Exploiting Metal-Organic Coordination Polymers as Highly Efficient

ARTICLE pubs.acs.org/ac

Exploiting Metal-Organic Coordination Polymers as Highly Efficient Immobilization Matrixes of Enzymes for Sensitive Electrochemical Biosensing Yingchun Fu,† Penghao Li,† Lijuan Bu,† Ting Wang,† Qingji Xie,*,† Jinhua Chen,‡ and Shouzhuo Yao†,‡ †

Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, P. R. China ‡ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China

bS Supporting Information ABSTRACT: We report on the exploitation of metal organic coordination polymers (MOCPs) as new and efficient matrixes to immobilize enzymes for amperometric biosensing of glucose or phenols. A ligand, 2,5-dimercapto-1,3,4-thiadiazole (DMcT), two metallic salts, NaAuCl4 and Na2PtCl6, and two enzymes, glucose oxidase (GOx) and tyrosinase, are used to demonstrate the novel concept. Briefly, one of the metallic salts is added into an aqueous suspension containing DMcT and one of the enzymes to trigger the metal organic coordination reaction, and the yielded MOCPs-enzyme biocomposite (MEBC) is then cast-coated on an Au electrode for biosensing. The aqueous-phase coordination polymerization reactions of the metallic ions with DMcT are studied by visual inspection as well as some spectroscopic, microscopic, and electrochemical methods. The thus-prepared glucose and phenolic biosensors perform better in analytical performance (such as sensitivity and limit of detection) than those prepared by the conventional chemical and/or electrochemical polymerization methods and most of the reported analogous biosensors, as a result of the improved enzyme load/activity and mass-transfer efficiency after using the MOCPs materials with high adsorption/encapsulation capability and unique porous structure. For instance, the detection limit for catechol is as low as 0.2 nM here, being order(s) lower than those of most of the reported analogues. The enzyme electrode was also used to determine catachol in real samples with satisfactory results. The emerging MOCPs materials and the suggested aqueous-phase preparation strategy may find wide applications in the fields of bioanalysis, biocatalysis, and environmental monitoring.

B

iosensing of target analytes is regarded as one of the most important issues in a series of academic and industrial fields, such as bioanalysis including point-of-care rapid tests, environmental monitoring, and food safety. The immobilization of biorecognition molecules is one of the key steps in developing high-performance biosensors, since they can largely affect the load/activity of the immobilized biorecognition molecules and the mass-transfer efficiency of the relevant species. In our opinion, there are two existing matrix-based immobilization strategies for biomolecules, namely, bioimmobilization solely on the matrix surface (2-D protocol) or in/on the matrix interior/surface (3-D protocol). The 2-D protocol (such as surface adsorption on selfassembled monolayer and nanomaterials1,2) favorably makes biorecognition molecules spatially open to its substrates, but it often suffers from limited load and total-activity of the immobilized biomolecules due to the limited 2-D surface area available for bioimmobilization and the relatively high susceptibility of the unprotected surface-tethered biomolecules to the environment. In contrast, the 3-D protocol (e.g., entrapment of biomolecules in and their simultaneous adsorption on polymers and sol gels r 2011 American Chemical Society

through chemical or electrochemical methods3,4) can load more biorecognition molecules in a 3-D manner and more efficiently retain their bioactivity due to the protection of the 3-D shell. However, the mass transfer of the substrates and products should be unavoidably hindered to a larger degree by the 3-D matrix comparing with the case that the biomacromolecules are immobilized on the thinner 2-D matrix; thus, the mass transfer efficiency inside a 3-D matrix should also be an important factor worth considering for high-performance biosensing, in addition to the load and activity of the immobilized biorecognition molecules. Therefore, exploiting new matrixes/strategies with improved immobilization efficiency of biomolecules is always of high importance and interest. Metal organic coordination polymers (MOCPs) with metal ions as the connectors and ligands as the linkers, also known as metal organic frameworks (MOFs), are an emerging class of Received: February 23, 2011 Accepted: July 23, 2011 Published: July 24, 2011 6511

