Article pubs.acs.org/cm
A Partially Graphitic Mesoporous Carbon Membrane with ThreeDimensionally Networked Nanotunnels for Ultrasensitive Electrochemical Detection Cong Fu,† Deliang Yi,† Chao Deng,† Xingdong Wang,‡ Weijia Zhang,† Yi Tang,† Frank Caruso,§ and Yajun Wang*,† †
Department of Chemistry and Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, Fudan University, Shanghai 200433, P. R. China ‡ CSIRO Manufacturing, Private Bag 10, Clayton South, Victoria 3169, Australia § ARC Centre of Excellence in Convergent Bio-Nano Science and Technology and Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia S Supporting Information *
ABSTRACT: A hierarchically porous partially graphitic carbon (HPGC) membrane with three-dimensionally networked nanotunnels is prepared and applied as a monolithic matrix for electrochemical (bio)sensing. The walls of the nanotunnels (∼40−80 nm in diameter) are composed of partially graphitic carbon with ordered mesopores (∼6.5 nm in diameter). After modification with polydopamine, the HPGC membrane can be decorated with nanoparticles (i.e., Au NPs) and subsequently functions as a three-dimensional matrix for enzyme (i.e., glucose oxidase) immobilization. The Au NPs can accelerate electron transfer between the membrane electrode and enzyme and synergistically work with the enzyme in catalyzing glucose oxidation, thus considerably enhancing the sensitivity of the sensor. The signal intensity of the HPGC monolithic membrane electrode is ∼700 times higher than that obtained from mesoporous carbon/Nafion paste electrode counterparts. The limit of detection of the biosensor is 0.48 × 10−11 M toward glucose detection, 4 orders of magnitude lower than that achieved by conventional nanostructured glucose sensors. The HPGC membrane shows potential in preparing monolithic electrodes for diverse electrochemical applications such as electrochemical detection, energy storage, and electrochemical catalysis. in electrochemical biosensing applications.9−11 As part of the immobilization process, undertaking certain procedures, such as cross-linking, encapsulating, and embedding, is essential for appropriately adhering the enzyme to conventionally prepared (mesoporous carbon/Nafion paste) electrodes.12,13 However, such processes often lower the activity of the enzyme and obstruct active sites embedded in paste electrodes, thereby suppressing the performance of the biosensor.14 Herein, we report a facile strategy for preparing a hierarchically porous partially graphitic carbon (HPGC) membrane (Figure 1) and applying the HPGC membrane as a monolithic electrode matrix for constructing highly sensitive electrochemical (bio)sensors (Figure 2). A three-dimensionally assembled silica nanowire (NW) membrane, as we recently reported,15 is applied as a hard template to tune the
1. INTRODUCTION Electrochemical detection is an important technology that is used for the sensitive determination of molecules through monitoring of their specific electrochemical reactions on the interface of an electrode matrix.1 As an example of an electrochemical biosensor, enzymes are typically immobilized on a matrix. The biosensor can be applied as a biocatalyst for specific analyte reaction and detection.2 To achieve a rapid and sensitive response, electrochemical biosensors should ideally feature properties such as chemical inertness, enzyme compatibility, a large high specific surface area, and high porosity for realizing high loadings and fast diffusion rates of analytes.3 In the past 25 years, mesoporous materials have attracted much attention in many applications because of their extremely large surface areas, ordered pore structures, and diverse morphologies and compositions.4−8 The immobilization of enzymes in mesoporous materials, particularly mesoporous carbon, because of its conductivity, has been widely investigated © 2017 American Chemical Society
Received: April 7, 2017 Revised: May 25, 2017 Published: May 25, 2017 5286
DOI: 10.1021/acs.chemmater.7b01423 Chem. Mater. 2017, 29, 5286−5293
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Chemistry of Materials
macroscopic dimensions (size and thickness) of the HPGC membrane and generate three-dimensionally networked nanotunnels (∼40−80 nm in diameter) inside the membrane. Nonionic surfactant Pluronic F127 is used as a porogen, which is added to the carbon precursor solution, to produce ordered mesopores (∼6.5 nm in diameter) in the carbon walls of the nanotunnels. The hierarchical pore structure endows the HPGC membrane with properties that are desirable for designing highly efficient sensors. (1) The chemical inertness and high conductivity of the partially graphitic carbon matrix make it an ideal electrode material. (2) The mesoporous walls and abundant nanotunnels allow the incorporation of large amounts of functional species. (3) The three-dimensional (3D) nanotunnels allow fast transportation of analyte molecules through the membrane. Recently, polydopamine (PDA) has been widely investigated because of its useful properties,16−18 such as excellent film forming and adhesive ability, nontoxicity, high biocompatibility, and semiconductivity,19 as well as its noble metal ion reducing ability for preparing noble metal nanoparticles (NPs).18 Noble metal NPs, especially gold NPs, have been widely used as catalysts, peroxidase mimics,20,21 and affinity matrices to conjugate various biomolecules (e.g., DNA and proteins).22 There have also been reports that the Au NPs could enhance the electron transfer between the electrode and the enzyme.23 Herein, a thin layer of PDA is deposited in situ on the surface of the mesopores to endow the HPGC membrane with high biocompatibility and functional properties after further decoration of the PDA-modified membrane with Au NPs and enzymes for (bio)sensor construction. The deposited PDA enhances the stability of the sensor and also facilitates the formation of Au NPs.
