Active Pt Nanoparticles Stabilized with Glucose Oxidase - American

13846

J. Phys. Chem. C 2008, 112, 13846–13850

Active Pt Nanoparticles Stabilized with Glucose Oxidase Pierre Karam,† Yan Xin,‡ Sarah Jaber,† and Lara I. Halaoui*,† Chemistry Department, American UniVersity of Beirut, Beirut 110236, Lebanon and National High Magnetic Field Laboratory, Florida State UniVersity, Tallahassee, Florida 32310, USA ReceiVed: January 26, 2008; ReVised Manuscript ReceiVed: May 25, 2008

We report the growth of Pt nanoparticles (NP) coupled and stabilized in situ with glucose oxidase (GOx). The enzyme-capped-Pt nanoparticles were formed from K2PtCl6 (aq) reduced with H2 (g) in the presence of GOx at a molar ratio of 889/1 Pt(IV)/GOx, creating a direct-linkage between NP and protein during particle growth. Transmission electron microscopy imaging revealed the formation of fcc Pt nanocrystals in the presence of the enzyme with an average dimension ) 4.05 ( 0.62 nm, whereas in its absence Pt black precipitated from solution. This is evidence that the glycoprotein terminated propagation of Pt growth and stabilized the NP surface. Transfer of Pt NP-GOx to an electrode by assembly on poly(diallyldimethylammonium chloride) allowed amperometric glucose determination with a low limit of detection of ca. 30 µM. Introduction Bonding metal or semiconductor nanoparticles (NP) to enzymes gains significant interest for creating functional hybrid nanostructures in which a NP electronic, catalytic, or photonic property is coupled to a protein-specific recognition and biocatalytic activity. This can lead to fast communication between an enzymatic process and a NP response for signal transduction in biosensing or for catalytic cascade reactions. We report here a one-pot solution preparation of Pt NP coupled and stabilized in situ with glucose oxidase (GOx) during particle growth. The Pt NP-GOx hybrid will be of particular interest in glucose nanoscale biosensors, and the method can possibly be generalized to link other NPs and proteins. At GOx-modified Pt electrodes, the enzyme catalyzes the oxidation of β-D-glucose by molecular oxygen to gluconic acid and H2O2, and H2O2 is catalytically oxidized at Pt.1,2a,3,4 Miniaturizing the electrode offers the advantage of an enhanced signal-to-noise ratio and possibly stability and also lowers sample size requirement, which motivates the use of Pt NPs, Pt black, and ultra-microelectrodes (UME).1-4 Attaching enzymes to NPs may also protect against enzyme denaturation (on flat surfaces) in some systems.5 An enzyme-capped-NP can be seen as the ultimate miniaturized biosensing element. Enzymes have been bonded to other NPs or nanotubes following nanomaterial preparation. For example, Lin et al. coupled GOx to the tips of carbon nanotubes via amide bonds,6 Chen et al.7a and Bestman et al.7b used 1-pyrene butanoic acid succinimidyl ester to link various proteins to the sidewalls of SWCNT by covalent and van der Waals forces, and Willner et al. partially implanted Au NPs in GOx or glucose dehydrogenase for direct wiring to electrodes by reconstitution of the apoenzyme on cofactor-functionalized Au NPs.8 GOx/glucose and tyrosinase/tyrosine have also been shown to mediate growth of Au NPs by action of enzymatically produced reducing agents.9 Other biomolecules have been reported to functionalize NP surfaces. Polsky et al. used nucleic acids to modify the surface * Corresponding author e-mail: [email protected]. † American University of Beirut. ‡ Florida State University.