dx.doi.org/10.1021/ac200471v | Anal. Chem. 2011, 83, 6511–6517

Analytical Chemistry inorganic organic porous hybrid materials having high porosity and highly desirable frameworks via regulating the molecular building blocks. MOCPs with very large surface area, tunable pore size, high thermal stability, as well as tempting magnetic, electrical, optical, and catalytic properties have attracted considerable attentions and shown application potential in gas purification and separation,5 7 catalysis,8,9 chemical sensing,10 and drug delivery/ release.11,12 For example, Horcajada et al. reported on the adsorption of the model molecule ibuprofen in the rigid MOCPs MIL100 and MIL-101 (MIL = Material Institut Lavoisier) with very large pores, which exhibit an unprecedentedly high drug storage capacity of 1.4 g of drug per gram of porous solid.12 Imaz et al. presented MOCPs’ micro/nanospheres of Zn(II) and 1,4-bis(imidazol-1-ylmethyl)benzene as novel functional encapsulating matrixes of magnetic nanoparticles, quantum dots, and organic dyes for functional nanomaterials with excellent magnetic and optical properties.13 Therefore, the MOCPs that possess impressive adsorbability, encapsulation capability, and high porosity may be exploited as efficient matrixes to immobilize biorecognition molecules with high load/activity and high mass-transfer efficiency for high-performance biosensing; however, as we are aware, such attempts have not been reported yet. N-Heterocyclic thiones containing a thioamide group are intriguing ligands in coordination chemistry, which can achieve versatile coordination modes using exocyclic sulfur and endocyclic nitrogen atoms.14,15 These ligands also show prototropic tautomerism, acid base equilibrium, and redox reactions based on mercapto-disulfido conversion. Among N-heterocyclic thiones, 2,5-dimercapto-1,3,4-thiadiazole (DMcT, see Scheme 1 for its chemical structure), a commonly known bismuthiol, is regarded as a particularly interesting ligand, since its donor sites (mainly two exocyclic sulfur and two endocyclic nitrogen atoms) enable coordination to two or more metal ions and thus form various MOCPs in organic solvents.16 18 However, the coordination chemistry of DMcT to form MOCPs in aqueous solution, as we know, has not been investigated due perhaps to the limitation of the low solubility of DMcT in water. Phenolic compounds are widely used in wood preservatives, textiles, herbicides, and pesticides, and they are released into the ground and surface water and have been regarded as one of the most common and toxic species in the environment. Therefore, the identification and quantification of these compounds are of great importance in environment monitoring. Electrochemical biosensors,19 29 especially amperometric biosensors,20 29 have been considered as one of the most powerful tools for the in situ detection of phenolic compounds due to the high sensitivity, simplicity, and ease to miniaturization. The tyrosinase-immobilized electrodes have been suggested for phenolic biosensing, and various materials, such as sol gel,24,27 clays,23 nanomaterials,21,22,25,30 polymers,24,26,28,29 have been used as the enzyme-immobilization matrixes. Even so, exploiting new enzyme-immobilization materials with high enzyme load/ activity and mass-transfer efficiency for phenolic biosensing remains an interesting and important research topic. Here, we report on the exploitation of MOCPs as new and efficient immobilization matrixes of enzymes in aqueous suspensions to develop glucose oxidase (GOx)-based glucose biosensors and tyrosinase-based phenolic biosensors. The aqueous-phase coordination polymerization reactions of the metallic ions with DMcT are studied by visual inspection as well as UV vis, FT-IR, and Raman spectroscopy, microscopic techniques, electrochemistry methods, and the quartz crystal microbalance (QCM) method. The MOCPs can immobilize enzymes with high load/activity and

ARTICLE

Scheme 1. Illustration of the Preparation of the MOCPsenzyme Biocomposites (MEBCs)-Based Biosensor and the Biosensing Mechanism (with MEBC 3 As an Example)

mass-transfer efficiency, and the thus-prepared glucose and phenolic biosensors perform better than those prepared by the conventional chemical and/or electrochemical polymerization methods and most of the reported analogues.

’ EXPERIMENTAL SECTION Instrumentation and Chemicals. All electrochemical experiments were conducted on a CHI660C electrochemical workstation (CH Instrument Co.) with a conventional three-electrode electrolytic cell. The Au electrode with 2.0 mm diameter (area = 0.031 cm2) served as the working electrode, unless otherwise specified, a KCl-saturated calomel electrode (SCE) as the reference electrode, and a carbon rod as the counter electrode. All potentials reported here are cited versus SCE. A computerinterfaced HP4395A impedance analyzer (Hewlett-Packard) was employed in the QCM experiments, and 9 MHz QCM Au electrodes of a keyhole configuration and 0.29 cm2 area (Beijing Chenjing Electronic Co., China) were used. Atomic force microscopy (AFM) images were collected on a PicoPlus atomic force microscope (Agilent) with the tap mode using a SiN tip. Transmission electron microscopy (TEM) images were collected on a JEM-2100F transmission electron microscope (Japan). UV vis spectrophotometry was conducted on a UV-2450 UV vis spectrophotometer (Shimadzu, Japan). FT-IR spectra were collected on a pressed pellet with KBr in the transmission mode on a Nexus-670 Fourier transform-infrared spectrophotometer (Nicolet Instrument Co.) controlled by Ominic software. Raman experiments were conducted on a LABRAM-010 laser confocal Raman instrument (HORIBA Jobin Yvon Co., France), 632.8 nm He Ne laser excitation (0.1 mW), and a 50 long working-distance objective (8 mm). The width of the slit and the size of the pinhole were set as 100 and 1000 μm, respectively, and the integration time was 5 s. GOx (EC 1.1.3.4; type II from Aspergillus niger, activity ≈ 150 kU g 1) and tyrosinase (EC 1.14.18.1, 2870 U mg 1, from mushroom) was purchased from Sigma. DMcT was a product of Alfa Aesar. pH 7.0 phosphate buffer solution (PBS), 0.1 M KH2PO4 K2HPO4 + 0.1 M K2SO4, was used. All other chemicals were of analytical grade or better quality and used as received. MilliQ ultrapure water (Millipore, g 18 MΩ cm) was used throughout. All experiments were performed at room temperature. Fabrication and Characterization of the Enzyme Electrodes. First, Au electrodes were rinsed thoroughly according to a reported procedure with minor modifications.31 Then, the biosensors were prepared as illustrated in Scheme 1. To an ultrasonically dispersed 0.5 mL of PBS suspension of 0.5 mg mL 1 DMcT was added 1 mg of tyrosinase (2.0 mg mL 1) or 2 mg of GOx (4.0 mg mL 1), 6512

