Article pubs.acs.org/ac
Inorganic/Organic Doped Carbon Aerogels As Biosensing Materials for the Detection of Hydrogen Peroxide Sheying Dong,*,† Nan Li,† Gaochao Suo,† and Tinglin Huang‡ †
College of Sciences, Xi′an University of Architecture and Technology, Xi′an 710055, People’s Republic of China School of Environmental and Municipal Engineering, Xi′an University of Architecture and Technology, Xi′an 710055, People’s Republic of China
‡
S Supporting Information *
ABSTRACT: In this article, three different inorganic/organic doped carbon aerogel (CA) materials (Ni-CA, Pd-CA, and Ppy-CA) were, respectively, mixed with ionic liquid (IL) to form three stable composite films, which were used as enhanced elements for an integrated sensing platform to increase the surface area and to improve the electronic transmission rate. Subsequently, the effect of the materials performances such as adsorption, specific surface area and conductivity on electrochemistry for myoglobin (Mb) was discussed using N2 adsorption−desorption isotherm measurements, scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). Moreover, they could act as sensors toward the detection of hydrogen peroxide (H2O2) with lower detection limits (1.68 μM, 1.02 μM, and 0.85 μM, for Ni-CA/IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and PpyCA/IL/Mb-CPE, respectively) and smaller apparent Michaelis−Menten constants KM. The results indicated that the electroconductibility of the doped CA materials would become dominant, thus playing an important role in facilitating the electron transfer. Meanwhile, the synergetic effect with [BMIm]BF4 IL improved the capability of the composite inorganic/ organic doped CA/IL matrix for protein immobilization. This work demonstrates the feasibility and the potential of a series of CA-based hybrid materials as biosensors, and further research and development are required to prepare other functional CAs and make them valuable for more extensive application in biosensing.
T
and graphene-based materials, CA and CA-based hybrid materials have a great potential in electrocatalysis, especially for fabricating electrochemical sensors based on enzymes or proteins. Hence, it will have a potential research value to bring them in electrochemical sensors to immobilize enzymes and proteins. In the construction of carbon-based hybrid materials, metal nanoparticles such as Ni and Pd were always preferred for their high electrocatalytic behavior to biomolecules in sensing application.5,6 Meanwhile, conducting polymers can be exploited as an excellent substance for the preparation of nanocomposites with nanoscaled biomolecules. Thereinto, polypyrrole (Ppy) is one of the most extensively used conducting polymers in the design of bioanalytical sensors7 because of its sufficient conductibility, selectivity, stability, sensitivity, and biological affinity. Accordingly, it may be beneficial and practical to prepare different inorganic/organic functionalized CA nanocomposites as biosensing materials. When the CA is doped with Ni, Pd, or Ppy, functionalization
he development of materials science and nanotechnology has brought a great momentum to bioelectroanalysis and analysts. In the recent decades, scientists are always enthusiastic about finding materials with good biocompatibility to improve the behavior of biosensors.1 Generally, the immobilization of biomolecules in designer-made nanoscale structures can significantly improve the performance of biocatalytic processes. From the viewpoint of the immobilization of enzymes and proteins possessing relatively high molecular weight and large molecular diameter, mesoporous carbon, with the diameter in the range from 2 to 50 nm, may be a potential candidate to accommodate these biomolecules. Recently, carbon aerogel (CA), as an important three-dimension nanomaterial, has attracted tremendous scientific and technological attention since it was first patented by Pekala.2 To date, most of the CA studies have focused on electric double layer of supercapacitors.3 There is only limited research on the immobilization of large biomolecules by CA.4 Electrodes modified by CA and a class of CA-based hybrid materials for biosensors have not received sufficient attention. In view of the versatile properties of CA including outstanding electrical conductivity, large surface area, fine pore size, and high porosity, which are similar to those of other carbon nanomaterials such as graphene © 2013 American Chemical Society
Received: May 19, 2013 Accepted: November 27, 2013 Published: November 27, 2013 11739
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Scheme 1. Schematic Diagram of the Procedure to Construct the Ni-CA/IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/ Mb-CPE
conventional three-electrode system comprised of a platinum wire auxiliary, a saturated calomel electrode (SCE) reference, and the modified carbon paste electrode (CPE) working electrode. The morphology of the electrode surface was determined by SEM (Quanta 600FEG) at an accelerating voltage of 15 kV. Ultraviolet−visible (UV−vis) spectra were recorded on a Nicolet Evolution 300 spectrophotometer in 0.1 M PBS, and the IR spectra (KBr) were recorded on an IRspectrometer FTIR-8400S (Shimadzu) at room temperature. CA was prepared under ambient conditions according to the method reported in the literature.16 However, microwaveassisted heating was adopted during the resorcinol-formaldehyde reaction in this study to shorten the reaction time by 4 days, compared with using conventional heating. The nanosized Ni doped CA (Ni-CA), Pd doped CA (Pd-CA), and Ppy doped CA (Ppy-CA) nanocomposites were successfully fabricated via chemical precipitation17 and chemical redox polymerization,18 which are described in the Supporting Information. Preparation of the Modified Electrodes. CPE was fabricated as follows: 0.6 g of liquid paraffin and 3.4 g of graphite powder were hand-mixed to produce a homogeneous paste. The prepared carbon paste was then firmly packed into a PVC tube (3 mm internal diameter). A copper wire (1.5 mm external diameter) was introduced into the other end for electrical contact. The CPE surface was carefully smoothed on a weighing paper before use. The enzyme electrode was prepared according to the following procedure: first, 5 μL of Mb solution (5 mg/mL) was casted onto the surface of a freshly polished CPE, and the modified electrode was dried in a refrigerator at 4 °C to obtain Mb-CPE. Second, 5 mg of doped CA (Ni-CA, Pd-CA, and PpyCA) and 10 μL of [BMIm]BF4 IL were dispersed into 1 mL of 0.1 M PBS (pH 7.0). After sonication for 30 min, 10 μL of the above suspension was pipetted onto Mb-CPE to obtain dopedCA/IL/Mb-CPE (Ni-CA/IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/Mb-CPE), which was subsequently evaporated in a refrigerator at 4 °C to form a stable film. Finally, the modified electrode was rinsed twice or thrice with doubly distilled water to remove the unimmobilized Mb molecules. When not in use, the enzyme electrode was stored in 0.1 M PBS (pH 7.0) at 4 °C in a refrigerator. In addition, the enzyme electrode was also constructed in simpler ways, and the preparation process was described in the Supporting Information. Electrochemical Measurements. EIS measurements were carried out in 5.0 mM K3 Fe(CN) 6 /K 4 Fe(CN) 6 (1:1) containing 0.1 M KCl, while the applied perturbation amplitude was 0.005 V, the frequencies swept from 105 to 10−2 Hz.
effects occur, leading to the formation of doped materials with high conductivity. Most importantly, these factors should be responsible for the improvement of electrocatalytic performance of CA nanomaterials to some extent. This research encouraged us to explore appropriate CA-based hybrid materials to improve the performance of biocatalytic processes. In addition, previous research demonstrated that ionic liquid (IL) is a kind of ideal matrix that can disperse the nanomaterials into the modified layers to improve the stability and promote the electrocatalysis of protein.8 In recent years, enzyme/protein-modified electrodes for the determination of H2O2 have been frequently applied,9 owing to the advantages such as simple equipment, low operating expense, and fast analytical time. The modified electrode materials including metals,10 transition metal oxides,11 redox dyes,12 conducting polymers,13 and graphene14 were used to construct biosensors for H2O2. Nevertheless, it has still some deficiencies such as enzymes or proteins denaturation, low reproducibility, and stability.15 In the current study, we describe the use of three different doped CAs (Ni-CA, Pd-CA, and Ppy-CA) as the sensing platform in the detection of H2O2. Performance metrics such as adsorption and electrochemical conductivity of materials modified with Mb were reported along with a N 2 adsorption−desorption isotherm, scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). As expected, the synergistic performance of the proteins, inorganic/organic doped CA materials, as well as IL provides an extensive platform and opens up a new avenue for fabricating excellent electrochemical biosensors. The modification process for the Mb modified electrodes is shown in Scheme 1.