of 4.0 nm Pt NP prepared by citrate reduction, and the nucleic acid-functionalized Pt NPs were utilized as labels for DNA and thrombin recognition.10 In this paper, we demonstrate that a protein molecule can modify and stabilize the surface of a growing nanocrystal, creating direct linkage in situ. GOx-stabilized Pt NPs were prepared from PtCl62- (aq) solution in the presence of GOx at a molar ratio of 889:1 of Pt(IV)/GOx under a reducing H2(g) atmosphere, leading to the growth of fcc Pt NP of 4.05 ( 0.62 nm average dimension. The enzyme-stabilized Pt NPs are shown to maintain their electrocatalytic activity for several processes catalyzed at Pt, including H2O2 oxidation, and the enzyme retains catalytic activity for glucose oxidation. A proof-ofconcept experiment showed that an assembled film of GOx-Pt NPs in a cationic polyelectrolyte on an electrode surface allows amperometric glucose determination with a detection limit ca. 30 µM, by electrocatalytic oxidation at the Pt NP surface of H2O2 produced in the enzyme catalyzed reaction of glucose and oxygen. Experimental Methods Materials. Potassium hexachloroplatinate(IV), K2PtCl6 (Acros Organics); glucose oxidase (GOx) Aspergillus niger type X-S, 150 Units/mg (Sigma Aldrich); poly(diallyldimethylammonium chloride), PDDA, MW ∼200-350 kDa, 20 wt % in water (Sigma Aldrich); sodium chloride (Fluka Chemika); sodium phosphate monobasic monohydrate (Acros); sodium phosphate dibasic (Acros); ammonium hydroxide, 28-30 wt % (Acros); hydrogen peroxide 35 wt % (Fluka); ethanol 98% (Merck); D-(+)-Glucose, 99.5% (Sigma Aldrich); and double distilled (dd) water were used in this study. Synthesis of GOx Stabilized Pt NPs. A 6 mL volume of 0.3 mg/mL GOx aqueous solution was added to 100 mL of 0.1 mM K2PtCl6 (aq) solution previously aged for 12 h. The Pt salt/ enzyme solution (the pH was adjusted to 7.0 with 1 M NaOH) was aged for another 12 h at 4 °C. The solution was then deoxygenated by bubbling argon for 20 min, followed by vigorously bubbling H2 (g) for 7 min, and the sealed reaction was left to proceed in the dark at RT for 6 h, resulting in a golden brown solution. The solution pH was adjusted to 7.0 with NaOH immediately after completion of reaction, and used

10.1021/jp800779c CCC: $40.75  2008 American Chemical Society Published on Web 08/13/2008

Pt Nanoparticles Stabilized with Glucose Oxidase for the assembly. In the control experiment, the same synthetic procedure was followed in the absence of GOx. All glassware was incubated at 65 °C and cooled to RT before use. TEM Imaging. TEM images were acquired after precipitation and resuspension of Pt NPs. The NPs were centrifuged at 4 °C for 10 min at 15 000 rpm using a Sorvall Discovery 100SE ultracentrifuge and then suspended in the same volume of dd water by sonication. A 10 µL drop was solvent cast on a C/Cu grid (SPI) and air-dried. TEM images and electron diffraction patterns were acquired on a JEOL-2011 high-resolution transmission electron microscope (NHMFL) operated at 200 kV with a point resolution of 0.23 nm and a lattice resolution of 0.14 nm. The d spacings were measured from the diffraction pattern using d ) A/D, where D is the diameter of the diffraction rings and A is a constant determined using an Al polycrystal as an external calibration standard at a fixed camera length of the pattern. The different rings were assigned to the corresponding fcc Pt lattice planes. Electrochemical Measurements. Pt NP-GOx were assembled on In:SnO2 electrodes (ITO, coated one side, Rs ) 8-12 Ω; Delta Technologies, Ltd.). The ITO substrates were cleaned in boiling ethanol for 30 min and then in a warm 7:3 (v/v) 28% NH3/35% H2O2 mixture for 10 min, rinsed 3 × 2 min in dd water, and air-dried. To assemble a bilayer of PDDA/Pt NP-GOx, the electrode was dipped in 10 mM PDDA/0.4 M NaCl (aq) solution (the concentration of PDDA is for the monomer unit) for 30 min, rinsed 2 × 1 min in water, and airdried for 10 min. The PDDA-modified surface was then immersed for 60 min in the as-prepared Pt NP-GOx solution after raising its pH to 7.0 (with 0.05 M NaOH), rinsed with dd water for 1 min, and air-dried. To assemble a bilayer of PDDA/ GOx film on ITO, the PDDA-modified ITO electrode was dipped in a solution of GOx (1.8 mg/106 mL, same concentration as used for the Pt NP preparation) at pH 7 for 60 min, rinsed with dd water for 1 min, and air-dried. Amperometric measurements were collected with a CHI630A (CH instruments) electrochemical workstation in a 3-electrode cell with a Pt wire or gauze (Alfa Aesar) as the auxiliary electrode, a Ag/AgCl (CH instruments) as the reference electrode, and 0.1 M phosphate buffer solution pH 7 (PBS) as supporting electrolyte. Solutions of D-(+)-Glucose (0.1 and 1 M) in 0.1 M PBS were left to mutarotate for at least 12 h at 4 °C before measurements. Volumes of glucose solutions were injected into the air-saturated (unless otherwise indicated) and continuously stirred (with a magnetic stirrer) PBS electrolyte, and the current was recorded at 0.6 V bias applied to the PDDA/ Pt NP-GOx electrode to detect the oxidation of H2O2 produced by the enzymatic reaction of glucose with oxygen. When studying the film response in deaerated medium, the PBS solution and the glucose solution were bubbled with N2 (g) for 30 min, and a N2 blanket was maintained near the solution surface during measurements. The background current was permitted to reach a steady state before measurement. The current response in the calibration plot was computed from the raw amperometric data as the average signal 15 s after injection until the next glucose addition (∆t ≈ 35 s) after subtraction of background current. The magnitude of the noise here was computed as the standard deviation of the current response during the same time interval. The background current was averaged in the 100 s prior to the first injection. UV-Visible Measurements. Quartz substrates (G. M. Associates, Inc.) were cleaned in piranha solution (3:7 (v/v) 35% H2O2/98% H2SO4) for 30 min, rinsed 5 × 2 min in dd water and dried. n bilayers of PDDA/Pt NP-GOx were as-