dx.doi.org/10.1021/ac200471v |Anal. Chem. 2011, 83, 6511–6517

Analytical Chemistry followed by addition of 20 μL of 0.1 M NaAuCl4 (or 2.4 mM Na2PtCl6) with slow stirring, and the suspension was allowed to stay for 5 min (10 min in the Na2PtCl6 case) to form MOCPsenzyme biocomposites (MEBCs). Note that the GOx-involved MEBCs are defined here as MEBCs 1 (using NaAuCl4) and 2 (using Na2PtCl6), and the tyrosinase-involved MEBCs as MEBCs 3 (using NaAuCl4) or 4 (using Na2PtCl6), respectively. In addition, enzyme-free MOCPs 1 (using NaAuCl4 in the absence of enzyme) and MOCPs 2 (using Na2PtCl6 in the absence of enzyme) were similarly prepared. The MEBCs suspensions were then centrifuged and washed with PBS three times, and the finally yielded precipitates were redispersed in 100 μL water, 1 μL of which was carefully cast onto the Au electrode and dried at room temperature. The yielded MEBCs suspension could be stored at 4 °C in PBS for 2 weeks with negligible bioactivity loss. When not in use, the biosensors were stored in 0.1 M pH 7.0 PBS at 4 °C. Amperometric Measurements. As shown in Scheme 1, the tyrosinase catalyzes the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the further oxidation to o-quinones (diphenolase activity) in the presence of molecular oxygen,24 and the electrochemical reduction of the quinone product gives the biosensing signal for phenolic determination. Measurements of the prepared enzyme electrodes were carried out at 0.1 V for phenols under solution-stirred conditions in pH 7.0 PBS, and the responses were marked with the change values between the steady-state currents after addition of substrates and the initial background currents without the substrates. Detection of glucose was conducted similarly at 0.7 V under solution-stirred conditions in pH 7.0 PBS.

’ RESULTS AND DISCUSSION Coordination Polymerization Chemistry of DMcT and Metallic Ions in Aqueous Suspension and Characterization of MEBCs.

Two classes of polymerization reactions (Scheme S-1 in the Supporting Information) between NaAuCl4 (or Na2PtCl6) and DMcT are foreseeable, namely, the coordination polymerization reaction of metal ions with DMcT and the redox polymerization reaction. The redox polymerization reaction shows the characteristic that NaAuCl4 (or Na2PtCl6) is reduced by DMcT to produce metal nanoparticles, and DMcT is chemically oxidized to form poly(DMcT) (PDMcTc) via S S linking, as reported previously.32 Here, we employ visual inspection as well as UV vis, Raman, and FT-IR spectrophotometry, microscopic techniques, QCM, and electrochemistry methods to obtain the insights into and make clear the involved reactions. Figure 1 shows digital pictures of the PBS suspensions of DMcT (sample 1), DMcT + GOx (sample 2), DMcT + GOx + H2O2 (sample 3, the product is PDMcTc-GOx composites), DMcT + GOx + NaAuCl4 (sample 4, MEBCs 1), and DMcT + GOx + Na2PtCl6 (sample 5, MEBCs 2), respectively. Rather homogeneous suspensions of samples 1 and 2 could be obtained via facile ultrasonication treatment. When NaAuCl4 or Na2PtCl6 was added into the suspension containing DMcT and the enzyme (samples 4 and 5), brown and loose floccules were observed immediately or in ∼10 min. For comparison, H2O2 as a metal-free oxidant was added to chemically oxidize the SH of DMcT to form PDMcT (PDMcTc) via the S S linking (sample 3),32 and a few light-yellow precipitates were found after 20 min of reaction, indicating a rather slow redox kinetic as reported previously.32 Also, the apparent amounts of the precipitates of samples 4 or 5 were much more than that in sample 3. The obvious differences of precipitation-rate and precipitatemorphology should imply that the polymerization mechanism for samples 4 and 5 is most likely different from that for sample 3

ARTICLE

Figure 1. Digital picture of several PBS suspensions (1.0 mL) containing DMcT (1), DMcT + GOx (2), PDMcTc-GOx (3), MEBCs 1 (4), and MEBCs 2 (5), respectively. Concentrations: DMcT, 2.5 mg mL 1 for suspension 3 and 5 mg mL 1 for others; GOx, 4.0 mg mL 1; H2O2, 15 mM; NaAuCl4, 4 mM; Na2PtCl6, 2.4 mM. The arrow indicates the light-yellow precipitates in sample 3.