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EXPERIMENTAL SECTION Reagents and Apparatus. Bovine myoglobin (Mb, MW 17 800) was purchased from Sigma Chemical Co., and [BMIm]BF4 IL was purchased from Hangzhou Chemer Chemical Limited Company. High purity graphite powder was obtained from China National Medicine Corporation. K3Fe(CN)6, K4Fe(CN)6, KCl, K2HPO4, KH2PO4, KOH, Na2CO3, FeCl3·6H2O, NiCl2·6H2O, C6H6O2, HCHO (40%), H2O2 (30%), and liquid paraffin were obtained from Xi’an Chemical Reagent Corporation. C4H5N, PdCl2, and sodium dodecylsulfate (SDS) were obtained from China National Medicine Corporation. All chemicals were of analytical reagent grade. A 0.1 M phosphate buffer solution (PBS, pH 7.0) was used. All solutions were made up with twice-distilled water. Electrochemical measurements were performed with a CHI660B workstation (Shanghai Chenhua Co., China) with a 11740
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Figure 1. (A) N2 adsorption−desorption isotherms and (B) the resultant pore size distribution of CA, Ni-CA, Pd-CA, and Ppy-CA.
particles ranged from 15 to 30 nm in Figure 2A. It was found in Figure 2B,C that the skeleton particles with Ni and Pd nanoparticles doped CA materials were larger and more compact than CA, which were attributed to the reintegration of metal particles when doped CA was calcined at a high temperature. While the micrograph of Ppy-CA in Figure 2D showed porosity with wide size range. The surface morphologies of these CA-based hybrid materials were composed of microbead particles, which resulted from the interlinked micelles formed in the presence of a surfactant. From the surface morphology of different modified electrodes, it could be observed that the surface of CPE was formed by irregularly shaped flakes, whereas the scrappy particles in Figure 2B′, C′, and D′ derived from the adsorption of Mb onto the surface of inorganic/organic doped CA. Obviously, compared with the SEM images of CA and inorganic/organic doped CA materials, the stable composite films of the modified electrodes in all cases exhibited higher hierarchy and porosity, which could provide a specific interface for the adsorption and immobilization of protein molecules. UV−Visible and Fourier Transform-Infrared (FT-IR) Spectra Results. To investigate qualitatively the biological activity of Mb before and after immobilization on the inorganic/organic doped CA/IL composite films, the changes in the Soret band were observed by monitoring the absorption band in the prosthetic heme-group region of the composite films.19 The discussion of UV−vis (Figure S1 in the Supporting Information) and some references indicated that the presence of graphite and [BMIm]BF4 IL did not denature the Mb. Moreover, as shown in Figure 3A, the Soret bands of Mb in different solutions were 408.9 nm (CA/IL/Mb), 408.6 nm (NiCA/IL/Mb), 408.7 nm (Pd-CA/IL/Mb), and 408.4 nm (PpyCA/IL/Mb). Compared with Mb in the buffer (408.4 nm), Mb retained its native structure in the presence of inorganic/ organic doped CA and [BMIm]BF4. The CA/IL/Mb, Ni-CA/ IL/Mb, Pd-CA/IL/Mb, and Ppy-CA/IL/Mb films were also analyzed by FT-IR. As shown in Figure 3B, the spectrum of free Mb (d) was similar to that of Mb in CA/IL (a′), Ni-CA/IL (a), Pd-CA/IL (b), and Ppy-CA/IL (c). These results confirmed that the secondary structure of Mb retained the essential feature of its native state.20 The UV−vis and FT-IR results supported the conclusion that the native structure of Mb had been retained. Thus, inorganic/organic doped CA and [BMIm]BF4 IL composite matrixes provided an excellent platform for the further study of the direct electrochemistry of Mb. EIS Characterization. Alternating current impedances were used to show the impedance change while preparing different
Amperometric experiments were performed in a constantly stirred cell with the successive addition of H2O2 into 20.0 mL of 0.1 M PBS supporting electrolyte (pH 7.0) at 25 °C. The electrode potential was set at −0.35 V vs RE (SCE). Experimental solutions were deoxygenated by purging with high pure nitrogen for 30 min and maintained under nitrogen atmosphere during the measurements.
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RESULTS AND DISCUSSION Textural Properties of CA and Inorganic/Organic Doped CA. The textural properties of CA and three doped CA materials were examined by N2 adsorption−desorption isotherm measurements. A typical adsorption−desorption isotherm (Figure 1A) exhibited type IV according to IUPAC with H2 hysteresis loops, indicating the presence of mesopores. Meanwhile, the samples exhibited a narrow mesopore size distribution, as indicated by a steep capillary condensation step of the isotherms and the Barrett−Joyner−Halenda (BJH) pore size distribution (Figure 1B). On this basis, Table 1 compares Table 1. Textual Properties of CA and Three Doped CA Materials samples
BET surface area (m2/g)
average pore diameter (nm)
pore volume (cm3/g)
CA Ni-CA Pd-CA Ppy-CA
747 682 583 485
7.9 8.3 8.3 8.0
0.61 0.58 0.52 0.43
the textural properties of CA and three doped CA materials. From the results in Figure 1 and Table 1, it can be concluded that the surface areas of three CA-based hybrid materials increase in the order Ni-CA > Pd-CA > Ppy-CA. Although Brunauer−Emmett−Teller (BET) surface area and pore volume slightly decreased after the impregnation with Ni, Pd, and Ppy nanoparticles, the textural properties of CA were retained. Characterization of the Electrodes Modified by Inorganic/Organic Doped CA. To investigate the effects of CA and inorganic/organic doped CA materials microstructure on the fabrication of the modified electrodes, SEM images of the CA hybrid materials and their corresponding modified electrodes were determined in Figure 2. The SEM images showed the CA skeletal structure consisted of interconnected nanosized carbon ligaments that defined a continuous polyporous network. The diameter of most of the spherical 11741
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Figure 2. SEM images of (A) CA, (B) Ni-CA, (C) Pd-CA, (D) Ppy-CA, (A′) CPE, (B′)Ni-CA/IL/Mb-CPE, (C′) Pd-CA/IL/Mb-CPE, and (D′) Ppy-CA/IL/Mb-CPE.
Figure 3. (A) UV−vis absorption spectra of (a′) CA/IL/Mb, (a) Ni-CA/IL/Mb, (b) Pd-CA/IL/Mb, (c) Ppy-CA/IL/Mb, (d) Mb in 0.1 M PBS solution at pH 7.0. (B) FT-IR spectra of (a′) CA/IL/Mb, (a) Ni-CA/IL/Mb, (b) Pd-CA/IL/Mb, (c) Ppy-CA/IL/Mb, (d) Mb film.
the excellent conductivity, the Rct of modified electrodes NiCA/IL-CPE, Pd-CA/IL-CPE, and Ppy-CA/IL-CPE further decreased compared with that of CA/IL-CPE when Ni nanoparticle, Pd nanoparticle, and Ppy were doped onto the surface of CA (Figure 4A). However, the presence of Mb led to the increase of the impedance of the CA/IL/Mb-CPE, Ni-CA/ IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/Mb-CPE (Figure 4B), further confirming that Mb was effectively immobilized in the composite films. Direct Electrochemistry of the Mb Modified Electrodes. The typical CVs of different electrodes in 0.1 M PBS (pH 7.0) were obtained. No obvious electrochemical responses occurred at CPE, IL-CPE, and electrodes modified with different doped CA materials. These results indicated that neither IL nor doped CA was electroactive in the examined range. As shown in Figure 5A, no obvious electrochemical responses occurred after Mb was casted on the CPE electrode (e). The fact illustrated that the Mb molecules were not completely immobilized on the CPE surface. For IL/Mb-CPE (d), however, a couple of ill-defined and asymmetric redox peaks were observed, indicating a slow electron transfer process. For CA/IL/Mb-CPE (a′), the peak current values increased slightly and a smaller peak-to-peak separation was obtained. By contrast, the responses of Mb at inorganic/organic doped-CA/IL/Mb-CPE (a−c) were apparently enhanced and
electrodes. The EIS results of the different electrodes were described in Figure 4. The resistance (Rct) controlled the electron transfer kinetics of the redox probe at the electrode interface. The high Rct of CPE indicated an inefficient electron transfer process at the surface of CPE, whereas the highly conductive IL contributed to the lower Rct of IL-CPE. Given
Figure 4. EIS of CPE, IL-CPE, CA/IL-CPE, CA/IL/Mb-CPE, Ni-CA/ IL-CPE, Ni-CA/IL/Mb-CPE, Pd-CA/IL-CPE, Pd-CA/IL/Mb-CPE, Ppy-CA/IL-CPE, and Ppy-CA/IL/Mb-CPE. Supporting solution: 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) containing 0.1 M KCl. 11742
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Figure 5. (A) Cyclic voltammograms of (a′) CA/IL/Mb-CPE, (a) Ni-CA/IL/Mb-CPE, (b) Pd-CA/IL/Mb-CPE, (c) Ppy-CA/IL/Mb-CPE, (d) IL/ Mb-CPE, and (e) Mb-CPE. Supporting solution, 0.1 M PBS solution (pH 7.0); scan rate, 0.1 V s−1. Cyclic voltammograms of (B) Ni-CA/IL/MbCPE, (C) Pd-CA/IL/Mb-CPE, (D) Ppy-CA/IL/Mb-CPE in 0.1 M pH 7.0 PBS with scan rates from 0.02 to 1.00 V s−1. Insets show the plots of cathodic and anodic peak currents vs (a) scan rates and the (b) square root of scan rates.
