyme from 1500 to 1800 cm−1 using both ssp (s-polarized SFG signal, s-polarized visible input, and p-polarized input IR) and ppp (p-polarized SFG signal, p-polarized visible input, and p-polarized input IR) polarization combinations,20−24 which can be used to determine the enzyme orientation. The SFG ssp spectra were fitted using the a standard spectral fitting method. Preparation of a Nanochannel Reactor with Joint Gas− Solid−Liquid Interfaces and Controlled Wettability. A thin gold film with about 100 nm thickness, determined from SEM images, was first sputtered onto one face of the PAA membrane at 5 mA for 3 min. The resulting gold film coated PAA membrane was then incubated with 0.01 mM NDM in ethanol overnight, washed with ethanol 3 times, and dried in N2. After that, the electrochemical deposition of gold nanoparticles was performed at −0.2 V for 20 min by dipping the side of the PAA membrane with the gold film into 2.4 mM HAuCl4 solution in 100 mM PBS, pH 7.4. The PAA membrane sputtered gold film with gold nanoparticle electrodeposition (AuNPs/NDM/Au/
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RESULTS AND DISCUSSION Figure 1 shows the typical approach to construct a nanochannel reactor with joint gas−solid−liquid interfaces with enzyme. The PAA membrane was coated with a gold film via sputtering (Au/ PAA) and then immersed in NDM solution to obtain a hydrophobic surface (NDM/Au/PAA). After that, Au nanoparticles (AuNPs) were electrochemically deposited on NDM/ Au/PAA to form AuNPs/NDM/Au/PAA. The wettability was further controlled by self-assembly of NDM (NDM/AuNPs/ NDM/Au/PAA) or MUA (MUA/AuNPs/NDM/Au/PAA). Finally, GOx was immobilized at the as-prepared interface. This modified PAA membrane as working electrode (WE) was fixed between two chambers of a homemade electrolytic cell with the GOx side in contact with electrolyte and oxygen diffusing directly from the gas phase to the GOx active sites through the nanochannels of the membrane. This device has the merits of allowing free diffusion of substrates from the liquid-phase and oxygen from the gas-phase to boost the kinetics of reactions. The electrochemical performance and morphology of the nanochannels with gas−solid−liquid interface are demonstrated by electrochemical current response and SEM images, respectively. This interface depends on the pore diameter of Au/PAA and the electrodeposition time of AuNPs at NDM/ Au/PAA. The current increases with the pore diameter and reaches a plateau at 90 nm (Figure 2a). Then, the PAA membrane with 90 nm pores (channel length of 100 ± 20 μm) is verified by scanning electron microscopy (SEM), As shown in Figure 2b, the gold sputtering step forms a covering gold film with a thickness of 100 ± 5 nm on the PAA surface; no gold nanoparticles are observed in the channels of the PAA. To prevent liquid outflow from the nanochannels and provide hydrophobic nanochannels for gas molecule diffusion, the PAA membrane is modified to be hydrophobic with NDM deposition producing a water contact angle (CA) of 133° ± 4.1° (NDM/Au/PAA) (Figure 2b, inset). In the next step, the 10442
DOI: 10.1021/jacs.7b05249 J. Am. Chem. Soc. 2017, 139, 10441−10446
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Journal of the American Chemical Society
the active sites of GOx. For the oxygen-mediated enzymatic reaction, the O2 molecules temporarily residing on the protein surface is likely the first step for O2 diffusing to the active sites. It was previously reported that the hydrophobic residues of a protein surface are responsible for hosting O2 molecules and carrying O2 molecules inside the protein through several additional paths.25 Thus, the wettability environment of a protein surface is beneficial for encouraging oxygen molecule residence, which allows the O2 molecules to easily reach the interior of the protein and enhance the reactivity of the protein. After immobilization, different conformations and orientations of GOx are displayed at the hydrophobic or hydrophilic interface. When protein is adsorbed at a solid−liquid interface in an aqueous