J. Phys. Chem. C, Vol. 112, No. 36, 2008 13847

Figure 1. (A) TEM image and (B) size distribution histogram of Pt NPs formed by stabilization with GOx.

sembled by repeating the same procedure described above n times after a 10 min drying in air following each bilayer. Absorption spectra were collected using a Jasco V-570 UV-visible spectrophotometer. Results and Discussion GOx-stabilized Pt NPs were prepared from PtCl62- (aq) solution in the presence of GOx at a molar ratio of 889:1 of Pt(IV)/GOx under a reducing H2(g) atmosphere. A 6 mL volume of 0.3 mg/mL GOx (aq) solution (Apergillus niger, 150 units/ mg) was added to 100 mL of 0.1 mM K2PtCl6 (aq) previously aged for 12 h at RT, and the mixture (at pH 7.0) was aged for another 12 h at 4 °C with the rationale of allowing the binding of the enzyme functional groups with the Pt complex. Following deoxygenation with Ar, H2 (g) was bubbled into the mixture, and the sealed reaction proceeded at RT for 6 h. The solution turned golden-brown, indicative of the formation of Pt NPs. In a control experiment, the same procedure in the absence of GOx resulted in a blue solution tint and the precipitation of Pt black (cf. Supporting Information Figure S1). Size measurements from transmission electron microscopy (TEM) images (Figure 1) showed an average dimension of 4.05 ( 0.62 nm for 200 particles chosen randomly when appearing as non aggregates. HRTEM (Figure 2, and Supporting Information Figure S2) and electron diffraction (Figure 2) revealed the growth of fcc Pt nanocrystals in the presence of GOx with a lattice constant a ) 3.93 Å, in agreement with bulk Pt (a ) 3.92 Å). The size distribution histogram shown in Figure 1 agreed with dimensional analysis from HRTEM images of 21 nanocrystals whose lattice planes were resolved, yielding dimensions of 3.72 ( 0.69 nm and 3.95 ( 1.11 nm, parallel and perpendicular to the (111) planes, respectively.

13848 J. Phys. Chem. C, Vol. 112, No. 36, 2008

Figure 2. (A) HRTEM image, and (B) ED pattern of fcc Pt NP formed in the presence of GOx.

Figure 3. (A) Cyclic voltammograms in deoxygenated 0.1 PBS pH 7 at PDDA/Pt NP-GOx bilayer on ITO (i) and at unmodified ITO (ii). The inset shows the CV at the former electrode showing the region of Hupd at Pt NPs. (B) Amperometric response to H2O2 addition (3.3 mM) to air-saturated and stirred 0.1 M PBS solution at PDDA/Pt NP-GOx bilayer on ITO (i), unmodified ITO (ii), and a PDDA/GOx film on ITO (iii).

GOx is a homodimer glycoprotein with a molecular weight of 160 kDa and 16% carbohydrate content.11 The enzyme evidently terminated propagation of Pt particle growth, which is attributed to surface groups (for example, COOH of aspartic and glutamic acid, NH2 of lysine and arginine,11a or other O-H or CdO groups) binding to the Pt(IV)-complex or to the surface of growing Pt nanoparticles. The pH was initially 7.0, making the enzyme significantly negatively charged with Z = -63 (estimated from Figure 3 in ref 12) but dropped to 3.2 after 6 h (due to H+ production), causing a net surface charge reversal (pI ) 4.2). The effect of GOx surface charge on NP growth has yet to be studied. GOx has been reported (from XRD data) to have a dimer ellipsoidal shape with dimensions 7.0 × 5.5 × 8.0 nm, monomer dimensions of 6.0 × 5.2 × 3.7 nm, and a long narrow contact area.11 STM images of GOx on Au13a and HOPG13b showed the “butterfly” shape (each monomer as a “wing”) characteristic