shifted to more negative potentials, resulting in an increase of the peak separation between the anodic and cathodic peaks. The peak separation at higher scan rate can be used to estimate the heterogeneous electron transfer rate constant (ks). The ks as well as the transfer coefficient (α) were calculated based on the following Laviron equation:
their reversibility was significantly improved. From the comparison of the electrochemical behavior of Mb at different modified electrodes, it was clearly seen that inorganic/organic doped CA materials were vital in facilitating the direct electron transfer between Mb and the underlying electrode. The CVs of Mb at the Ni-CA/IL/Mb-CPE, Pd-CA/IL/MbCPE, and Ppy-CA/IL/Mb-CPE at different scan rates are shown in Figure 5B−D. Inset of Figure 5B-a, C-a, and D-a indicated that the anodic and cathodic peak currents increased linearly with the increase of scan rate from 0.02 to 1.00 V s−1, suggesting the electrochemical reactions of the three composite film modified electrodes were all surface-controlled process.21 For a surface-controlled process, the integration of reduction peaks gives nearly constant charge (Q) at different scan rates. The average surface coverage (Γ*) of the electroactive Mb on the modified electrodes was estimated at a slow scan rate according to Faraday’s law: Q = nAFΓ*, where Q is the charge involved in the reaction, n is the number of electron transferred, F is Faraday’s constant, and A is the effective surface area of the electrode.8 The Γ* values (Table S1, Supporting Information) are significantly higher than the theoretical monolayer coverage (1.89 × 10−11 M cm−1)22 and also higher than that of the electrode modified by the LaF3-DP-CeO2/IL composite.23 This meant that the multilayers of Mb were entrapped in the threedimensional composite film of doped CA and participated in the electron transfer process. At higher scan rate, the peak currents (ip) were proportional to the square root of the scan rate (v1/2) in the inset of Figure 5B-b, C-b, and D-b. On the other hand, the oxidation peaks shifted to more positive potentials and the reduction peaks
Ep,a = E°′ + RT ln v /(1 − α)nF
(1)
Ep,c = E°′ − RT ln v /αnF
(2)
log ks = α log(1 − α) + (1 − α)log α − log(RT /nFv) − (1 − α)αnF ΔEp/2.3RT
(3)
where n is the number of electron transfer, ΔEp is the peak to peak potential separation, R, T, and F are symbols that have conventional meanings. The estimated α and ks are shown in Table S1 in the Supporting Information. The values of ks are significantly larger than the values obtained from other carbon composite electrodes, such as silver nanoparticles doped carbon nanotubes film24 and Nafion/MWCNTs/CILE.25 The results suggested that the combination of IL and doped CA improved the electrochemical transfer. Possible Mechanism and Effects of the Material Performance on Electrochemistry. The modified electrodes were constructed by different methods, and the corresponding results were supplied in the Supporting Information. More importantly, when the electrodes were constructed as shown in the schematic and described in the Experimental Section by the graphite instead of CA and doped CA, it was found that the peak current of Mb and reversibility of the electrode decreased. 11743
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In the meantime, the Mb molecules would soon fall off from the Mb-CPE when the modified electrode was in the PBS solution for the CV measurement. These phenomena suggested that the interaction of the CA or doped CA with Mb may play a significant role in the immobilization of Mb. In the process of constructing an electrode in the paper, the doped-CA/IL acted as a thin film or barrier atop the Mb-CPE and Mb was encapsulated within the composite material.26 Then a sandwich-like network of coated doped-CA/IL provides more sites and channels for the immobilization of Mb, aiding in the prevention of the denaturation and leakage of Mb and enhancing the electron conductivity. On the other hand, the results of spectra and electrochemical experiments demonstrated that Mb molecules were entrapped in the CA/IL film. Especially, a pair of stable well-defined redox peaks of Mb appears on the doped CA modified electrodes. Therefore, CA and CA-based hybrid materials were preferable electrode modified materials for immobilizing enzyme or protein. The effect of doped CA type on electrochemistry cannot be ignored. A comparison of the different properties of doped CA is shown in Figure 6. The BET surface areas of the three CA-
Figure 7. Amperometric current−time response curves of Ni-CA/IL/ Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/Mb-CPE at −0.35 V (vs SCE) upon successive addition of H2O2 into 0.1 M PBS solution (pH 7.0). Inset: calibration curve of steady-state currents vs H2O2 concentration, respectively.
doped CA modified electrodes all exhibited rapid responses. The apparent Michaelis−Menten constant (KM), which is an indicator of the enzyme−substrate kinetics, can be obtained from the Lineweaver−Burk equation (1/Iss = 1/Imax + KM/Imax × 1/c, where Iss is the steady-state current after the addition of substrate and c is the bulk concentration of the substrate). For the as-prepared biosensor, the calculated KM is shown in Table 2. A small KM indicates the Mb entrapped in the doped-CA/IL composite possessed a high affinity to H2O2. Upon the addition of H2O2 into a continuously stirred PBS at −0.35 V (vs SCE), the current responses of Pd-CA/IL/MbCPE and Ppy-CA/IL/Mb-CPE increased dramatically compared with that of Ni-CA/IL/Mb-CPE. Table 2 compares performances of H2O2 biosensors obtained in this work with those previously reported in the literature. It can be seen that the prominent electrocatalytic ability of inorganic/organic doped CA/IL/Mb-CPE were superior to the reported H2O2 sensors. These characteristics may be attributed to the higher number of protein-binding sites provided by the inorganic/ organic doped CA/IL composite as well as to the short diffusion distance for H2O2 to access the immobilized Mb. Real Sample Analysis. To evaluate the feasibility of the proposed method for routine analysis, the Ni-CA/IL/Mb-CPE sensor was used in the determination of H2O2 in a disinfectant. The disinfectant sample (1.0 μL) was added into 25 mL of 0.1 M PBS (pH 7.0). Recovery studies on the samples were completed by adding H2O2 standard solutions. The obtained results are shown in Table 3. Stability and Reproducibility of the H2O2 Biosensor. The stabilities of the Ni-CA/IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/Mb-CPE electrodes were first evaluated by examining the CV peak currents of Mb after continuous scanning for 50 cycles. No decrease in the voltammetric response was observed, suggesting that the Ni-CA/IL/Mb, PdCA/IL/Mb, and Ppy-CA/IL/Mb composite film modified electrodes were stable in PBS. The stabilities of the composite membrane modified electrodes were also determined by measuring the current response. The three inorganic/organic doped CA/IL/Mb-CPE all retained >95% of their initial responses after in 0.1 M PBS (pH 7.0) at 4 °C for 2 weeks and maintained 87% of their initial activities after 4 weeks. The fabrication reproducibilities of eight electrodes, which were fabricated using the same procedure, was acceptable and
Figure 6. Comparison of properties for three doped CAs and their electrodes.
