J. Phys. Chem. C 2008, 112, 14781–14785
14781
Adsorption of Laccase on the Surface of Nanoporous Gold and the Direct Electron Transfer between Them Huajun Qiu,† Caixia Xu,† Xirong Huang,*,† Yi Ding,† Yinbo Qu,‡ and Peiji Gao‡ Key Laboratory of Colloid and Interface Chemistry of the Ministry of Education of China and State Key Laboratory of Microbial Technology of China, Shandong UniVersity, Jinan 250100, People’s Republic of China ReceiVed: June 25, 2008; ReVised Manuscript ReceiVed: July 23, 2008
Nanoporous gold (NPG) with different pore sizes was obtained by simple dealloying and thermal annealing methods. The morphology of the NPG was characterized by scanning electron microscopy and nitrogen adsorption technique. Laccase was immobilized on the surface of the NPG by physical adsorption. Detailed studies were made on the effect of the pore size on laccase immobilization. NPG with pore size of 40-50 nm was demonstrated to be a suitable support for laccase immobilization. Compared with free enzyme, the optimum pH of immobilized laccase did not change; the optimum temperature, however, rose from 40 to 60 °C. Both thermal and storage stabilities of laccase improved markedly via the immobilization. Laccase immobilized on NPG (100 nm in thickness) was used for enzyme electrode construction. Direct electrochemistry of laccase on NPG supported by glassy carbon electrode (NPG/GC) was achieved with high efficiency due to the outstanding physicochemical characteristics of the NPG. The laccase-loaded NPG/GC electrode also exhibited a strong electrocatalytic activity toward O2 reduction. When stored at 4 °C for 1 month, the electrode showed no obvious changes in its response. All results presented in the paper indicated that NPG was an excellent carrier for laccase immobilization and would have potential applications in biofuel cell and/or biosensor areas. Introduction Laccase is a multicopper oxidase which contains four copper ions classified into three types (T1, T2, T3) in accordance with their spectroscopic characteristics. During the enzymatic reaction, one electron from the substrate is first transferred to the copper ion at the T1 site, the primary electron acceptor of the enzyme; then it is transferred through an intramolecular electron transfer mechanism via a His-Cys-His bridge to the copper ions at the T2/T3 site, where oxygen is reduced to water.1 Laccase is able to oxidize a large number of organic and inorganic substrates with concomitant reduction of molecular oxygen to water.2 So it has great potential applications in the fields of biosensor and biofuel cell, etc. Immobilization of laccase on an appropriate support is a crucial step for the construction of mediator-free biosensor or biofuel cells,3-5 and many attempts have been made in this field.6-9 In recent years, the use of nanomaterials for enzyme immobilization and enzyme electrode construction has attracted a great attention. Among the nanomaterials, gold nanoparticle has been widely used due to its biocompatibility, high specific surface area, and reaction activity, etc.10-12 However, it has some demerits when used for this purpose. For example, it is difficult to separate it from the bulk solution even by ultracentrifugation, and it tends to agglomerate over time under operating conditions. This tendency results in a reduction of its active surface area and the lifetime of a constructed device even if the nanoparticle was covalently adsorbed on the surface of electrodes.13,14 Nanoporous gold (NPG) is a new kind of nanomaterials. This nanomaterial can be obtained by etching commercially available * To whom correspondence should be addressed. Telephone: +86 531 88365433. Fax: +86 531 88564750. E-mail:
[email protected]. † Key Laboratory of Colloid and Interface Chemistry of the Ministry of Education of China. ‡ State Key Laboratory of Microbial Technology of China.