(the S S linking mechanism to yield PDMcTc), as discussed below, and the precipitates with loose (porous) morphology in samples 4 and 5 may be beneficial to the immobilization of more enzymes with minor activity-loss (conformation change). UV vis, FT-IR, and Raman spectroscopy and TEM experiments were conducted to identify the products, and the latter two are focused on the S and N atoms of DMcT. The UV vis spectrophotometric results are shown in Figure S-1 in the Supporting Information. DMcT presented a prominent peak at 320 nm.33 However, after full reaction of NaAuCl4 and DMcT after ∼1 min, both the mixture and the suspension of the centrifugationisolated precipitates did not show the peak of DMcT monomer at 320 nm, and the relatively small whole-wavelength absorption with inflection points at ∼400 nm might be ascribed to the light scattering of the insoluble MEBCs, as reported previously,18 while the supernatant showed no peak at wavelength longer than 250 nm, implying that the depletion of DMcT after its full reaction with NaAuCl4. We did not observe absorption peaks at wavelength >500 nm, which are characteristic of Au nanoparticles,34 indicating a coordination polymerization-dominant mechanism rather than a redox polymerization one. This conclusion is also confirmed by TEM results, as shown in Figure S-2 in the Supporting Information, which show that the Au nanoparticles resulted from the reduction of NaAuCl4 by DMcT (redox polymerization mechanism) are quite few or negligible. The FT-IR spectrophotometric results are shown in Figure S-3 in the Supporting Information. The stretching vibration peak of S H of DMcT at 2474 cm 1 was obvious,18 which was not observed for PDMcTc and MOCPs. The peak of S S stretching was observed at 478 cm 1 for PDMcTc, indicating its S S linking structure after SH oxidation.35 However, the S S peak was minor or invisible in the MOCPs, suggesting that a nonredox polymerization reaction predominates the formation of MOCPs. In comparison with DMcT, we observed a minor shift of the peak of CdS for the PDMcTc but obvious negative shifts of 15 20 cm 1 for the MOCPs. In addition, we observed two peaks characteristic of the stretching vibrations of Au S and Pt S at 380 and 392 cm 1 in the Raman spectra (Figure S-4 in the Supporting Information), respectively. The above data should indicate the occurrence of sulfur coordination.36,37 In addition, the peaks characteristic of the stretching vibrations of Au N and Pt N were found at 546 and 555 cm 1, respectively, which were not found for DMcT and PDMcTc, implying the occurrence of nitrogen coordination.36,37 In conclusion, the above experiments solidly support that the MOCPs were produced in the neutral aqueous suspensions by the coordination polymerization mechanism involving the coordination of excocyclic S and endocyclic N atoms of DMcT with the metal ions. 6513