based hybrid materials decreased in the order Ni-CA/IL-CPE > Pd-CA/IL-CPE > Ppy-CA/IL-CPE. However, their conductivities, Γ*, and peak currents increased in above order. The change trend of the BET surface area is not consistent with that of Γ*, confirming that the synergetic effect with [BMIm]BF4 IL improved the capability of the composite inorganic/organic doped CA/IL matrix for protein immobilization. Although the value of Γ* for Ppy-CA/IL-CPE slightly increased compared with that of Pd-CA/IL-CPE, its peak current clearly increased. These results further confirm that the electron conductibility of various guests such as Ni, Pd, and Ppy are important. Therefore, the type of doped CA, which is a vital constituent of the composite, serves an important function in immobilizing protein and in facilitating the electron transfer. Electrocatalytic Activities of the Modified Electrodes to H2O2. It is well-known that proteins containing hemegroups, such as hemoglobin, Mb, and horseradish peroxidase, can electrocatalyze the reduction of H2O2.27 Figure 7 represents the typical current−time curves of the sensors based on the NiCA/IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/MbCPE upon the addition of an aliquot concentration of H2O2 into a continuously stirred 0.1 M PBS (pH 7.0), and the three 11744
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Table 2. Performances of Different H2O2 Biosensors biosensor
detection range (μM)
detection limit (μM)
KM (mM)
ref
Hb-chi/nanoCaCO3-chi/CILE Ti/TiO2/Au/HRP/GCE Au-SPAN/HRP/GCE GCE/Chi/CoFe2O4/HRP Au/GS/HRP/CS Ni-CA/IL/Mb-CPE Pd-CA/IL/Mb-CPE Ppy-CA/IL/Mb-CPE
5.0−1300 4.0−400 10−2000 30−8000 5.0−5130 5.0−975 3.0−815 2.5−1060
1.6 2.0 1.6 2.0 1.7 1.68 1.02 0.85
0.81
28 29 30 31 32 this work this work this work
University of Architecture and Technology (Grant No. ZC1004).
Table 3. Results of Recovery Test for the Biosensor with the Ni-CA/IL/Mb-CPE
a
samples of disinfectant
content c/(μM)
added c/(μM)
total after addition c/(μM)a
recovery (%)
1 2 3
10.3 9.2 10.8
10.0 10.0 10.0
21.8 18.2 19.8
107.4 94.8 95.2
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yielded RSD of 4.5%, 4.2%, and 3.9%, respectively, for the current determination of 100 μM H2O2 in 0.1 M PBS (pH 7.0). The results indicated that the three proposed biosensors all exhibited high stability and reproducibility and were therefore suitable for H2O2 determination.
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CONCLUSIONS We demonstrated the feasibility of using three inorganic/ organic functional CA hybrid materials as matrixes to improve the performance of bioelectrocatalytic processes significantly. The results show lower H2O2 detection limits and smaller apparent Michaelis−Menten constants. As expected, the network of coated doped-CA/IL provides more sites and channels for the immobilization of Mb, aiding in the prevention of the denaturation and leakage of Mb and enhancing the electron conductivity. The results provide an extensive platform and open up a new avenue for fabricating high-performance electrochemical biosensors. Further research and development are required to prepare other functional CAs and to improve their performance for a more extensive application in biosensing. ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
(1) Freeman, M. H.; Hall, J. R.; Leopold, M. C. Anal. Chem. 2013, 85, 4057−4065. (2) Pekala, R. W. J. Mater. Sci. 1989, 24, 3221−3227. (3) Wang, X. Y.; Wang, X. Y.; Liu, L.; Yi, L. H.; Hu, C. Y.; Zhang, X. Y.; et al. Synth. Met. 2011, 161, 1725−1730. (4) Yu, Z. H.; Su, T. T.; Ren, C. H.; Li, F.; Xia, D. G.; Cheng, S. Y. Acta Phys.-Chim. Sin. 2012, 28, 2867−2873. (5) Chen, Q. W.; Zhang, L. Y.; Chen, G. Anal. Chem. 2012, 84, 171− 178. (6) Naruse, J.; Hoa, L. Q.; Sugano, Y.; Ikeuchi, T.; Yoshikawa, H.; Saito, M.; et al. Biosens. Bioelectron. 2011, 30, 204−210. (7) Lawal, A. T.; Adeloju, S. B. Biosens. Bioelectron. 2013, 40, 377−84. (8) Dong, S. Y.; Zhang, P. H.; Liu, H.; Li, N.; Huang, T. L. Biosens. Bioelectron. 2011, 26, 4082−4087. (9) Cheng, X. H.; Guo, W. Dyes Pigments 2007, 72, 372−377. (10) Mathew, M.; Sandhyarani, N. Biosens. Bioelectron. 2011, 28, 210−215. (11) Yang, J.; Xiang, H.; Shuai, L.; Gunasekaran, S. Anal. Chim. Acta 2011, 708, 44−45. (12) Salomi, B. S. B.; Mitra, C. K. Biosens. Bioelectron. 2007, 22, 1825−1829. (13) Goncalves, A. R.; Ghica, M. E.; Brett, C. M. A. Electrochim. Acta 2011, 56, 3685−3692. (14) Chen, Q. W.; Zhang, L. Y.; Chen, G. Anal. Chem. 2012, 84, 171−178. (15) Yang, Y. F.; Mu, S. L. J. Electroanal. Chem. 1997, 432, 71−78. (16) Wu, D. C.; Fu, R. W.; Zhang, S. T.; Dresselhaus, M. S. Carbon 2004, 42, 2033−2039. (17) Lee, Y. J.; Park, S.; Seo, J. G.; Yoon, J. R.; Yi, J.; Song, I. K. Curr. Appl. Phys. 2011, 11, 631−635. (18) Lu, X. F.; Mao, H.; Zhang, W. J. Polym. Compos. 2009, 30, 847− 854. (19) George, P.; Hanania, G. I. H. J. Biochem. 1953, 55, 236−243. (20) Kauppinen, J. K.; Moffat, D. J.; Mantsch, H. H.; Cameron, D. G. Appl. Spectrosc. 1981, 35, 271−276. (21) Murray, R. W. Chemically Modified Electrodes. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker, Inc.: New York, 1984; pp 191−368. (22) Wang, S. F.; Chen, T.; Zhang, Z. L.; Shen, X. C.; Lu, Z. X.; Pang, D. W.; Wong, K. Y. Langmuir 2005, 21, 9260−9266. (23) Dong, S. Y.; Li, N.; Huang, T. L.; Tang, H. S.; Zheng, J. B. Sens. Actuators, B 2012, 173, 704−709. (24) Liu, C. Y.; Hu, J. M. Biosens. Bioelectron. 2010, 25, 1447−1453. (25) Sun, W.; Li, X. Q.; Wang, Y.; Li, X.; Zhao, C. Z; Jiao, K. Bioelectrochemistry 2009, 75, 170−175. (26) Shiddiky, M. J. A.; Torriero, A. A. J. Biosens. Bioelectron. 2011, 26, 1775−1787. (27) Liu, X. Q.; Zhang, J. M.; Liu, S. H.; Zhang, Q. Y.; Liu, X. H.; Wong, D. K. Y. Anal. Chem. 2013, 85, 4350−4356. (28) Zhao, H. Y.; Zheng, J. B.; Sheng, Q. L. J. Chin. Chem. Soc. 2011, 58, 346−52.
Average measured five times.
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2.21 12.2 2.61 0.52 0.39 0.29
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Phone: (+86)-29-82201203. Fax: (+86)-29-82205332. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors appreciate the support from the National Natural Science Foundation of China (Grant No. 50830303), the Projects in the National Science & Technology (Grant No. 2012BAC04B02), the Overall Innovation Project of Science & Technology in Shaanxi Province (Grant No. 2011KTCG0307), and the Research Achievements Foundation of Xi′an 11745
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(29) Kafi, A. K. M.; Wu, G. S.; Chen, A. C. Biosens. Bioelectron. 2008, 24, 566−571. (30) Chen, X. J.; Chen, Z. X.; Zhu, J. W.; Xu, C. B.; Yan, W.; Yao, C. Bioelectrochem. 2011, 82, 87−94. (31) Yardımcı, F. S.; Şenel, M.; Baykal, A. Mater. Sci. Eng., C 2012, 32, 269−275. (32) Zhou, K. F.; Zhu, Y. H.; Yang, X. L.; Luo, J.; Li, C. Z.; Luan, S. R. Electrochem. Acta 2010, 55, 3055−3060.