Figure 1. Schematic illustration of the HPGC membrane preparation and its functionalization for electrochemical detection. (i) The silica NW membrane was assembled from discrete silica NWs. (ii) The resol (carbon) precursor solution (containing F127 and the iron complex) infiltrated the silica NW membrane. (iii) The heat process was used to graphitize the resol to partially graphitic mesoporous carbon, and subsequent HF etching to remove the silica template produced the HPGC membrane. (iv) PDA modification of the HPGC membrane was achieved via a dopamine self-polymerization technique. (v) In situ deposition of Au NPs inside the PDA-modified HPGC membrane was performed. (vi) GOx immobilization in the Au NP−PDA-modified HPGC membrane was performed. The diagram is not drawn to scale.
2. MATERIALS AND METHODS 2.1. Reagents and Chemicals. Poly(ethylene oxide)-blockpoly(propylene oxide)-block-poly(ethylene oxide) Pluronic F127 (F127, MW = 12600 Da), Fe(NO3)3·9H2O, tetraethoxysilane (TEOS), polyvinylpyrrolidone (PVP, MW = 40 kDa), trimethoxy(octadecyl)silane (TMOCS), dopamine, gold(III) chloride trihydrate (HAuCl4, ≥49%, Au basis), and tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma-Aldrich Inc. Phenol (99.98 wt %), formalin (37.0−40.0 wt %), hydrogen chloride (HCl, 36.0−38.0 wt %), an ammonium solution (25 wt % NH3), sodium hydroxide (NaOH), ethanol, and n-pentanol were obtained from Shanghai Chemical Co. Water used in all experiments was distilled and deionized. Unless indicated otherwise, all reagents used were of analytical grade and used without purification. 2.2. Silica NW Membrane Assembly. The vacuum filtration method was used to prepare the free-standing NW membranes. The silica NW colloids were prepared using a method described previously.15 An appropriate amount of a diluted silica NW suspension was filtered through a cellulose acetate filter paper (pore size of 0.45 μm) to form an interwoven silica membrane. A custom mold with multiple holes (diameter of 4.0 mm) on the plate was mounted on the filter paper to tune size of the assembled silica NW membrane. The obtained silica NW membrane was then calcined at 550 °C for 6 h in an air atmosphere to reinforce the membrane structure. 2.3. Fabrication of HPGC Membrane Electrodes. The procedure employed for HPGC membrane electrode fabrication is shown in Figure 1. The silica NW membrane was immersed in a carbon precursor solution reported to prepare partially graphitic mesoporous carbon materials.24 Briefly, phenol (8.0 g), a NaOH solution (1.7 g, 20 wt %), and formalin (14.1 g) were mixed and stirred at 70 °C for 1 h. Water was removed by vacuum evaporation at 48 °C. The mixture solution was then cooled to room temperature, adjusted to neutral pH by addition of a HCl solution (2 M), and finally
Figure 2. HPGC membrane electrode fabrication: (a) HPGC membrane, (b) copper rod, (c) HPGC membrane adhered to the flat end of the copper rod with silver conductive paint, (d) heatshrinkable tube, and (e) HPGC membrane-adhered copper rod wrapped with the heat-shrinkable tube shown in panel d.