environment, protein residues will interact with exposed functional groups on the solid surface, and the interactions between protein and solid surfaces dominate the protein immobilization and determine the catalytic activities of protein.18,26−34 For a hydrophobic interface, the interaction between protein residues and the exposed hydrophobic groups leads to unfolding or adoption of a non-native conformation of the protein to expose the interior hydrophobic residues to the interface. For a hydrophilic interface, the protein also exposes the interior hydrophilic residues to the surface to interact with the hydrophilic functional groups. Because the interfaces with different wettability possess different affinity for oil droplets in water,35 the different conformations of GOx at hydrophobic and hydrophilic interfaces can be verified by monitoring the contact angles of oil droplets in water. The CAs of oil droplets in water are 11.3° ± 1.9° and 143.6° ± 2.8° for hydrophobic and hydrophilic interfaces without enzyme coating, respectively (Figure S5). After the adsorption of GOx, the CAs of oil droplets in water are 35.4° ± 2.7° and 139.8° ± 3.4° for the hydrophobic and hydrophilic interfaces, respectively (Figure 3a). At the hydrophobic interface, the interaction between the hydrophobic amino acids on the GOx surface and the modified NDM on the PAA leads to the hydrophobic residues of the immobilized GOx being exposed, resulting in a CA of the oil droplet in water representative of an oleophilic surface. After GOx adsorbs on a hydrophilic surface, the interaction between the immobilized GOx and MUA on the PAA leads to the hydrophilic residues of the immobilized GOx being exposed, resulting in a CA of the oil droplet in water being representative of an oleophobic surface. These results confirm that the two CAs of oil droplets in water represent the different conformations of the GOx at hydrophobic and hydrophilic interfaces. We reason that the hydrophobic surface made by ndodecanethiol may promote unfolding of GOx due to the interaction between hydrophobic groups in the interior of GOx and the alkyl chain of n-dodecanethiol.31−34 Therefore, the adsorption of GOx to hydrophobic surface is due to hydrophobic interactions, which leads to structural changes in the protein upon adsorption. The nonnative conformation of GOx at interfaces with different wettability is explored by sum frequency generation spectroscopy (SFG).36−38 SFG spectra with ssp (s-polarized SF output, s-polarized visible input, and ppolarized infrared input) polarization are fitted by the standard spectral fitting method.21,22 The GOx immobilization at the hydrophobic interface shows an absorbance peak located at 1622 cm−1 in the amide I spectra area (Figure 3b, blue dot).23,24 Previous studies demonstrated that unfolding of a native protein was involved in the conversion of a soluble protein into a β-sheet-rich structured aggregate,38−41 which is
Figure 2. Characterization of nanochannels with gas−solid−liquid interface by electrochemical current response and SEM images. (a) Current increases along with the pore diameter and reaches a plateau at 90 nm; the inset displays the pore diameter of Au/PAA. Scale bar = 100 nm. (b) Side-view SEM images of PAA covered with gold film (Au/PAA); the inset shows that the CA of a water droplet on NDM/ Au/PAA is 133° ± 4.1°. Scale bar = 100 nm. (c) With an extension of the electrodeposition time, the surface density of the gold nanoparticles increases and the current response rises. As the deposition time extends beyond 1200 s, the current decreases sharply. Inset shows the size of electrodeposited AuNPs. Scale bar = 100 nm. (d) SEM images of AuNPs/NDM/Au/PAA with insets showing a CA of 23.8° ± 3.2° for MUA/AuNPs/NDM/Au/PAA (top) and 133.7° ± 1.5° for NDM/AuNPs/NDM/Au/PAA (bottom). Scale bar = 100 nm. (e) Thin GOx coated film at the as-prepared interface. Scale bar = 100 nm. (f) Mechanism of GOx reactivity at the as-prepared interface: oxygen from the gas phase passes through the nanochannels at this interface to reach the active sites of GOx.