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Inorganic/Organic Doped Carbon Aerogels As Biosensing Materials for the Detection of Hydrogen Peroxide Sheying Dong,*,† Nan Li,† Gaochao Suo,† and Tinglin Huang‡ †
College of Sciences, Xi′an University of Architecture and Technology, Xi′an 710055, People’s Republic of China School of Environmental and Municipal Engineering, Xi′an University of Architecture and Technology, Xi′an 710055, People’s Republic of China
‡
S Supporting Information *
ABSTRACT: In this article, three different inorganic/organic doped carbon aerogel (CA) materials (Ni-CA, Pd-CA, and Ppy-CA) were, respectively, mixed with ionic liquid (IL) to form three stable composite films, which were used as enhanced elements for an integrated sensing platform to increase the surface area and to improve the electronic transmission rate. Subsequently, the effect of the materials performances such as adsorption, specific surface area and conductivity on electrochemistry for myoglobin (Mb) was discussed using N2 adsorption−desorption isotherm measurements, scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). Moreover, they could act as sensors toward the detection of hydrogen peroxide (H2O2) with lower detection limits (1.68 μM, 1.02 μM, and 0.85 μM, for Ni-CA/IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and PpyCA/IL/Mb-CPE, respectively) and smaller apparent Michaelis−Menten constants KM. The results indicated that the electroconductibility of the doped CA materials would become dominant, thus playing an important role in facilitating the electron transfer. Meanwhile, the synergetic effect with [BMIm]BF4 IL improved the capability of the composite inorganic/ organic doped CA/IL matrix for protein immobilization. This work demonstrates the feasibility and the potential of a series of CA-based hybrid materials as biosensors, and further research and development are required to prepare other functional CAs and make them valuable for more extensive application in biosensing.
T
and graphene-based materials, CA and CA-based hybrid materials have a great potential in electrocatalysis, especially for fabricating electrochemical sensors based on enzymes or proteins. Hence, it will have a potential research value to bring them in electrochemical sensors to immobilize enzymes and proteins. In the construction of carbon-based hybrid materials, metal nanoparticles such as Ni and Pd were always preferred for their high electrocatalytic behavior to biomolecules in sensing application.5,6 Meanwhile, conducting polymers can be exploited as an excellent substance for the preparation of nanocomposites with nanoscaled biomolecules. Thereinto, polypyrrole (Ppy) is one of the most extensively used conducting polymers in the design of bioanalytical sensors7 because of its sufficient conductibility, selectivity, stability, sensitivity, and biological affinity. Accordingly, it may be beneficial and practical to prepare different inorganic/organic functionalized CA nanocomposites as biosensing materials. When the CA is doped with Ni, Pd, or Ppy, functionalization
he development of materials science and nanotechnology has brought a great momentum to bioelectroanalysis and analysts. In the recent decades, scientists are always enthusiastic about finding materials with good biocompatibility to improve the behavior of biosensors.1 Generally, the immobilization of biomolecules in designer-made nanoscale structures can significantly improve the performance of biocatalytic processes. From the viewpoint of the immobilization of enzymes and proteins possessing relatively high molecular weight and large molecular diameter, mesoporous carbon, with the diameter in the range from 2 to 50 nm, may be a potential candidate to accommodate these biomolecules. Recently, carbon aerogel (CA), as an important three-dimension nanomaterial, has attracted tremendous scientific and technological attention since it was first patented by Pekala.2 To date, most of the CA studies have focused on electric double layer of supercapacitors.3 There is only limited research on the immobilization of large biomolecules by CA.4 Electrodes modified by CA and a class of CA-based hybrid materials for biosensors have not received sufficient attention. In view of the versatile properties of CA including outstanding electrical conductivity, large surface area, fine pore size, and high porosity, which are similar to those of other carbon nanomaterials such as graphene © 2013 American Chemical Society
Received: May 19, 2013 Accepted: November 27, 2013 Published: November 27, 2013 11739
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Scheme 1. Schematic Diagram of the Procedure to Construct the Ni-CA/IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/ Mb-CPE
conventional three-electrode system comprised of a platinum wire auxiliary, a saturated calomel electrode (SCE) reference, and the modified carbon paste electrode (CPE) working electrode. The morphology of the electrode surface was determined by SEM (Quanta 600FEG) at an accelerating voltage of 15 kV. Ultraviolet−visible (UV−vis) spectra were recorded on a Nicolet Evolution 300 spectrophotometer in 0.1 M PBS, and the IR spectra (KBr) were recorded on an IRspectrometer FTIR-8400S (Shimadzu) at room temperature. CA was prepared under ambient conditions according to the method reported in the literature.16 However, microwaveassisted heating was adopted during the resorcinol-formaldehyde reaction in this study to shorten the reaction time by 4 days, compared with using conventional heating. The nanosized Ni doped CA (Ni-CA), Pd doped CA (Pd-CA), and Ppy doped CA (Ppy-CA) nanocomposites were successfully fabricated via chemical precipitation17 and chemical redox polymerization,18 which are described in the Supporting Information. Preparation of the Modified Electrodes. CPE was fabricated as follows: 0.6 g of liquid paraffin and 3.4 g of graphite powder were hand-mixed to produce a homogeneous paste. The prepared carbon paste was then firmly packed into a PVC tube (3 mm internal diameter). A copper wire (1.5 mm external diameter) was introduced into the other end for electrical contact. The CPE surface was carefully smoothed on a weighing paper before use. The enzyme electrode was prepared according to the following procedure: first, 5 μL of Mb solution (5 mg/mL) was casted onto the surface of a freshly polished CPE, and the modified electrode was dried in a refrigerator at 4 °C to obtain Mb-CPE. Second, 5 mg of doped CA (Ni-CA, Pd-CA, and PpyCA) and 10 μL of [BMIm]BF4 IL were dispersed into 1 mL of 0.1 M PBS (pH 7.0). After sonication for 30 min, 10 μL of the above suspension was pipetted onto Mb-CPE to obtain dopedCA/IL/Mb-CPE (Ni-CA/IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/Mb-CPE), which was subsequently evaporated in a refrigerator at 4 °C to form a stable film. Finally, the modified electrode was rinsed twice or thrice with doubly distilled water to remove the unimmobilized Mb molecules. When not in use, the enzyme electrode was stored in 0.1 M PBS (pH 7.0) at 4 °C in a refrigerator. In addition, the enzyme electrode was also constructed in simpler ways, and the preparation process was described in the Supporting Information. Electrochemical Measurements. EIS measurements were carried out in 5.0 mM K3 Fe(CN) 6 /K 4 Fe(CN) 6 (1:1) containing 0.1 M KCl, while the applied perturbation amplitude was 0.005 V, the frequencies swept from 105 to 10−2 Hz.
effects occur, leading to the formation of doped materials with high conductivity. Most importantly, these factors should be responsible for the improvement of electrocatalytic performance of CA nanomaterials to some extent. This research encouraged us to explore appropriate CA-based hybrid materials to improve the performance of biocatalytic processes. In addition, previous research demonstrated that ionic liquid (IL) is a kind of ideal matrix that can disperse the nanomaterials into the modified layers to improve the stability and promote the electrocatalysis of protein.8 In recent years, enzyme/protein-modified electrodes for the determination of H2O2 have been frequently applied,9 owing to the advantages such as simple equipment, low operating expense, and fast analytical time. The modified electrode materials including metals,10 transition metal oxides,11 redox dyes,12 conducting polymers,13 and graphene14 were used to construct biosensors for H2O2. Nevertheless, it has still some deficiencies such as enzymes or proteins denaturation, low reproducibility, and stability.15 In the current study, we describe the use of three different doped CAs (Ni-CA, Pd-CA, and Ppy-CA) as the sensing platform in the detection of H2O2. Performance metrics such as adsorption and electrochemical conductivity of materials modified with Mb were reported along with a N 2 adsorption−desorption isotherm, scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). As expected, the synergistic performance of the proteins, inorganic/organic doped CA materials, as well as IL provides an extensive platform and opens up a new avenue for fabricating excellent electrochemical biosensors. The modification process for the Mb modified electrodes is shown in Scheme 1.