Au/Ag alloy with concentrated nitric acid.15-17 The NPG thus obtained has the following characteristics: (1) it is a conductor with large surface area; (2) it is truly support-free; (3) the preparation methodology is simple and reproducible; (4) the pore size is tunable in a wide range from a few nanometers to several microns; (5) its nanostructured surfaces are very clean. In the present work, an NPG sheet was used to immobilize laccase and then to construct a laccase electrode. In addition to the general enzymatic properties, the electrochemical and electrocatalytic behaviors of the immobilized laccase were also investigated. Experimental Section Chemicals. Laccase from Trametes Versicolor, Nafion, and 2,6-dimethoxyphenol (DMP) were purchased from Sigma. Au/ Ag alloy (50:50, wt %) sheets with thickness of 25 µm and 100 nm were purchased from Changshu Noble Metal Co. Coomassie brilliant blue G-250 was a product of Sanland-chem International Inc. Other chemicals used were of analytical grade. A 0.1 M phosphate-citric acid buffer solution and triply distilled water were used throughout the experiments. Preparation and Characterization of NPG. Dealloying of Au/Ag alloy was carried out in concentrated nitric acid for a certain period of time. The resulted samples were then washed to the neutral pH with triply distilled water. The microstructure of NPG was characterized with a JEOL JSM-6700F field emission scanning electron microscope, which was equipped with an Oxford INCA x-sight energy-dispersive X-ray spectrometer (EDS) for compositional analysis. The surface area of NPG used was measured with Quadrasorb SI-MP (Quantachrome Instrument) using the BET method. Immobilization of Laccase in NPG. NPG was kept in 2 mL of 7 mg mL-1 laccase solution at 4 °C for 24 h; then it was rinsed 5 times with 10 mL buffer solution (pH 4.4) to remove
10.1021/jp805600k CCC: $40.75 2008 American Chemical Society Published on Web 08/30/2008
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TABLE 1: Changes of the Amount of Immobilized Laccase and the Specific Activity with the Pore Size of NPGa
a
no.
pore size/nm
specific surface area/(m2 g-1)
amount of immobilized laccase/(mg g-1)
specific activity (U/(mg of protein))
sample 1 sample 2 sample 3
10-20 40-50 90-100
20.9 ( 0.2 14.4 ( 0.2 8.7 ( 0.2
7.2 ( 0.3 15.5 ( 0.3 10.6 ( 0.3
0.43 0.79 0.81
NPG with thickness of 25 µm.
weakly adsorbed enzyme on the outer surface and stored at 4 °C in a refrigerator. The amount of the enzyme immobilized on the support was calculated on the basis of the difference between the amount of protein added and that recovered in the supernatant and washing buffer. Protein concentration was determined by the Bradford method, using bovine albumin as a standard. Determination of Laccase Activity. The activity of the free and immobilized laccase was determined spectrophotometrically on the basis of the absorbance change at 470 nm at 30 °C. An appropriate amount of laccase (5 µL of free enzyme or 5 mg of laccase-loaded NPG (in fragments)) was mixed with stirring with 1 mL buffer solution (pH 2.4-7.0) and 100 µL of 10 mM DMP. After 5 min, the absorbance of the supernatant was determined using a Shimadzu UV-2550 spectrophotometer. The molar extinction coefficient of the oxidation of DMP at 470 nm was 49.6 mM-1 cm-1. One unit of activity was defined as the amount of enzyme required to have 1 µmol of DMP oxidized in 1 min. Thermal Stability and Reusability. The thermal stability was evaluated by incubating free and immobilized laccase in buffer solution (pH 4.4) at 50 °C. At given time intervals, 5 µL of free enzyme or a separate cuvette containing 5 mg of laccaseloaded NPG was taken out and left for 1 h at room temperature. The residual activity was then assayed at 30 °C. The optimal activity of laccase was taken as 1. For repeated use, the immobilized enzyme (5 mg) was taken out from the reaction mixture (pH 4.4) after 10 min reaction at 30 °C and rinsed three times with buffer solution (pH 4.4). Then the immobilized enzyme was added into the same but fresh reaction solution for the next use. Each time, the enzyme was taken out, the reacted solution was immediately poured into a cuvette for absorbance measurement at 470 nm. Leaching Test. A 5 mg laccase-loaded NPG of different pore sizes was added into 1 mL buffer solution (pH 4.4) and incubated for 1 h; then the NPG was removed, and 100 µL