dx.doi.org/10.1021/ac200471v |Anal. Chem. 2011, 83, 6511–6517

Analytical Chemistry The direct electrochemistry of PDMcT on its mercaptodisulfido conversion is quasi-reversible, as examined previously.32 Hence, cyclic voltammetry (CV) was also adopted to exclude the direct chemical polymerization of DMcT through S S linking (redox-based) and prove the coordination polymerization without S S formation. The PDMcTc, MOCPs 1, and MOCPs 2 were cast-coated onto glassy carbon electrodes (GCE), cyclic voltammetry (CV) was then used to investigate the electrochemistry of each film, as shown in Figure S-5 in the Supporting Information, and CV curves of bare GCE in PBS in the absence or presence of DMcT are also given as controls. For the CV of GCE in the DMcT suspension, we found two obvious peaks at 0.15 and 0.25 V in the positive scan, which are corresponding to the oxidation of DMcT to oligomers and then the further oxidation of oligomers to PDMcT via linkage of SH to be S S , as well as 0.45 V for the reduction of PDMcT in the negative scan via cleavage of S S , which agree well with our previous results.32 For CV of the PDMcTc electrode, we found a cathodic peak at 0.35 V in the negative scan and an anodic peak at 0.2 V in the positive scan, which are ascribed to the reduction of S S to SH and its reoxidation to S S, respectively. However, there were no similar peaks observed for the MOCPs 1 and 2, proving negligible S S bonding and highlighting the coordination polymerization chemistry of DMcT with the metallic salts in the MOCPs. UV vis spectrophotometry was also used to quantify the GOx in the centrifugation-isolated supernatant of the suspensions of MEBCs 1 and 2, and the GOx solution and the centrifugation-isolated supernatant of PDMcTc-GOx suspension were used as controls, all with identical initial concentration of GOx of 4 mg mL 1. The results are shown in Figure 2. The peak absorbance of GOx at ∼280 nm in the supernatant of PDMcTcGOx was 0.10 ( 0.01 (n = 3, same as below), being 67% of that in the GOx solution (0.15 ( 0.01). In contrast, the absorbance of GOx in the supernatants of MEBCs 1 and 2 were decreased to be 0.029 ( 0.004 and 0.061 ( 0.008, being 19% and 41% of that in the GOx-solution case, respectively. We can thus calculate the enzyme loads in the MEBCs 1 and 2 to be 6.5 and 4.7 mg per mg DMcT, which are 12- and 8.9-fold of that for PDMcTc-GOx (0.53 mg GOx per mg DMcT), respectively, suggesting that MOCPs with high surface area and porosity are much more efficient in immobilizing enzyme than the polymer synthesized by conventional chemical oxidation. When the concentrations of GOx and NaAuCl4 were increased during the preparation, the GOx loads were even improved to 8.3 ( 0.5 and 7.2 ( 0.4 mg per mg of DMcT (Figure S-6 in the Supporting Information), respectively, highlighting the significant load capability of the MOCPs. However, the best biosensing performance occurred when the concentrations of GOx and NaAuCl4 were 4 mg mL 1 and 4 mM (Table S-1 in the Supporting Information), respectively, suggesting that the overloading of GOx might lead to disadvantageous conformation of immobilized GOx and mass transfer efficiency. Note here the load of enzyme in the MEBCs 1 is 1.4-fold of that in the MEBCs 2, which, we speculate, may be ascribed to the relatively lower encapsulation efficiency of MEBCs 2 with lower coordination polymerization rate (experimentally 1 and 10 min for reaction equilibrium of MEBCs 1 and 2 at identical moles of reactants, respectively). Mass transfer of the substrate and product of an enzyme reaction inside the enzyme film is one of the key factors in determining the analytical performance of an amperometric enzyme electrode. Here, we investigated the mass transfer efficiency of MEBCs 1

ARTICLE

Figure 2. UV vis spectra for 0.2 mg mL 1 GOx solution (1) and the centrifugation-isolated supernatant of the suspension of PDMcTc-GOx (2), MEBCs 1 (3), or MEBCs 2 (4). The initial concentrations of GOx were all 4 mg mL 1, and all samples were 20-fold diluted before the measurements. Concentrations: DMcT, 2.5 mg mL 1 for suspension 2 and 0.5 mg mL 1 for others; GOx, 4.0 mg mL 1; H2O2, 15 mM; NaAuCl4, 4 mM; Na2PtCl6, 2.4 mM.

Figure 3. Chronoamperometric responses on bare QCM (1), MEBCs 1/QCM (2), MEBCs 2/QCM (3), PDMcTc-GOx/QCM (4), and PDMcTe-GOx/QCM (5) electrodes at 0.7 V in pH 7.0 PBS to incremental additions of 1 mM H2O2. The scales are for all curves.

and 2 films on QCM Au electrodes (defined as MEBCs 1/QCM and MEBCs 2/QCM) to H2O2 through detecting its oxidation current at 0.7 V, with bare, electrosynthesized polymer of DMcT and GOx (PDMcTe-GOx) modified, and PDMcTc-GOx modified QCM electrodes (PDMcTe-GOx/QCM, PDMcTc-GOx/QCM) as controls. Since the current responses of the film-modified QCM electrodes should depend on the electrode-surface concentration of H2O2, which is dependent on the mass transfer of H2O2 from the bulk solution to the electrode surface, the mass transfer efficiency of various films could be appropriately characterized and compared by the current responses of relevant electrodes to H2O2 at identical bulk concentrations. Note that the mass of each loaded polymer was identical, as characterized by QCM through monitoring the frequency shifts before and after the polymer loading (∼ 3 kHz). The steady-state current responses are shown in Figure 3. We obtained current responses of 85 ( 6 and 76 ( 5 μA (n = 3, same as below) on the MEBCs 1/QCM and MEBCs 2/QCM, being as high as 65% and 58% of that on the bare electrode (130 ( 4 μA), respectively. They are 2.5- and 2.2-fold of that on the PDMcTc-GOx/QCM (34 ( 3 μA), 7.7- and 6.9-fold of that on the PDMcTe-GOx/QCM (11 ( 1 μA), respectively, 6514

dx.doi.org/10.1021/ac200471v |Anal. Chem. 2011, 83, 6511–6517

Analytical Chemistry

ARTICLE

Table 1. Performance of Various Glucose and Phenolic Biosensors (r2 Values for LDR Are All >0.99) biosensor MEBCs 1/Au

substrate

1

cm 2)

LDR (μM for glucose, nM for phenols)

LOD (nM)