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EXPERIMENTAL SECTION Reagents and Apparatus. Bovine myoglobin (Mb, MW 17 800) was purchased from Sigma Chemical Co., and [BMIm]BF4 IL was purchased from Hangzhou Chemer Chemical Limited Company. High purity graphite powder was obtained from China National Medicine Corporation. K3Fe(CN)6, K4Fe(CN)6, KCl, K2HPO4, KH2PO4, KOH, Na2CO3, FeCl3·6H2O, NiCl2·6H2O, C6H6O2, HCHO (40%), H2O2 (30%), and liquid paraffin were obtained from Xi’an Chemical Reagent Corporation. C4H5N, PdCl2, and sodium dodecylsulfate (SDS) were obtained from China National Medicine Corporation. All chemicals were of analytical reagent grade. A 0.1 M phosphate buffer solution (PBS, pH 7.0) was used. All solutions were made up with twice-distilled water. Electrochemical measurements were performed with a CHI660B workstation (Shanghai Chenhua Co., China) with a 11740
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Figure 1. (A) N2 adsorption−desorption isotherms and (B) the resultant pore size distribution of CA, Ni-CA, Pd-CA, and Ppy-CA.
particles ranged from 15 to 30 nm in Figure 2A. It was found in Figure 2B,C that the skeleton particles with Ni and Pd nanoparticles doped CA materials were larger and more compact than CA, which were attributed to the reintegration of metal particles when doped CA was calcined at a high temperature. While the micrograph of Ppy-CA in Figure 2D showed porosity with wide size range. The surface morphologies of these CA-based hybrid materials were composed of microbead particles, which resulted from the interlinked micelles formed in the presence of a surfactant. From the surface morphology of different modified electrodes, it could be observed that the surface of CPE was formed by irregularly shaped flakes, whereas the scrappy particles in Figure 2B′, C′, and D′ derived from the adsorption of Mb onto the surface of inorganic/organic doped CA. Obviously, compared with the SEM images of CA and inorganic/organic doped CA materials, the stable composite films of the modified electrodes in all cases exhibited higher hierarchy and porosity, which could provide a specific interface for the adsorption and immobilization of protein molecules. UV−Visible and Fourier Transform-Infrared (FT-IR) Spectra Results. To investigate qualitatively the biological activity of Mb before and after immobilization on the inorganic/organic doped CA/IL composite films, the changes in the Soret band were observed by monitoring the absorption band in the prosthetic heme-group region of the composite films.19 The discussion of UV−vis (Figure S1 in the Supporting Information) and some references indicated that the presence of graphite and [BMIm]BF4 IL did not denature the Mb. Moreover, as shown in Figure 3A, the Soret bands of Mb in different solutions were 408.9 nm (CA/IL/Mb), 408.6 nm (NiCA/IL/Mb), 408.7 nm (Pd-CA/IL/Mb), and 408.4 nm (PpyCA/IL/Mb). Compared with Mb in the buffer (408.4 nm), Mb retained its native structure in the presence of inorganic/ organic doped CA and [BMIm]BF4. The CA/IL/Mb, Ni-CA/ IL/Mb, Pd-CA/IL/Mb, and Ppy-CA/IL/Mb films were also analyzed by FT-IR. As shown in Figure 3B, the spectrum of free Mb (d) was similar to that of Mb in CA/IL (a′), Ni-CA/IL (a), Pd-CA/IL (b), and Ppy-CA/IL (c). These results confirmed that the secondary structure of Mb retained the essential feature of its native state.20 The UV−vis and FT-IR results supported the conclusion that the native structure of Mb had been retained. Thus, inorganic/organic doped CA and [BMIm]BF4 IL composite matrixes provided an excellent platform for the further study of the direct electrochemistry of Mb. EIS Characterization. Alternating current impedances were used to show the impedance change while preparing different
Amperometric experiments were performed in a constantly stirred cell with the successive addition of H2O2 into 20.0 mL of 0.1 M PBS supporting electrolyte (pH 7.0) at 25 °C. The electrode potential was set at −0.35 V vs RE (SCE). Experimental solutions were deoxygenated by purging with high pure nitrogen for 30 min and maintained under nitrogen atmosphere during the measurements.
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RESULTS AND DISCUSSION Textural Properties of CA and Inorganic/Organic Doped CA. The textural properties of CA and three doped CA materials were examined by N2 adsorption−desorption isotherm measurements. A typical adsorption−desorption isotherm (Figure 1A) exhibited type IV according to IUPAC with H2 hysteresis loops, indicating the presence of mesopores. Meanwhile, the samples exhibited a narrow mesopore size distribution, as indicated by a steep capillary condensation step of the isotherms and the Barrett−Joyner−Halenda (BJH) pore size distribution (Figure 1B). On this basis, Table 1 compares Table 1. Textual Properties of CA and Three Doped CA Materials samples
BET surface area (m2/g)
average pore diameter (nm)
pore volume (cm3/g)
CA Ni-CA Pd-CA Ppy-CA
747 682 583 485
7.9 8.3 8.3 8.0
0.61 0.58 0.52 0.43
the textural properties of CA and three doped CA materials. From the results in Figure 1 and Table 1, it can be concluded that the surface areas of three CA-based hybrid materials increase in the order Ni-CA > Pd-CA > Ppy-CA. Although Brunauer−Emmett−Teller (BET) surface area and pore volume slightly decreased after the impregnation with Ni, Pd, and Ppy nanoparticles, the textural properties of CA were retained. Characterization of the Electrodes Modified by Inorganic/Organic Doped CA. To investigate the effects of CA and inorganic/organic doped CA materials microstructure on the fabrication of the modified electrodes, SEM images of the CA hybrid materials and their corresponding modified electrodes were determined in Figure 2. The SEM images showed the CA skeletal structure consisted of interconnected nanosized carbon ligaments that defined a continuous polyporous network. The diameter of most of the spherical 11741
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Figure 2. SEM images of (A) CA, (B) Ni-CA, (C) Pd-CA, (D) Ppy-CA, (A′) CPE, (B′)Ni-CA/IL/Mb-CPE, (C′) Pd-CA/IL/Mb-CPE, and (D′) Ppy-CA/IL/Mb-CPE.
Figure 3. (A) UV−vis absorption spectra of (a′) CA/IL/Mb, (a) Ni-CA/IL/Mb, (b) Pd-CA/IL/Mb, (c) Ppy-CA/IL/Mb, (d) Mb in 0.1 M PBS solution at pH 7.0. (B) FT-IR spectra of (a′) CA/IL/Mb, (a) Ni-CA/IL/Mb, (b) Pd-CA/IL/Mb, (c) Ppy-CA/IL/Mb, (d) Mb film.
the excellent conductivity, the Rct of modified electrodes NiCA/IL-CPE, Pd-CA/IL-CPE, and Ppy-CA/IL-CPE further decreased compared with that of CA/IL-CPE when Ni nanoparticle, Pd nanoparticle, and Ppy were doped onto the surface of CA (Figure 4A). However, the presence of Mb led to the increase of the impedance of the CA/IL/Mb-CPE, Ni-CA/ IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/Mb-CPE (Figure 4B), further confirming that Mb was effectively immobilized in the composite films. Direct Electrochemistry of the Mb Modified Electrodes. The typical CVs of different electrodes in 0.1 M PBS (pH 7.0) were obtained. No obvious electrochemical responses occurred at CPE, IL-CPE, and electrodes modified with different doped CA materials. These results indicated that neither IL nor doped CA was electroactive in the examined range. As shown in Figure 5A, no obvious electrochemical responses occurred after Mb was casted on the CPE electrode (e). The fact illustrated that the Mb molecules were not completely immobilized on the CPE surface. For IL/Mb-CPE (d), however, a couple of ill-defined and asymmetric redox peaks were observed, indicating a slow electron transfer process. For CA/IL/Mb-CPE (a′), the peak current values increased slightly and a smaller peak-to-peak separation was obtained. By contrast, the responses of Mb at inorganic/organic doped-CA/IL/Mb-CPE (a−c) were apparently enhanced and
electrodes. The EIS results of the different electrodes were described in Figure 4. The resistance (Rct) controlled the electron transfer kinetics of the redox probe at the electrode interface. The high Rct of CPE indicated an inefficient electron transfer process at the surface of CPE, whereas the highly conductive IL contributed to the lower Rct of IL-CPE. Given
Figure 4. EIS of CPE, IL-CPE, CA/IL-CPE, CA/IL/Mb-CPE, Ni-CA/ IL-CPE, Ni-CA/IL/Mb-CPE, Pd-CA/IL-CPE, Pd-CA/IL/Mb-CPE, Ppy-CA/IL-CPE, and Ppy-CA/IL/Mb-CPE. Supporting solution: 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) containing 0.1 M KCl. 11742
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Figure 5. (A) Cyclic voltammograms of (a′) CA/IL/Mb-CPE, (a) Ni-CA/IL/Mb-CPE, (b) Pd-CA/IL/Mb-CPE, (c) Ppy-CA/IL/Mb-CPE, (d) IL/ Mb-CPE, and (e) Mb-CPE. Supporting solution, 0.1 M PBS solution (pH 7.0); scan rate, 0.1 V s−1. Cyclic voltammograms of (B) Ni-CA/IL/MbCPE, (C) Pd-CA/IL/Mb-CPE, (D) Ppy-CA/IL/Mb-CPE in 0.1 M pH 7.0 PBS with scan rates from 0.02 to 1.00 V s−1. Insets show the plots of cathodic and anodic peak currents vs (a) scan rates and the (b) square root of scan rates.