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DOI: 10.1021/acs.chemmater.7b01423 Chem. Mater. 2017, 29, 5286−5293
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Chemistry of Materials
Figure 3. SEM images of the (a) silica NW membrane template and (b−d) replicated HPGC membrane. The insets in panels a and b are digital photographs of the respective membranes (scale bar of 1 mm). (e−h) SEM images and (f and h) gold (indicated as red dots) elemental mapping profiles of the (e and f) outer surface and (g and h) cross section of the HPGC−PDA−Au membrane. (i) Energy-dispersive X-ray spectroscopy linear scans showing the distribution of the gold element within the surface (black line) and central area of the cross section (red line) of the HPGC−PDA−Au membrane. TEM images of the (j) HPGC−PDA and (k and l) HPGC−PDA−Au−GOx membranes. dissolved in ethanol (20 wt %) to form solution I. To prepare solution II, Pluronic F127 (1.6 g) and Fe(NO3)3·9H2O (0.11 g) were dissolved in a methanol solution (6 g) at 40 °C. To prepare solution III, TEOS (2.08 g) and a HCl solution (1.0 g, 0.2 M) were mixed at 40 °C for 0.5 h. All three solutions were then mixed together and concentrated under vacuum at 40 °C until the volume was reduced to approximately one-quarter of the total volume to form a carbon precursor resin. The membrane was then subjected to infiltration with the resin. The process was performed under vacuum to ensure complete infiltration. The resin-infiltrated membrane was then dried at room temperature and calcined at 900 °C for 2 h in a nitrogen atmosphere. After removal of the silica template with hydrofluoric acid (HF) and the ferrous components with an aqua regia solution (Caution: Extreme care should be taken when handling HF and the aqua regia solution), a carbon membrane with an interconnected, hierarchically nanoporous structure was obtained. The HPGC membrane was polished gently with a 600 grit sand paper (to remove the relatively dense mesoporous carbon layer on the membrane surface) before it was adhered to the end of a copper rod with silver conductive paint. Then, a heatshrinkable, insulating polyester tube was used to seal the HPGC membrane-adhered copper rod to obtain the HPGC membrane-based electrode. PDA modification of the electrodes was performed by immersing the electrodes in a 1 mM dopamine solution for 1 h, followed by immersion in 1 mM Tris buffer (pH 8.5) for 10 min. Au NPs were deposited by immersing the electrodes in a 1 mM HAuCl4
solution for 10 min and subsequent thorough washing with water. To load GOx, the electrodes were immersed in 1 mL of a GOx solution (1 mg/mL) for 3 h. After that, the electrodes were removed from the incubation solution and air-dried at ambient temperature. Ultraviolet− visible (UV−vis) spectrophotometry was used to monitor the GOx loading by measuring the absorbance of the enzyme solution at 276 nm. The electrode was stored at 4 °C prior to use. 2.4. Synthesis of the Mesoporous Carbon/Nafion Paste Electrodes. First, one piece of the HPGC−PDA−Au−GOx membrane was crumbled into tiny pieces and added to a Nafion solution (100 μL, 0.5%) and wax oil to form an electrode paste. Then, the paste was applied to the end of a copper rod and dried at room temperature. The paste was evenly spread to form a thin layer, which could cover the whole end of the copper rod. 2.5. Electrochemical Measurements. Cyclic voltammetry and differential pulse voltammetry measurements were performed on a CHI 660D electrochemical analyzer (Shanghai Chenhua Limited Co.) under ambient conditions in a phosphate-buffered saline (PBS) solution containing 0.2 mol/L KCl. A three-electrode system, which consisted of the HPGC membrane (∼3.5 mm diameter) as the working electrode, a KCl-saturated Ag/AgCl electrode as the reference electrode, and a Pt wire electrode as the auxiliary electrode, was used. Electrochemical impedance spectroscopy was performed on an AutoLab PGSTAT302 instrument (Eco Chemie, Utrecht, The Netherlands) in a 0.01 M [Fe(CN)6]3−/4− solution. The impedance 5288
DOI: 10.1021/acs.chemmater.7b01423 Chem. Mater. 2017, 29, 5286−5293
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Chemistry of Materials
Figure 4. (a) N2 sorption isotherms, (b) XRD patterns, and (c) Raman spectra of the HPGC (I), HPGC−PDA (II), HPGC−PDA−Au (III), and HPGC−PDA−Au−GOx (IV) membranes. (d) Percentage amount of GOX retained in the HPGC (I), HPGC−PDA (II), and HPGC−PDA−Au (III) membranes after soaking the enzyme-loaded membranes in water for different periods of time. measurements were performed in the frequency range from 100 mHz to 100 kHz. The experimental impedance data were analyzed by the Kramers−Kronig procedure to confirm the frequency dependence of the impedance. 2.6. Instrumentation. X-ray diffraction (XRD) measurements were taken on a Rigaku Dmax-3C diffractometer with Cu Kα radiation (40 kV, 40 mA, λ = 0.15405 nm). The crystal size of the Au NPs was estimated using the Scherrer equation: size = kλ/B cos θ, where B is the peak width (full width at half-height in radians) and k is a constant (0.89). Transmission electron microscopy (TEM) images were obtained on a JEOL 2011 microscope operating at 200 kV. Scanning electron microscopy (SEM) measurements were conducted on a Nova NanoSem 450 microscope operating at 5 kV. Raman spectra were collected on a Super LabRam Raman spectrometer, using a He−Ne laser with an excitation wavelength of 633 nm. N2 adsorption− desorption isotherms were measured at 77 K on a Quantachrome NOVA 4000e analyzer. The Brunauer−Emmett−Teller method was used to calculate the specific surface areas. The pore volume and pore size distribution for the mesopores were derived from the adsorption branch of the N2 sorption isotherm using the Barrett−Joyner− Halenda model. The pore volume and pore size distribution of the nanopores were also measured using a low-field nuclear magnetic resonance (NMR) freeze−thaw pore analyzer (Niumag NMRC12010V) at 32 K. Inductively couple plasma measurements were taken on a Hitachi Z-5000 Zeeman atomic absorption analyzer.