80

0.025 1092

15

MEBCs 2/Au

52

0.075 700

75

PDMcTc-GOx/Au

27

0.25 145

230

MEBCs 3/Au

MEBCs 4/Au

glucose

sensitivity (μA mM

catechol

6780

0.2 15000

0.2

p-cresol

5070

2 25000

1

phenol

1583

10 14400

6

catechol

1430

2.5 44000

1.9

p-cresol phenol

1253 239

5 14400 20 41200

4 13

suggesting much higher mass transfer efficiencies of the MEBCs than those of the commonly used polymer matrix synthesized through either chemical or electrochemical oxidation with relatively compact structure. AFM images of the surfaces of MEBCs 1/Au and MEBCs 2/Au electrodes are shown in Figure S-7 in the Supporting Information, and we observed obviously rough surfaces and macropores as well as lots of nanoparticles that might be the MEBCs composites. Here, the impressive mass-transfer efficiency of MEBCs should be ascribed to the inherent high porosity of the MEBCs, which should be notably beneficial to the enzyme reaction and the biosensing performance. Biosensing Performance. At first, the biosensing performance for glucose was examined, since the well-known GOx-glucose system has been well established for amperometric biosensing. Under optimized conditions (Table S-1 in the Supporting Information), the performances of the MEBCs 1/Au and MEBCs 2/ Au electrodes were examined, with the PDMcTc-GOx/Au electrode for comparison. The amperometric responses and calibration curves are shown in Figure S-8 in the Supporting Information, and the sensitivity, linear detection range (LDR), and limit of detection (LOD) are listed in Table 1. The MEBCs 1/Au, MEBCs 2/Au, and PDMcTcGOx/Au electrodes presented sensitivities of 186-, 121-, and 63fold of that based on the direct electropolymerization protocol as reported in our previous study (0.43 μA cm 2 mM 1),32 respectively. The sensitivities of the three electrodes were also much superior to those reported in the literature, which are generally lower than 10 μA cm 2 mM 1.38 42 These data should solidly prove the obvious superiority of immobilization of enzymes through the in situ one-pot entrapment strategies upon the interfacial direct-immobilization ones, as also proved in our previous studies.32,43,44 More importantly, although all benefited from the in situ pre-entrapments, the MEBCs 1/Au and MEBCs 2/Au electrodes possessed sensitivities of 3- and 1.9-fold of that of the PDMcTc-GOx/Au electrode, respectively. The outstanding performance of the MEBCs-involved enzyme electrodes may be ascribed to the inherent porous structure and high adsorbability of MEBCs, since (1) the porous MEBCs can provide obviously larger space to in situ entrap more enzyme molecules by the one-pot protocol and may offer more favored microsurrounding to efficiently avoid bioactivity loss (conformation change) than conventional polymers and (2) the porous structure of MEBCs can largely facilitate the mass transfer of the analytes and products, thus promote the enzyme catalysis efficiency and the performance of biosensors. On the basis of the understandings of MEBCs-based glucose biosensors, we similarly developed MEBCs 3/Au and MEBCs 4/Au electrodes to detect three phenols, catechol, p-cresol, and

Figure 4. Chronoamperometric responses and calibration curves (insert) of the MEBCs 3/Au (1) and MEBCs 4/Au (2) electrodes to successive additions of catechol at 0.1 V in pH 7.0 PBS.

phenol. The typical steady-state amperometric responses to catechol (as an example) are shown in Figure 4, the calibration curves for detection of p-cresol and phenol are given in Figure S-9 in the Supporting Information, and the sensitivities, LDR, and LOD are listed in Table 1. The data suggest that the prepared biosensors can rapidly and sensitively detect phenols with LDR extending to 3 5 orders of magnitude and LOD down to nanomolar or even sub-nanomolar level. For example, the MEBCs 3/Au could detect catechol down to 0.2 nM, and the inspiring result was lower than that of most reported analogues by an order of magnitude or more, which were reported generally to be higher than 5 nM, as listed in Table S-2 in the Supporting Information. As discussed above, the tempting merits of the MEBCs should be responsible for the satisfactory performance of thus-prepared biosensors. To further investigate the performance of the MEBCs-based biosensors, we investigated the repeatability, reproducibility, stability, selectivity, and application feasibility, with the MEBCs 3/Au electrode and catechol as an example. The repeatability was examined by the successive determinations of 5 μM catechol, and a relative standard deviation (RSD) of 4.3% was obtained for 10 determinations. The fabrication reproducibility was also estimated (10 independent biosensors), and we obtained a RSD of 6515