shifted to more negative potentials, resulting in an increase of the peak separation between the anodic and cathodic peaks. The peak separation at higher scan rate can be used to estimate the heterogeneous electron transfer rate constant (ks). The ks as well as the transfer coefficient (α) were calculated based on the following Laviron equation:
their reversibility was significantly improved. From the comparison of the electrochemical behavior of Mb at different modified electrodes, it was clearly seen that inorganic/organic doped CA materials were vital in facilitating the direct electron transfer between Mb and the underlying electrode. The CVs of Mb at the Ni-CA/IL/Mb-CPE, Pd-CA/IL/MbCPE, and Ppy-CA/IL/Mb-CPE at different scan rates are shown in Figure 5B−D. Inset of Figure 5B-a, C-a, and D-a indicated that the anodic and cathodic peak currents increased linearly with the increase of scan rate from 0.02 to 1.00 V s−1, suggesting the electrochemical reactions of the three composite film modified electrodes were all surface-controlled process.21 For a surface-controlled process, the integration of reduction peaks gives nearly constant charge (Q) at different scan rates. The average surface coverage (Γ*) of the electroactive Mb on the modified electrodes was estimated at a slow scan rate according to Faraday’s law: Q = nAFΓ*, where Q is the charge involved in the reaction, n is the number of electron transferred, F is Faraday’s constant, and A is the effective surface area of the electrode.8 The Γ* values (Table S1, Supporting Information) are significantly higher than the theoretical monolayer coverage (1.89 × 10−11 M cm−1)22 and also higher than that of the electrode modified by the LaF3-DP-CeO2/IL composite.23 This meant that the multilayers of Mb were entrapped in the threedimensional composite film of doped CA and participated in the electron transfer process. At higher scan rate, the peak currents (ip) were proportional to the square root of the scan rate (v1/2) in the inset of Figure 5B-b, C-b, and D-b. On the other hand, the oxidation peaks shifted to more positive potentials and the reduction peaks
Ep,a = E°′ + RT ln v /(1 − α)nF
(1)
Ep,c = E°′ − RT ln v /αnF
(2)
log ks = α log(1 − α) + (1 − α)log α − log(RT /nFv) − (1 − α)αnF ΔEp/2.3RT
(3)
where n is the number of electron transfer, ΔEp is the peak to peak potential separation, R, T, and F are symbols that have conventional meanings. The estimated α and ks are shown in Table S1 in the Supporting Information. The values of ks are significantly larger than the values obtained from other carbon composite electrodes, such as silver nanoparticles doped carbon nanotubes film24 and Nafion/MWCNTs/CILE.25 The results suggested that the combination of IL and doped CA improved the electrochemical transfer. Possible Mechanism and Effects of the Material Performance on Electrochemistry. The modified electrodes were constructed by different methods, and the corresponding results were supplied in the Supporting Information. More importantly, when the electrodes were constructed as shown in the schematic and described in the Experimental Section by the graphite instead of CA and doped CA, it was found that the peak current of Mb and reversibility of the electrode decreased. 11743
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In the meantime, the Mb molecules would soon fall off from the Mb-CPE when the modified electrode was in the PBS solution for the CV measurement. These phenomena suggested that the interaction of the CA or doped CA with Mb may play a significant role in the immobilization of Mb. In the process of constructing an electrode in the paper, the doped-CA/IL acted as a thin film or barrier atop the Mb-CPE and Mb was encapsulated within the composite material.26 Then a sandwich-like network of coated doped-CA/IL provides more sites and channels for the immobilization of Mb, aiding in the prevention of the denaturation and leakage of Mb and enhancing the electron conductivity. On the other hand, the results of spectra and electrochemical experiments demonstrated that Mb molecules were entrapped in the CA/IL film. Especially, a pair of stable well-defined redox peaks of Mb appears on the doped CA modified electrodes. Therefore, CA and CA-based hybrid materials were preferable electrode modified materials for immobilizing enzyme or protein. The effect of doped CA type on electrochemistry cannot be ignored. A comparison of the different properties of doped CA is shown in Figure 6. The BET surface areas of the three CA-
Figure 7. Amperometric current−time response curves of Ni-CA/IL/ Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/Mb-CPE at −0.35 V (vs SCE) upon successive addition of H2O2 into 0.1 M PBS solution (pH 7.0). Inset: calibration curve of steady-state currents vs H2O2 concentration, respectively.
doped CA modified electrodes all exhibited rapid responses. The apparent Michaelis−Menten constant (KM), which is an indicator of the enzyme−substrate kinetics, can be obtained from the Lineweaver−Burk equation (1/Iss = 1/Imax + KM/Imax × 1/c, where Iss is the steady-state current after the addition of substrate and c is the bulk concentration of the substrate). For the as-prepared biosensor, the calculated KM is shown in Table 2. A small KM indicates the Mb entrapped in the doped-CA/IL composite possessed a high affinity to H2O2. Upon the addition of H2O2 into a continuously stirred PBS at −0.35 V (vs SCE), the current responses of Pd-CA/IL/MbCPE and Ppy-CA/IL/Mb-CPE increased dramatically compared with that of Ni-CA/IL/Mb-CPE. Table 2 compares performances of H2O2 biosensors obtained in this work with those previously reported in the literature. It can be seen that the prominent electrocatalytic ability of inorganic/organic doped CA/IL/Mb-CPE were superior to the reported H2O2 sensors. These characteristics may be attributed to the higher number of protein-binding sites provided by the inorganic/ organic doped CA/IL composite as well as to the short diffusion distance for H2O2 to access the immobilized Mb. Real Sample Analysis. To evaluate the feasibility of the proposed method for routine analysis, the Ni-CA/IL/Mb-CPE sensor was used in the determination of H2O2 in a disinfectant. The disinfectant sample (1.0 μL) was added into 25 mL of 0.1 M PBS (pH 7.0). Recovery studies on the samples were completed by adding H2O2 standard solutions. The obtained results are shown in Table 3. Stability and Reproducibility of the H2O2 Biosensor. The stabilities of the Ni-CA/IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/Mb-CPE electrodes were first evaluated by examining the CV peak currents of Mb after continuous scanning for 50 cycles. No decrease in the voltammetric response was observed, suggesting that the Ni-CA/IL/Mb, PdCA/IL/Mb, and Ppy-CA/IL/Mb composite film modified electrodes were stable in PBS. The stabilities of the composite membrane modified electrodes were also determined by measuring the current response. The three inorganic/organic doped CA/IL/Mb-CPE all retained >95% of their initial responses after in 0.1 M PBS (pH 7.0) at 4 °C for 2 weeks and maintained 87% of their initial activities after 4 weeks. The fabrication reproducibilities of eight electrodes, which were fabricated using the same procedure, was acceptable and
Figure 6. Comparison of properties for three doped CAs and their electrodes.