current response of NDM/AuNPs/NDM/Au/PAA with different electrodeposition time of AuNPs is investigated. The extension of the electrodeposition time causes an increase in the surface density of the gold nanoparticles (Figure S2) and a rise in response current. However, if the deposition time is over-extended, the current decreases sharply (Figure 2c), and the water CA of electrodes after different electrodeposition times of AuNPs are shown in Figure S3. The best deposition time observed for our system is 1200 s. After electrodeposition, many separated gold nanoparticles could be observed on the NDM/Au/PAA with a size of 100 ± 10 nm (Figure 2d and Figure S1c). The AuNPs/NDM/Au/PAA is further modified with NDM or MUA to either form hydrophobic or hydrophilic surfaces with water CA of 133.7° ± 1.5° for NDM/AuNPs/ NDM/Au/PAA and 23.8° ± 3.2° for MUA/AuNPs/NDM/ Au/PAA (Figure 2d insets), respectively. Thereafter, the GOx is immobilized at the as-prepared interface to form a hydrophobic interface (GOx/NDM/AuNPs/NDM/Au/PAA) or hydrophilic interface (GOx/MUA/AuNPs/NDM/Au/ PAA). As shown in Figure 2e, the SEM images of the GOx composite film coated at the as-prepared interface show that the composite film is separated from the substrate by the electrodeposited AuNPs. This architecture facilitates free oxygen diffusion from air and glucose from the liquid phase to the gas−solid−liquid nanochannel interface. The water CA after GOx immobilization is 105.5° ± 2.5° for the hydrophobic interface and 46.7° ± 1.9° for the hydrophilic interface (Figure S4). The change in CA after GOx immobilization is due to the different interaction between GOx and a solid surface. Figure 2f is also presented to reveal mechanism of oxygen crossing the nanochannels to the gas−solid−liquid interface and arriving at 10443
DOI: 10.1021/jacs.7b05249 J. Am. Chem. Soc. 2017, 139, 10441−10446
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Journal of the American Chemical Society
Figure 3. (a) The contact angles of an oil droplet in water at different wettability interfaces with GOx immobilization reveal the conformation of GOx rearrangement. In water, the hydrophobic interface is oleophilic, and the hydrophilic interface is oleophobic. (b) The SFG spectra of GOx at hydrophobic (blue dot) and hydrophilic interface (red dot). GOx immobilized at the hydrophobic interface shows an absorbance peak located at 1622 cm−1 in the amide I spectra area. In contrast, there is no signal at the hydrophilic interface. These results indicate that GOx immobilized at the hydrophobic interface displays a more favorable conformation for catalytic reactivity. (c) The measurements of gas bubble adhesion at hydrophobic (blue dot) and hydrophilic interfaces (red dot) in water. ΔF is the stretch force between the interface and air bubble. The hydrophobic interface presents higher air adhesion to gas molecules residing on it.
Figure 4. Electrochemical performance of controlled wettability gas− solid−liquid nanochannels interface for β-D-glucose at various concentrations. (a) CVs of GOx/NDM/AuNPs/NDM/Au/PAA measured in β-D-glucose (concentrations from 0 to 0.06 M). (b−d) Calibration plot derived from the LSV experiments related to the oxidation currents with β-D-glucose substrate at 0.55 V (vs SCE), after GOx immobilization at the hydrophobic interface (blue line) or hydrophilic interface (red line). The l and g represent the liquid chamber and gas chamber, respectively. All the gas chambers are measured against an atmospheric pressure. (b) In air (l)/N2 (g) (solid dot) or in N2 (l)/air (g) (open dot). The GOx is adsorbed at a hydrophobic interface (c) and hydrophilic interface (d) under different conditions. The GOx immobilized at the hydrophobic interface with oxygen from the gas phase exhibits higher oxidation currents with the β-D-glucose substrate. All the oxidation currents with β-D-glucose substrate are acquired at 0.55 V (vs SCE).