of 10 mM DMP was added. The light adsorption at 470 nm was recorded for 2 min to characterize the leached amount of laccase. Electrode Preparation. The laccase-loaded NPG/GC electrode was made by affixing the laccase-loaded NPG sheet (100 nm in thickness) to a GC electrode (3 mm in diameter). After that, 2 µL of 5% (wt %) Nafion was spread on the outer surface to avoid any enzyme leaching and NPG sheet falling off. The modified electrode was left to dry at 4 °C and stored at 4 °C in a refrigerator when not in use. The laccase-modified gold sheet electrode was prepared in the same way for control experiment. Electrochemical Measurement. Cyclic voltammetric experiments were performed on a CHI 1130A electrochemical workstation (CH Instrument Co, Shanghai, China) at room temperature (∼20 °C). A three-electrode system was used, including a modified electrode as working electrode, a platinum gauze as counter electrode and a saturated calomel electrode (SCE) as reference electrode. The test solutions were thoroughly deoxygenated with pure nitrogen for about 30 min prior to each experiment. All potential values were vs SCE unless otherwise specified.
Results and Discussion Characterization of NPG. During the dealloying process, upon sliver dissolution, gold atoms left behind would selforganize into an interconnected network of pores and ligaments. One important characteristic of NPG was that its structural unit (pore/ligament size) was tunable by varying the initial alloy composition or etching time or by employing thermal annealing after dealloying. This porous framework was highly desirable for free transport of small molecules in three dimensions. Also it may be a sound support for the immobilization of biomacromolecule,18 e.g., enzyme, nucleic acid, etc. In the present work, NPGs with uniformly distributed pore sizes of 10-20 (sample 1) and 40-50 nm (sample 2) were obtained by submerging Au/Ag alloy (25 µm in thickness) in concentrated nitric acid for about 1 and 17 h, respectively. NPG with pore size of 90-100 nm (sample 3) was obtained by thermal annealing of sample 2 at 200 °C for 1 h. The compositional analysis showed except for sample 1 (its atom ratio of Au to Ag was 27:1), Ag atoms in samples 2 and 3 were almost completely removed. For enzyme electrode construction, the Au/Ag alloy with a thickness of 100 nm was used and a time interval of 1.5 h was set for dealloying to obtain an NPG with a pore size of 40-50 nm. Scanning electron microscopy (SEM) showed that this NPG had the same morphology as sample 2. Effect of the Pore Size of NPG on the Immobilization of Laccase. As shown in Table 1, NPG with smaller pore size had larger specific surface area. However, the least amount of immobilized laccase and specific activity were found in sample 1. The main reason was that the pore size in sample 1 was not large enough for easy entrance of free hydrated laccase (its average diameter was around 7 nm).19 On the other hand, the laccase immobilized on the outer surface would block free laccase from accessing the inner surface of the NPG. After the immobilization of laccase, the pore size of sample 1 was reduced to several nanometers; the diffusion of the substrate DMP in and its oxidation product out of the pores would be more difficult, which resulted in the lowest specific activity in sample 1. The fact that the specific activities in samples 2 and 3 were almost equal indicated that there was no obvious mass-transfer resistance in sample 2 as compared with sample 3. In addition, more laccase was immobilized in sample 2 due to its larger specific surface area than sample 3. So sample 2 seemed to be a suitable support for laccase immobilization. Figure. 1 showed the SEM images of sample 2 before and after laccase immobilization. Due to the partial coverage of laccase on the outer surface of NPG, the brightness of Figure 1b was a little lower than that of Figure 1a. Compared with bare NPG (Figure 1a), laccase-loaded NPG (Figure 1b) also had smaller pore sizes and exhibited a relatively smoother surface morphology, suggesting a preferential immobilization of laccase over the ligament site with high radial curvatures. A similar phenomenon was reported elsewhere.20
Adsorption of Lacrosse on Nanoporous Gold Surface
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Figure 1. SEM images of sample 2 before (a) and after (b) laccase immobilization.