based hybrid materials decreased in the order Ni-CA/IL-CPE > Pd-CA/IL-CPE > Ppy-CA/IL-CPE. However, their conductivities, Γ*, and peak currents increased in above order. The change trend of the BET surface area is not consistent with that of Γ*, confirming that the synergetic effect with [BMIm]BF4 IL improved the capability of the composite inorganic/organic doped CA/IL matrix for protein immobilization. Although the value of Γ* for Ppy-CA/IL-CPE slightly increased compared with that of Pd-CA/IL-CPE, its peak current clearly increased. These results further confirm that the electron conductibility of various guests such as Ni, Pd, and Ppy are important. Therefore, the type of doped CA, which is a vital constituent of the composite, serves an important function in immobilizing protein and in facilitating the electron transfer. Electrocatalytic Activities of the Modified Electrodes to H2O2. It is well-known that proteins containing hemegroups, such as hemoglobin, Mb, and horseradish peroxidase, can electrocatalyze the reduction of H2O2.27 Figure 7 represents the typical current−time curves of the sensors based on the NiCA/IL/Mb-CPE, Pd-CA/IL/Mb-CPE, and Ppy-CA/IL/MbCPE upon the addition of an aliquot concentration of H2O2 into a continuously stirred 0.1 M PBS (pH 7.0), and the three 11744
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Table 2. Performances of Different H2O2 Biosensors biosensor
detection range (μM)
detection limit (μM)
KM (mM)
ref
Hb-chi/nanoCaCO3-chi/CILE Ti/TiO2/Au/HRP/GCE Au-SPAN/HRP/GCE GCE/Chi/CoFe2O4/HRP Au/GS/HRP/CS Ni-CA/IL/Mb-CPE Pd-CA/IL/Mb-CPE Ppy-CA/IL/Mb-CPE
5.0−1300 4.0−400 10−2000 30−8000 5.0−5130 5.0−975 3.0−815 2.5−1060
1.6 2.0 1.6 2.0 1.7 1.68 1.02 0.85
0.81
28 29 30 31 32 this work this work this work
University of Architecture and Technology (Grant No. ZC1004).
Table 3. Results of Recovery Test for the Biosensor with the Ni-CA/IL/Mb-CPE
a
samples of disinfectant
content c/(μM)
added c/(μM)
total after addition c/(μM)a
recovery (%)
1 2 3
10.3 9.2 10.8
10.0 10.0 10.0
21.8 18.2 19.8
107.4 94.8 95.2
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yielded RSD of 4.5%, 4.2%, and 3.9%, respectively, for the current determination of 100 μM H2O2 in 0.1 M PBS (pH 7.0). The results indicated that the three proposed biosensors all exhibited high stability and reproducibility and were therefore suitable for H2O2 determination.
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CONCLUSIONS We demonstrated the feasibility of using three inorganic/ organic functional CA hybrid materials as matrixes to improve the performance of bioelectrocatalytic processes significantly. The results show lower H2O2 detection limits and smaller apparent Michaelis−Menten constants. As expected, the network of coated doped-CA/IL provides more sites and channels for the immobilization of Mb, aiding in the prevention of the denaturation and leakage of Mb and enhancing the electron conductivity. The results provide an extensive platform and open up a new avenue for fabricating high-performance electrochemical biosensors. Further research and development are required to prepare other functional CAs and to improve their performance for a more extensive application in biosensing. ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
(1) Freeman, M. H.; Hall, J. R.; Leopold, M. C. Anal. Chem. 2013, 85, 4057−4065. (2) Pekala, R. W. J. Mater. Sci. 1989, 24, 3221−3227. (3) Wang, X. Y.; Wang, X. Y.; Liu, L.; Yi, L. H.; Hu, C. Y.; Zhang, X. Y.; et al. Synth. Met. 2011, 161, 1725−1730. (4) Yu, Z. H.; Su, T. T.; Ren, C. H.; Li, F.; Xia, D. G.; Cheng, S. Y. Acta Phys.-Chim. Sin. 2012, 28, 2867−2873. (5) Chen, Q. W.; Zhang, L. Y.; Chen, G. Anal. Chem. 2012, 84, 171− 178. (6) Naruse, J.; Hoa, L. Q.; Sugano, Y.; Ikeuchi, T.; Yoshikawa, H.; Saito, M.; et al. Biosens. Bioelectron. 2011, 30, 204−210. (7) Lawal, A. T.; Adeloju, S. B. Biosens. Bioelectron. 2013, 40, 377−84. (8) Dong, S. Y.; Zhang, P. H.; Liu, H.; Li, N.; Huang, T. L. Biosens. Bioelectron. 2011, 26, 4082−4087. (9) Cheng, X. H.; Guo, W. Dyes Pigments 2007, 72, 372−377. (10) Mathew, M.; Sandhyarani, N. Biosens. Bioelectron. 2011, 28, 210−215. (11) Yang, J.; Xiang, H.; Shuai, L.; Gunasekaran, S. Anal. Chim. Acta 2011, 708, 44−45. (12) Salomi, B. S. B.; Mitra, C. K. Biosens. Bioelectron. 2007, 22, 1825−1829. (13) Goncalves, A. R.; Ghica, M. E.; Brett, C. M. A. Electrochim. Acta 2011, 56, 3685−3692. (14) Chen, Q. W.; Zhang, L. Y.; Chen, G. Anal. Chem. 2012, 84, 171−178. (15) Yang, Y. F.; Mu, S. L. J. Electroanal. Chem. 1997, 432, 71−78. (16) Wu, D. C.; Fu, R. W.; Zhang, S. T.; Dresselhaus, M. S. Carbon 2004, 42, 2033−2039. (17) Lee, Y. J.; Park, S.; Seo, J. G.; Yoon, J. R.; Yi, J.; Song, I. K. Curr. Appl. Phys. 2011, 11, 631−635. (18) Lu, X. F.; Mao, H.; Zhang, W. J. Polym. Compos. 2009, 30, 847− 854. (19) George, P.; Hanania, G. I. H. J. Biochem. 1953, 55, 236−243. (20) Kauppinen, J. K.; Moffat, D. J.; Mantsch, H. H.; Cameron, D. G. Appl. Spectrosc. 1981, 35, 271−276. (21) Murray, R. W. Chemically Modified Electrodes. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker, Inc.: New York, 1984; pp 191−368. (22) Wang, S. F.; Chen, T.; Zhang, Z. L.; Shen, X. C.; Lu, Z. X.; Pang, D. W.; Wong, K. Y. Langmuir 2005, 21, 9260−9266. (23) Dong, S. Y.; Li, N.; Huang, T. L.; Tang, H. S.; Zheng, J. B. Sens. Actuators, B 2012, 173, 704−709. (24) Liu, C. Y.; Hu, J. M. Biosens. Bioelectron. 2010, 25, 1447−1453. (25) Sun, W.; Li, X. Q.; Wang, Y.; Li, X.; Zhao, C. Z; Jiao, K. Bioelectrochemistry 2009, 75, 170−175. (26) Shiddiky, M. J. A.; Torriero, A. A. J. Biosens. Bioelectron. 2011, 26, 1775−1787. (27) Liu, X. Q.; Zhang, J. M.; Liu, S. H.; Zhang, Q. Y.; Liu, X. H.; Wong, D. K. Y. Anal. Chem. 2013, 85, 4350−4356. (28) Zhao, H. Y.; Zheng, J. B.; Sheng, Q. L. J. Chin. Chem. Soc. 2011, 58, 346−52.
Average measured five times.
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2.21 12.2 2.61 0.52 0.39 0.29
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Phone: (+86)-29-82201203. Fax: (+86)-29-82205332. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors appreciate the support from the National Natural Science Foundation of China (Grant No. 50830303), the Projects in the National Science & Technology (Grant No. 2012BAC04B02), the Overall Innovation Project of Science & Technology in Shaanxi Province (Grant No. 2011KTCG0307), and the Research Achievements Foundation of Xi′an 11745
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(29) Kafi, A. K. M.; Wu, G. S.; Chen, A. C. Biosens. Bioelectron. 2008, 24, 566−571. (30) Chen, X. J.; Chen, Z. X.; Zhu, J. W.; Xu, C. B.; Yan, W.; Yao, C. Bioelectrochem. 2011, 82, 87−94. (31) Yardımcı, F. S.; Şenel, M.; Baykal, A. Mater. Sci. Eng., C 2012, 32, 269−275. (32) Zhou, K. F.; Zhu, Y. H.; Yang, X. L.; Luo, J.; Li, C. Z.; Luan, S. R. Electrochem. Acta 2010, 55, 3055−3060.
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