dx.doi.org/10.1021/ac200471v |Anal. Chem. 2011, 83, 6511–6517

Analytical Chemistry 8.5% for the steady-state current to 5 μM catechol (Figure S-10A in the Supporting Information). The long-term stability of the biosensors was investigated through three parallel measurements of three MEBCs 3/Au electrodes per day. We found that the amperometric response of these electrodes to 5 μM catechol remained almost constant for the first 15 days and decreased to about 85% of the initial values after 1 month (Figure S-10B in the Supporting Information). The good long-term stability seems to result from the favorable network structure of the MEBCs and the high adsorbability of MOCPs to effectively avoid enzyme from leakage and activity-loss. The selectivity of the biosensor was investigated by detecting the amperometric response of 0.1 mM ascorbic acid, 0.2 mM uric acid, 0.5 mM H2O2, and 10 mM glucose, which were lower than 1% of that to 10 μM catechol (Figure S-11 in the Supporting Information). The good selectivity of this biosensor should result mainly from the high specificity of tyrosinase and the low working potential ( 0.1 V). We also examined the application feasibility of the proposed biosensor through determining catachol of the real water sample from the Xiangjiang River using the standard addition method,22,45 and the results are listed in Table S-3 in the Supporting Information. We found that the results obtained in real water samples show good average recoveries from 95 to 106%, confirming that the proposed biosensor is applicable for practical phenolic detection.

’ CONCLUSIONS In summary, we have successfully exploited the MOCPs as novel and efficient matrixes for enzyme immobilization and amperometric biosensing of glucose and environmentally toxic phenols. DMcT can readily coordinate with metal ions through its exocyclic S and endocyclic N atoms to form MOCPs in neutral aqueous suspension, and the formed MEBCs of inherent and unique porosity present largely improved enzyme load/activity and mass-transfer efficiency than the conventional polymers synthesized by chemical or electrochemical oxidation. The MEBCs-based glucose and phenolic biosensors showed notably improved performance as compared with those constructed by conventional polymer materials/strategies. In our opinion, the new MEBCs material may be adaptive for batch preparation of screen-printed enzyme electrodes for studies and utilizations in the future. The new materials/strategy may create new avenues and find wide applications in the fields of biosensing, biofuels, biocatalysis, environmental monitoring, and human health. ’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone/Fax: +86 731 88865515. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grants 90713018 and 21075036), the State Special Scientific Project on Water Treatment (Grant 2009ZX07212-001-06), the Foundations of Hunan Provincial Education Department and Hunan Province (Grant 11JJ4014),

ARTICLE

Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province and State Key Laboratories of Chemo/Biosensing and Chemometrics (Grant 200902) and of Electroanalytical Chemistry. Fu and Li contributed equally to this work.

’ REFERENCES (1) Chaki, N. K.; Vijayamohanan, K. Biosens. Bioelectron. 2002, 17 1–12. (2) Jain, P. K.; Huang, X. H.; El-Sayed, I. H.; El-Sayed, M. A. Acc. Chem. Res. 2008, 41, 1578–1586. (3) Cosnier, S. Biosens. Bioelectron. 1999, 14, 443–456. (4) Dunn, B.; Zink, J. I. Acc. Chem. Res. 2007, 40, 747–755. (5) Li, J. R.; Kuppler, R. J.; Zhou, H. C. Chem. Soc. Rev. 2009, 38 1477–1504. (6) Long, J. R.; Yaghi, O. M. Chem. Soc. Rev. 2009, 38, 1213–1214. (7) Murray, L. J.; Dinca, M.; Long, J. R. Chem. Soc. Rev. 2009, 38 1294–1314. (8) Lee, J.; Farha, O. K.; Roberts, J.; Scheidt, K. A.; Nguyen, S. T.; Hupp, J. T. Chem. Soc. Rev. 2009, 38, 1450–1459. (9) Ma, L.; Abney, C.; Lin, W. Chem. Soc. Rev. 2009, 38, 1248–1256. (10) Chen, B.; Xiang, S. C.; Qian, G. D. Acc. Chem. Res. 2010, 43 1115–1124. (11) Rieter, W. J.; Pott, K. M.; Taylor, K. M. L.; Lin, W. J. Am. Chem. Soc. 2008, 130, 11584–11585. (12) Horcajada, P.; Serre, C.; Vallet-Regí, M.; Sebban, M.; Taulelle, F.; F
erey, G. Angew. Chem. 2006, 118, 6120–6124. (13) Imaz, I.; Hernando, J.; Ruiz-Molina, D.; Maspoch, D. Angew. Chem., Int. Ed. 2009, 48, 2325–2329. (14) Raper, E. S. Coord. Chem. Rev. 1997, 165, 475–567. (15) Raper, E. S. Coord. Chem. Rev. 1996, 153, 199–255. (16) Srivastava, T. N.; Srivastava, P. C.; Srivastava, K. J. Inorg. Nucl. Chem. 1975, 37, 1803–1804. (17) Gajendragad, M. R.; Agarwala, U. Z. Anorg. Allg. Chem. 1975, 415, 84–96. (18) Ortega, P.; Vera, L. R.; Guzm
an, M. E. Macromol. Chem. Phys. 1997, 198, 2949–2956. (19) Lei, Y.; Zhao, G.; Liu, M.; Xiao, X.; Tang, Y.; Li, D. Electroanalysis 2007, 19, 1933–1938. (20) Dai, Z.; Xu, X.; Wu, L.; Ju, H. Electroanalysis 2005, 17 1571–1577. (21) Wang, S.; Tan, Y.; Zhao, D.; Liu, G. Biosens. Bioelectron. 2008, 23, 1781–1787. (22) Lu, L.; Zhang, L.; Zhang, X.; Huan, S.; Shen, G.; Yu, R. Anal. Chim. Acta 2010, 665, 146–151. (23) Shan, D.; Cosnier, S.; Mousty, C. Anal. Chem. 2003, 75 3872–3879. (24) P
erez, J. P. H.; L
opez, M. S. P.; L
opez-Cabarcos, E.; L
opez-Ruiz, B. Biosens. Bioelectron. 2006, 22, 429–439. (25) Shan, D.; Zhu, M.; Han, E.; Xue, H.; Cosnier, S. Biosens. Bioelectron. 2007, 23, 648–654. (26) Lakshmi, D.; Bossi, A.; Whitcombe, M. J.; Chianella, I.; Fowler, S. A.; Subrahmanyam, S.; Piletska, E. V.; Piletsky, S. A. Anal. Chem. 2009, 81, 3576–3584. (27) Zhang, Y.; Ji, C. Anal. Chem. 2010, 82, 5275–5281. (28) Xue, H.; Shen, Z. Talanta 2002, 57, 289–295. (29) Mailley, P.; Cummings, E. A.; Mailley, S. C.; Eggins, B. R.; McAdams, E.; Cosnier, S. Anal. Chem. 2003, 75, 5422–5428. (30) Carralero, V.; Mena, M. L.; Gonzalez-Cort
es, A.; Y
a~ nez-Sede~ no, P.; Pingarr
on, J. M. Biosens. Bioelectron. 2006, 22, 730–736. (31) Zhang, J.; Song, S.; Wang, L.; Pan, D.; Fan, C. Nat. Protoc. 2007, 2, 2888–2895. (32) Fu, Y. C.; Zou, C.; Xie, Q. J.; Xu, X. H.; Chen, C.; Deng, W. F.; Yao, S. Z. J. Phys. Chem. B 2009, 113, 1332–1340. (33) Shouji, E.; Yokoyama, Y.; Pope, J. M.; Oyama, N.; Buttry, D. A. J. Phys. Chem. B 1997, 101, 2861–2866. (34) Daniel, M. C.; Astruc, D. Chem. Rev. 2004, 104, 293–346. 6516