Karam et al. of the native enzyme, having dimensions of 10 ( 1 nm × 6 ( 1 nm or 6.5 ( 1 nm × 5 ( 1 nm in different orientations on Au.13a The average Pt NP size is thus comparable to the smallest enzyme dimension. In addition to single nanocrystals, connected nanoparticles and clustered nanoparticles were also observed in HRTEM (Figure S2). To estimate the number of protein molecules associated on average with a NP we calculate that a Pt sphere of 4.05 nm diameter and the same density as bulk Pt contains ∼2.30 × 103 atoms, giving 2.6:1 GOx/NP (or 5.2:1 monomer/NP) correspondence. The stabilizer/Pt atom ratio (1:889) is significantly smaller than that used to prepare Pt NPs with small size capping agents.14 For example, 5:1 or 1:1 ratios of polyacrylate (PAC, MW ) 2100)/PtCl42- were used by El-Sayed et al. to synthesize ∼7-11 nm Pt NPs,14a whereas we used 31:1 PAC/ PtCl62- to prepare ∼2.5 nm Pt.14b Pt NPs modified with polyvinylpyrrolidone (PVP, MW ) 40 kDa) were prepared between 1.1 and 2.7 nm at a 9:1 ratio of PVP/PtCl62-14c or as ∼5 nm NPs at a 1:18 ratio of PVP/PtCl42-.14d 1.4 or 1.6 nm Pt NPs were encapsulated inside OH-terminated polyamidoamine dendrimers at 1 dendrimer/40 or 60 PtCl42-.14e 1.6 nm Pt NPs were also reportedly incorporated in glassy carbon using poly(phenylenediacetylene)/(PPh3)Pt(C2H4) (where Ph is phenyl) in a coordination ratio of 4:1 or 2:1.15 The small number of GOx molecules needed to stabilize Pt NP growth in this study must be a result of the large protein size. As a proof-of-concept experiment, we demonstrated that the GOx-Pt NP hybrid structure can have applicability in glucose nanoscale biosensors. The resulting Pt NPs and GOx from the preparation were shown in an electrochemical test to maintain activity for H2O2 oxidation and glucose oxidation, respectively. To transfer the particles to an electrode, an In:SnO2 substrate modified with poly(diallyldimethylammonium) chloride (PDDA), a cationic polyelectrolyte, was immersed for 1 h in the Pt NP-GOx solution at pH 7 (at this pH the enzyme is negatively charged), and UV-visible spectra indicated adsorption on PDDA (followed in multilayers, cf. Supporting Information Figure S3). PDDA has been shown to provide an adsorption matrix for assembly of polyacrylate modified-Pt NP by electrostatic and hydrophobic interactions14b and is shown here to allow adsorption of the negatively charged GOx-Pt NP as well, which is attributed to similar electrostatic interactions. Cyclic voltammetry at a PDDA/Pt NP-GOx bilayer on ITO showed features characteristic of Pt, providing evidence of assembly of Pt NPs and of retention of their surface catalytic activity and also indicating the feasibility of charge transport between the NPs and the PDDA-modified ITO electrode. The CV at PDDA/ Pt NP-GOx in PBS pH 7 (Figure 3A) shows a reduction current in the potential range -0.27 to -0.53 V and is attributed to hydrogen underpotential deposition (Hupd)16 at the Pt NPs, followed by hydrogen evolution with an onset at -0.53 V. On the reverse scan, hydrogen oxidation with a peak at -0.5 V followed by atomic hydrogen desorption (Hdes) was also detected. Comparison to the CV at an unmodified ITO electrode demonstrates that these electrocatalytic reactions are taking place at the Pt NPs surface. Oxidation/reduction peaks that would correspond to electrochemistry of the enzyme cofactor FAD (Flavin Adenine Dinucleotide) in the buried active site were not observed in the CV, therefore there was no evidence of direct electrical wiring of the enzyme to the electrode surface. The oxidation current onset at 0.3 V (cf. inset of Figure 3A) is attributed to OH adsorption or other Pt oxide formation, whereas the broad reduction peak on the negative sweep with an onset at 0.35 V is likely due to OH desorption or to the reduction of