Figure 3. Changes of the residual activity of laccase with the incubation time at 50 °C: -0-, immobilized enzyme; -9-, free enzyme.
Figure 2. Changes of absorbance at 470 nm with time for the oxidation of DMP by laccase from leaching solutions of three immobilized laccases: (a) laccse sample 3; (b) laccase sample 2; (c) laccase sample 1.
The leaching test showed that the amount of laccase leached from the three samples followed the order sample 3 > sample 2 > sample 1 (see Figure 2). Apparently, laccase was physically absorbed on the surface of NPG, but some chemical adsorption seemed to have occurred. It was well-accepted that, in addition to sulfur atoms, lateral amido of amino acid residue could bind strongly to the surface of nanoscale gold.21 In addition, a small pore size was an unfavorable factor for laccase escape from the inner surface. Thus, NPG with pore size of 40-50 nm was selected and used in the following experiments. Effect of Buffer pH and Temperature. The immobilized laccase and the free counterpart had similar pH-activity profiles with the same optimum at pH 4.4. However, at pH 4.4, the optimum temperature for the activity of the immobilized laccase was 60 °C, which was ca. 20 °C higher than the free enzyme. This result should be attributed to the protecting effect of the formed nanoscale pore channels on the conformation of laccase. Thermal Stability, Reusability, and Storage Stability. As shown in Figure 3, the activity of the free enzyme decreased dramatically after incubation at 50 °C and 2 h later, only 6% of its initial activity remained. Under the same conditions, however, the activity of the immobilized laccase decreased slowly and about 60% of initial activity remained after 2 h incubation at 50 °C. This might be due to the multipoint attachment between the enzyme molecule and the concave pore surface,22-24 thereby stabilizing the three-dimensional structure of enzyme. A similar stabilizing effect on acetylcholine esterase immobilized on nanoporous silica beads and nanoporous carbon was also observed by Sotiropoulou et al.24 The residue activity of the immobilized laccase after repeated use at 30 °C and pH 4.4 was shown in Figure 4. The repeated
Figure 4. Residual activity of the immobilized laccase after repeated uses at 30 °C and pH 4.4.
use decreased the activity of the immobilized enzyme, but ca. 65% of initial activity was retained after eight cycles. When stored at 4 °C for 1 month and then assayed at the optimum reaction conditions, the immobilized enzyme did not lose any activity, but the free enzyme lost 30% of its initial activity, indicating that the storage stability of laccase increased considerably after immobilization on NPG. In addition, the denatured laccase on NPG could be easily removed simply by immersing the NPG into concentrated nitric acid (ca. 1 min), thereby realizing the recycle of the carrier NPG. Electrochemical Behavior of Laccase on NPG/GC Electrode. To explore the electrochemical behavior of laccase on NPG, the cyclic voltammograms of both the laccase-loaded NPG/GC electrode and the laccase-modified gold sheet electrode were recorded. As shown in Figure 5, laccase-modified gold sheet electrode did not exhibit discernible peaks under the given condition. This was because, on the conventional gold sheet electrode, the electron transfer between laccase and the electrode surface was usually limited due to the deep entombing of electroactive group in the enzyme structure, the improper orientation of enzyme on the surface and/or the denaturization of enzyme when adsorbed on the surface. However, the laccaseloaded NPG/GC electrode produced a pair of well-defined and quasi-reversible peaks with the formal potential (E° ) (Epa + Epc)/2) and ∆E p of 309 and 73 mV, respectively, at a scan rate of 0.1 V s-1. Control experiments showed that, under the same
14784 J. Phys. Chem. C, Vol. 112, No. 38, 2008
Figure 5. Cyclic voltammograms of two different electrodes in 0.1 M phosphate-citric acid buffer solution (pH 4.4; scan rate, 0.1 V s-1): (a) laccase-loaded NPG/GC electrode; (b) laccase modified gold sheet electrode. The pH value of the supporting electrolyte was selected base on its influence on the peak current.