diameter of 80 nm, as observed from the SEM image (Figure 3a). First, the silica NW membrane imbibed a phenolic resin− Pluronic F127−silica/iron complex carbon precursor slurry (for catalytic graphitization).24 The carbonaceous precursor-imbibed silica NW membrane was then carbonized at 900 °C to graphitize the carbon framework. The silica template and iron species were subsequently removed to finally obtain the replicated HPGC membrane. The HPGC membrane had a diameter of 3.5 mm and a thickness of 0.3 mm (Figure 3b), which corresponds to a shrinkage of ∼10% in the diameter and ∼20% in the thickness of the silica NW template membrane, respectively. The hierarchical pore structure of the HPGC membrane could be observed from the SEM images (Figure 3c,d). Nanotunnels with a diameter of ∼80 nm were replicated from the silica NWs. The walls of the nanotunnels consisted of homogeneously distributed hexagonally ordered mesopores (∼6.5 nm in diameter). These pores formed upon removal of the Pluronic F127 porogen, which was added to the carbon precursor solution. The N2 sorption isotherms of the HPGC membrane showed a steep increase in the relative pressure (P/P0) range of 0.5−0.6 (Figure 4a), indicative of abundant ordered mesopores present in the membrane. The HPGC membrane had a high Brunauer−Emmett−Teller surface area of 1190 m2/g and a mesopore volume of 1.21 cm3/g for ordered mesopores with pore sizes centered at 6.8 nm (Figure S1). The volume for the nanotunnels was estimated to be 6.8 cm3/g (Figure S2a) using
3. RESULTS AND DISCUSSION The silica NW template membrane (4 mm in diameter and 0.38 mm in thickness) used in the study presented here consisted of interwoven silica NWs, which had an average 5289
DOI: 10.1021/acs.chemmater.7b01423 Chem. Mater. 2017, 29, 5286−5293
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Chemistry of Materials
Figure 5. Electrode performance. (a) Electrochemical impedance spectroscopy results of the electrodes for the different membranes prepared in the study: HPGC (I), HPGC−PDA (II), HPGC−PDA−Au (III), and HPGC−PDA−Au−GOx (IV). (b) DPV current response of H2O2 detection at varying H2O2 concentrations of 0.5, 0.8, 1, 3, 7, 10, 20, and 40 × 10−7 M from panel a to h, respectively, using the HPGC−PDA−Au electrode. The inset graph is the cyclic voltammetry (CV) response of 10−7 M H2O2. (c) Different pulse voltammetry response of H2O2 detection as a function of H2O2 concentration using the HPGC−PDA−Au electrode. (d) Cyclic voltammetry of 10−11 M glucose detection in the presence of 2 mM H2O2 in solution using the differently functionalized HPGC electrodes: HPGC−PDA−Au−GOx (1), HPGC−PDA−GOx (2), and HPGC (3). (e) DPV current response of 10−11 M glucose detection measured on different electrodes in the absence (0 mM) and presence (2 mM) of H2O2 in the solution. AuNPs/GOx and GOx represent the HPGC−PDA−Au−GOx and HPGC−PDA−GOx membrane electrodes, respectively. Carbon* denotes the electrode prepared using conventional methods (the mesoporous carbon paste electrode was prepared by casting the powdered HPGC−PDA−Au−GOx membrane with Nafion and wax oil on a copper rod). Carbon** and Carbon*** denote membrane electrodes that did not contain nanotunnels (the silica NW template was not removed during preparation) and did not contain mesopores, without the addition of the Pluronic F127 porogen (for mesopore generation), respectively. All other procedures are exactly the same as those used for the preparation of the HPGC−PDA−Au−GOx membrane electrode. (f) Different pulse voltammetry response to glucose concentration measured from the HPGC−PDA−Au−GOx electrode. The arrows mean the scanning direction during the investigation.