dx.doi.org/10.1021/ac200471v |Anal. Chem. 2011, 83, 6511–6517

Analytical Chemistry

ARTICLE

(35) Infrared and Raman Spectra of Inorganic and Coordination Compounds: Part A: Theory and Applications in Inorganic Chemistry, 6th ed.; Nakamoto, K., Ed.; Wiley-Interscience: New York, 2008; pp 216 220. (36) Zaidi, S. A. A.; Farooqi, A. S.; Varshney, D. K.; Islam, V.; Siddiqi, K. S. J. Inorg. Nucl. Chem. 1977, 39, 581–583. (37) Infrared and Raman Spectra of Inorganic and Coordination Compounds: Part B: Applications in Coordination, Organometallic, and Bioinorganic Chemistry, 6th ed.; Nakamoto, K., Ed.; Wiley-lnterscience: New York, 2008. (38) Raitman, O. A.; Katz, E.; Buckmann, A. F.; Willner, I. J. Am. Chem. Soc. 2002, 124, 6487–6496. (39) Nien, P.-C.; Tung, T.-S.; Hoa, K.-C. Electroanalysis 2006, 18 1408–1415. (40) McMahon, C. P.; Rocchitta, G.; Serra, P. A.; Kirwan, S. M.; Lowry, J. P.; O’Neill, R. D. Anal. Chem. 2006, 78, 2352–2359. (41) Ma, M. M.; Qu, L. T.; Shi, G. Q. J. Appl. Polym. Sci. 2005, 98 2550–2554. (42) Li, M. R.; Deng, C. Y.; Xie, Q. J.; Yang, Y.; Yao, S. Z. Electrochim. Acta 2006, 51, 5478–5486. (43) Fu, Y. C.; Li, P. H.; Xie, Q. J.; Xu, X. X.; Lei, L. H.; Chen, C.; Zou, C.; Deng, W. F.; Yao, S. Z. Adv. Funct. Mater. 2009, 19, 1784–1791. (44) Fu, Y. C.; Chen, C.; Xie, Q. J.; Xu, X. H.; Zou, C.; Zhou, Q. M.; Tan, L.; Tang, H.; Zhang, Y. Y.; Yao, S. Z. Anal. Chem. 2008, 80 5829–5838. (45) Liu, Z. J.; Liu, B. H.; Kong, J. L.; Deng, J. Q. Anal. Chem. 2000, 72, 4707–4712.

6517

dx.doi.org/10.1021/ac200471v |Anal. Chem. 2011, 83, 6511–6517