characterized by the appearance of nonnative spectral bands from 1627 to 1622 cm−1.42−44 After GOx adsorption at a hydrophobic interface, most of the helix loss is converted to intermolecular antiparallel β-sheet motifs.45,46 In contrast, no signal is observed from 1500 to 1800 cm−1 for GOx at the hydrophilic interface (Figure 3b, red dot) due to the loss of helices that are transformed into turns and random structures.4 The outcome further confirms that the conformation of GOx depends on the wettability of the solid surface. The interfaces with different wettability possess different adhesion to air bubbles;47−51 the corresponding properties for air bubble adhesion at different wettability interfaces are also examined. As can be seen from Figure 3c, the hydrophobic interface shows strong underwater air bubble adhesion with a CA of 119.1° ± 1.6° and a maximum stretch force (ΔF) of 86.7 ± 2.5 μN (Figure 3c, blue dot). In contrast, the hydrophilic interface displays low underwater air bubble adhesion with CA of 142.4° ± 1.3° and a ΔF of 15 ± 1.7 μN (Figure 3c, red dot). Therefore, the hydrophobic interface shows more affinity for gas molecules than the hydrophilic interface. The amount of absorption of GOx is determined to be 28.7 ± 3.3 U at the hydrophobic interface and 36.2 ± 4.2 U at the hydrophilic interface, which was measured by colorimetric measurement (Figure S6). The catalytic ability of the immobilized GOx at the gas−solid−liquid interface with different wettability is examined by electrochemical techniques, that is, cyclic voltamogram (CV) (Figure 4a and Figure S7) and linear sweep voltammetry (LSV) (Figure S8). The GOx
adsorbed at proposed interface displays no direct electrochemical behavior (Figure S9). In the presence of β-D-glucose (the substrate for GOx), the oxidation currents of GOx immobilized at the proposed interfaces are dramatically enhanced, and the oxidation current increase is associated with increasing concentration of β-D-glucose (Figure 4a). In addition, the oxidation current of GOx immobilized at the hydrophobic interface in a liquid chamber with an air saturated solution and a gas chamber full of N2 (air (l)/N2 (g)) is 6.19 μA for β-D-glucose substrate at a certain concentration (e.g., 40 mM), which is 2.7 times larger than that at the hydrophilic interface (2.284 μA, Figure 4b, solid circular dot). Interestingly, with air (l)/air (g), the catalytic currents are 24.71 μA and 13.92 μA for the hydrophobic interface and hydrophilic interface, respectively (Figure 4b,c, open circular dot), which are, respectively, 4 and 6.1 times larger than those found with air (l)/N2 (g). This result indicates that the reactivity of immobilized GOx can be enhanced by diffusing oxygen through the PAA nanochannels to the gas−solid−liquid interface. Control experiments are performed by using N2 (l)/(N2 or air) (g). No obvious oxidation currents are observed in N2 (l)/ N2 (g) for both the hydrophobic and hydrophilic interfaces (Figure 4b,c, triangle solid dot). In N2 (l)/air (g), a doseindependent oxidation current is observed, and the oxidation currents are 20.652 μA and 8.386 μA for the hydrophobic interface and hydrophilic interface (Figure 4b, open circular dot), respectively, which are respective increases of 3.3 and 3.6 times compared to air (l)/N2 (g); however, these currents are smaller than those for air (l)/air (g). These results illustrate 10444
DOI: 10.1021/jacs.7b05249 J. Am. Chem. Soc. 2017, 139, 10441−10446
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Journal of the American Chemical Society Table 1. Kinetic Parameters of Immobilized GOx at Controlled Wettability Interface under Different Conditionsa air saturated solutionb N2 Icat (μA) Kapp m (mM) Vmax (mM s−1) kcat (s−1) −1 −1 kcat/Kapp s ) m (M a
N2 saturated solution air
air
O2
NDM
MUA
NDM
MUA
NDM
MUA
NDM
MUA
6.19 64.5 28.86 15.3 238
2.28 100.6 2.47 1.3 13
24.71 21.84 58.46 42.3 1940
13.92 26.43 51.84 28.8 1090
20.65 31.9 45.1 33.5 1050
8.38 54.48 37.08 19.2 354
52.11 16.88 92.5 67 3970
39.99 20.56 88.9 49.18 2392
All values for the hydrophobic interface are better than those for the hydrophilic interface. bSolution is 100 mM PBS, pH 7.4.
substrate than GOx/NDM/AuNPs/NDM/Au/PAA. This further confirms that the hydrophobic environment that is supplied by NDM at the first hydrophobic modification allows the O2 molecules to transport through the nanochannels of the PAA membrane freely; then, the O2 molecules reside and reach the active site of GOx to enhance the enzymatic reactivity. Additionally, the observed oxidation current for GOx/NDM/ Au/PAA is also much smaller than that of GOx/NDM/ AuNPs/NDM/Au/PAA. These results illustrate that the constructed nanochannels with joint gas−solid−liquid interface and controlled wettability resolves the gas-deficit problem and supplies a nanochannel for gas molecules to enhance catalytic reaction of enzyme.