Figure 6. Cyclic voltammograms of laccase-loaded NPG/GC electrode in 0.1 M phosphate-citric acid buffer solution (pH 4.4). Scan rate: (aff). 25, 50, 75, 100, 125, and 150 mV s-1. Inset: plots of anodic and cathodic peak currents versus scan rate.
conditions, the unloaded NPG/GC electrode and laccase-loaded but denatured NPG/GC electrode did not show any peaks. Obviously, this difference in the direct electron transfer between the enzyme and the electrode surface was closely correlated with the surface properties of carrier materials. It was reported that nanoscale gold could promote the electron transfer between the electrode and enzymes because of its outstanding physicochemical characteristics. In addition to more loaded laccase on NPG, a sound microenvironment provided by nanoscale pore channels of NPG should be responsible for the effective direct electron transfer. Further investigation indicated that the electron-transfer process was a surface-controlled process. As shown in Figure 6, a perfect linear line was obtained when the anodic and cathodic peak currents were plotted separately against the scan rate from 0.025 to 0.15 V s-1. The regression coefficients for cathodic and anodic peak currents were 0.998 and 0.997, respectively. On the basis of the following equation,25 Q ) nFAΓ*, the average surface coverage (Γ*) of the electroactive laccase immobilized on NPG/GC was estimated to be 2.1 × 10-11 mol cm-2 (n ) 1). This value was larger than the
Qiu et al.
Figure 7. Cyclic voltammograms of laccase-loaded NPG/GC electrode in (a) deaerated and (b) air-saturated 0.1 M phosphate-citric acid buffer solution (pH 4.4). Scan rate: 0.1 V s-1.
theoretically calculated value (4.3 × 10-12 mol cm-2) for monolayer coverage of laccase. This result indicated that more laccase was immobilized on the NPG due to its large nanoscale surface area. Furthermore, NPG contained a large number of edge-plane-like defective sites and a good electron-conductive network, which would promote greatly the direct electron transfer between the immobilized laccase and the electrode. Electrocatalysis. As shown in Figure 7, the voltammograms were quite different in the absence and presence of oxygen. In air-saturated solution, the cathodic peak of the laccase-loaded NPG/GC electrode occurred at about 0.02 V. This potential value, which corresponded to the reduction of oxygen,26 was much higher than that on bare gold sheet electrode (-0.7 V) or on NPG/GC electrode (-0.55 V; figure not shown). In the case of peak currents, the order was as follows: the laccase-loaded NPG/GC . the unloaded NPG/GC > the bare gold sheet. All these results indicated that the present laccase-loaded NPG/GC electrode had strong electrocatalytic activity toward oxygen reduction. Stability of the Present Laccase Electrode. After scanning 200 cycles in succession, the voltammograms of the laccaseloaded NPG/GC electrode were almost unchanged. After 1 month storage at 4 °C, no obvious change was observed in the peak current and potential. For biosensor construction, the present strategy had some advantages. Conclusions NPG with pore size of 40-50 nm was demonstrated to be a suitable support for laccase immobilization. Both thermal and storage stabilities improved markedly via the immobilization. Compared with laccase-modified gold sheet electrode, direct electrochemistry was achieved on laccase-loaded NPG/GC due to the special physicochemical characteristic of NPG. The electroreduction of oxygen was efficiently promoted by the laccase-loaded NPG/GC electrode according to the changes of peak current and peak potential. When stored at 4 °C for 1 month, the present enzyme electrode showed no obvious changes in its response. Acknowledgment. This work was financially supported by the National Natural Science Foundation of China (Grant No.30570014) and National Basic Research Program of China (Grant No.2007CB93).
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