SEM images (Figure 2j and Figure S3). The graphitic structure of the carbon framework in the HPGC−PDA membrane was also retained, as evidenced by the Raman spectrum (Figure 4c). Au NPs were then deposited on the HPGC−PDA membrane to produce the Au NP-functionalized HPGC−PDA membrane (denoted as HPGC−PDA−Au) through immersion of the HPGC−PDA membrane in a HAuCl4 solution in the absence of a foreign reducing agent. The loading of the Au NPs was ∼20 wt %, as determined by the inductively coupled plasma method. Energy-dispersive X-ray spectroscopy data revealed the homogeneous distribution of the Au NPs (indicated by the red dots) throughout the outer surface (Figure 3e,f) and cross section (Figure 3g,h) of the membrane. However, as observed from Figure 3i, the loading of gold in the core of the membrane was lower by ∼60%. The larger amount of Au NPs deposited on the outer surface of the membrane was probably caused by the relatively thicker PDA layer deposited on the membrane surface. The presence of Au NPs in the membrane is evidenced by the XRD spectrum in Figure 4b (curve III), which featured peaks at 2θ values of 38.3°, 44.5°, 64.7°, and 77.7°; these could be indexed to the (111), (200), (220), and (311) planes, respectively, of a cubic unit cell of Au (JCPDS Card No. 653107). The calculated mean crystal size of the Au NPs was 9 nm using the Scherrer equation and was consistent with the TEM result (Figure 3k,l). Considering that the Au NPs are larger than the mesopores (∼6 nm), the Au NPs are likely to
a low-field NMR freeze−thaw pore analyzer. A bimodal pore distribution (5−10 and 40−80 nm) was also visible (Figure S2b). The total porosity (Φ) of the membrane was ∼90%, calculated on the basis of the density of graphitic carbon (2.25 g/cm3).25 The Raman spectrum of the HPGC membrane (Figure 4c, curve I) featured a band at 1597 cm−1, which could be ascribed to the vibration of sp2-bonded carbon atoms in a graphite layer (G-band). The band at 1335 cm−1 was ascribed to the vibration of carbon atoms with dangling bonds in planar terminations of a disordered graphite-like structure (D-band).24 The relative intensity of these two peaks (ID/IG) in the spectrum was close to 1:1. The crystalline properties of the HPGC membrane were further evidenced in the XRD pattern (Figure 4b), which displayed two specific peaks at 2θ values of 22° and 42° (ascribed to the D-band and G-band carbon, respectively). PDA modification resulted in an ∼50% increase in the weight of the membrane, indicative of successful deposition of PDA on the membrane. The surface area and mesopore volume of the PDA-modified HPGC (denoted as HPGC−PDA) membrane considerably decreased to 234 m2/g and 0.24 cm3/g, respectively. The considerable decreases in the surface area and mesopore volume of the HPGC−PDA membrane are likely caused by the PDA infiltration of the mesopores. The mesoporous carbon framework was retained after the PDA modification, as evidenced from the high-resolution TEM and 5290
DOI: 10.1021/acs.chemmater.7b01423 Chem. Mater. 2017, 29, 5286−5293
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Chemistry of Materials accumulate on the walls of the nanotunnels. This assumption could be further validated by the N2 sorption data, which revealed that the surface area (202 m2/g) and mesopore volume (0.23 cm3/g) of the HPGC−PDA membrane decreased only slightly after the deposition of Au NPs. The loading of the enzyme, glucose oxidase (GOx), in the HPGC−PDA and HPGC−PDA−Au membranes was comparable (i.e., 220 and 200 mg/g, respectively). However, the loading was higher than that in the pristine HPGC membrane (66 mg/ g). The immobilization of the enzyme in the HPGC−PDA−Au membranes was also evidenced by the surface-enhanced Raman scattering effects of the Au NPs. The Raman spectrum of the HPGC−PDA−Au−GOx membrane displayed numerous peaks in the range of 600−1200 cm−1, which are characteristic of amino acids of proteins (Figure 4c).26 Variation of the relative intensity of the ID/IG peaks is likely caused by the organic PDA and GOx loading. More importantly, the HPGC−PDA−Au and HPGC−PDA membranes retained 86 and 72%, respectively, of the initially loaded GOx after being soaked for 10 h in water while being continuously shaken (Figure 4d). In contrast, nearly all of the GOx was released from the HPGC membrane after it had been soaked for