that the reactivity of the enzyme is enhanced by oxygen diffusion from both the liquid and gas phases but that the latter phase is more effective. To confirm the influence of the concentration of oxygen, the gas chamber is filled with pure oxygen while the liquid chamber is saturated with N2 or air (Figure 4c, solid circle dot and open square dot), and the oxidation currents are further improved up to 2.5 and 13 times (Table 1 and Table S1), compared to those when the gas chamber contained air and N2 (Figure 4b, blue solid circular dot and blue open circular dot), respectively. This result suggests that the reactivity of GOx can be controlled by the accessibility of oxygen from the gas phase. To gain further insights, the kinetic parameters of the enzymatic reactions occurring at the interfaces with different wettability were evaluated. For each enzymatic reaction, the dose-dependent plots show that at high glucose concentrations a plateau response is obtained, which is a characteristic of the Michaelis−Menten kinetic mechanism (Figure 4).48 According to the Lineweaver−Burk equation,52 the parameters of the enzymatic reaction kinetics of the interfaces with different wettabilities, including the apparent Michaelis−Menten constant (Kmapp), catalytic reaction rate constant (kcat), and maximum reaction rate (Vmax) are summarized in Table 1. The Kapp m is a reflection of the enzymatic affinity for substrate, 24 and a high Kapp m value represents a weak affinity and vice versa. app In addition, the rate parameter kcat/Km can be used to monitor the enzymatic reaction.53 As can be seen from Table 1, the kcat/ Kapp m values of GOx immobilized at the hydrophobic interface in N2 (l)/air (g) (1050) is 80-fold higher than that at the hydrophilic interface in air (l)/N2 (g) (13). For the hydrophobic interface, the kcat/Kapp m values are ordered in the sequence 6256 (air (l)/O2 (g)) > 3970 (N2 (l)/O2 (g)) > 1940 (air (l)/air (g)) > 1050 (N2 (l)/air (g)) > 238 M−1 s−1 (air (l)/ N2 (g)). For hydrophilic interface, the kcat/Kapp m values are ordered in the sequence 3156 (air (l)/O2 (g)) > 2392 (N2 (l)/ O2 (g)) > 1090 (air (l)/air (g)) > 354 (N2 (l)/air (g)) > 13 M−1 s−1 (air (l)/N2 (g)) (Table 1 and Table S1). These results illustrate that the reactivity of GOx strongly depends on the O2 concentration, that high reaction kinetics can be effectively achieved by increasing the diffusion of O2 through the gas phase, and that the hydrophobic interface preferably enhances the reactivity of GOx. At high glucose concentration, the oxygen concentration in the gas phase determines the amount of the oxygen that reaches the active site, which is the pseudorate-limiting step for the overall enzymatic reaction. It should be noted that the hydrophobic molecules and electrodeposited AuNPs are also two critical elements for enhancing enzymatic reactions. Figure S11 shows that the GOx adsorption at NDM/AuNPs/Au/PAA (GOx/NDM/AuNPs/ Au/PAA) exhibits a lower oxidation current for the β-D-glucose
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CONCLUSIONS In summary, we have fabricated a nanochannel reactor with joint gas−solid−liquid interfaces and controlled wettability for boosting gas involving enzymatic reactions. The conformation and orientation of the immobilized enzyme at the proposed interface can be facilely manipulated with improved affinity for gases and the ability to allow the gas substrates to transport through the nanochannels to directly reach the active sites of enzymes from the gas phase. Our results show that in the presence of oxygen from air, the catalytic efficiency of the enzyme can be improved up to 80 times, compared with that in the presence of the oxygen from solution. Moreover, the accessibility of oxygen from the gas phase can be regulated to further improve the reaction efficiency. Therefore, this work may provide a novel strategy for potential applications in gas sensors and industrial catalysis with gas substrates.
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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/jacs.7b05249. Additional experimental details (methods, SEM, contact angle and electrochemistry), tables (Supporting Table 1), and figures (Supporting Figures 1−11) (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*
[email protected] *
[email protected] ORCID
Yuanjian Zhang: 0000-0003-2932-4159 Ye Tian: 0000-0002-1913-6333 Songqin Liu: 0000-0002-4686-5291 Lei Jiang: 0000-0003-4579-728X 10445
DOI: 10.1021/jacs.7b05249 J. Am. Chem. Soc. 2017, 139, 10441−10446
Article
Journal of the American Chemical Society Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully appreciate the support from National Natural Science Foundation of China (Grants 21635004, 21627806, and 21671194).
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DOI: 10.1021/jacs.7b05249 J. Am. Chem. Soc. 2017, 139, 10441−10446