Pt Nanoparticles Stabilized with Glucose Oxidase

Figure 4. (A) Continuous amperometry plot at PDDA/GOx-Pt NP on ITO at 0.6 V vs Ag/AgCl in response to glucose additions in PBS pH 7 (V ) 6 mL, air-saturated and stirred) shown for the first two injections: 1.5 µL of 0.1 M glucose (25 µM) and 2.5 µL (equivalent to an additional 42 µM). (B) Calibration plot of current density as a function of glucose concentration at PDDA/Pt NP-GOx (at 0.6 V) in 0.1 M PBS pH 7. The current density is relative to the cross-sectional area (∼1.6 cm2).

other Pt oxide. Further evidence of Pt NP assembly and electrocatalytic activity is provided in the significant amperometric response to H2O2 addition at the PDDA/Pt NP-GOx film biased at 0.6 V (vs Ag/AgCl) compared to the current measured at PDDA/GOx modified- or unmodified-ITO electrodes (cf. Figure 3B), indicating H2O2 electrocatalytic oxidation (H2O2 f 2H+ + 2e + O2) at the Pt surface. We note that it is possible that some remaining free-GOx coadsorbed with GOx-modified Pt NP on PDDA by this assembly procedure. We attempted to separate GOx-Pt NP from free-GOx (by ultracentrifugation), but this was not successful in apparently retaining the same surface modification as the precipitated NP (resuspended by sonication) did not assemble on PDDA.17 Continuous amperometric measurements were recorded in response to glucose additions into air-saturated and stirred 0.1 M phosphate buffer solution pH 7 (Figure 4, and Supporting Information Figure S4) at 1 bilayer of PDDA/Pt NP-GOx. A similar current response to glucose was not measured in deoxygenated PBS; this is shown in Supporting Information Figure S5 at the same PDDA/Pt NP-GOx film in air-saturated solution and after deaerating by bubbling N2 (g) (the glucose solution was also deoxygenated). In addition, cyclic voltammetry at the PDDA/Pt NP-GOx assembly did not provide evidence of direct electrical wiring of the enzyme, therefore the current response at the PDDA/Pt NP-GOx film to glucose must be due to the electrooxidation at Pt NPs of H2O2 produced by the enzyme-catalyzed reaction of glucose and O2. The significantly smaller current responses to H2O2 (Figure 3B) and to glucose (cf. Supporting Information Figure S6) at a PDDA/GOx film on ITO compared to the PDDA/Pt NP-GOx assembly demonstrated that the Pt NPs in this architecture electrocatalyze the oxidation of produced H2O2. Figure 4B shows a calibration plot of steady-state current versus glucose concentration at PDDA/Pt NP-GOx assembly

J. Phys. Chem. C, Vol. 112, No. 36, 2008 13849 biased at 0.6 V versus Ag/AgCl. A sensitivity equal to 0.42 µA mM-1 cm-2 was measured over a linear range from 0.56 to 16 mM (R2 ) 0.993), indicating considerable catalytic and biocatalytic activity at adsorbed Pt NPs and GOx. The lower sensitivity at higher concentrations results from surface site saturation. A higher rate was recorded from 25 µM to 0.56 mM, with a S/N ratio of 2 at 25 µM and 5 at the next injection of 42 µM (Figure 4), indicating a limit of detection (LOD) in the range of 30 µM. This is better than that reported at a 50-80 nm carbon nanotube array with GOx bonded to the tips (LOD 80 µM)6 or at a Prussian blue modified 100-500 nm carbon nanofiber (LOD 100 µM).18 Better detection limits were demonstrated at Pt UME modified with GOx, for instance, LOD values of 0.5 µM or 10 µM were reported at a platinized Pt UME (50-500 or 10 µm diam, respectively) fabricated by incorporating GOx (at 50 mg GOx in solution) into the micropores,3a and a LOD of 0.5 µM was reported at a 100 µm diameter platinized Pt UME by trapping GOx (at 30 mg/mL) while electrodepositing Pt black.3b In this latter report, a higher LOD of 50 µM was measured at 10 µm diameter UME. Reducing the radius of the active Pt surface to 76 nm while trapping GOx in an electropolymerized phenol/2-allylphenol, Hrapovic et al. demonstrated a LOD of 20 µM at a loading of 10 mg GOx/mL, whereas a LOD in the millimolar range was reported at 2.5 mg GOx/mL and attributed to low GOx loading.4 On the other hand, a GC electrode modified with 2-3 nm Pt NP (in Nafion) and GOx (by casting 3 µL at 20 mg/mL, and cross-linking) exhibited a higher LOD of 400 µM; codeposition of SWCNT improved detection to 0.5 µM.1b The 30 µM LOD at the PDDA/Pt NP-GOx assembly reported here was not optimized as a function of GOx and Pt NP loading, but notably this procedure utilizes significantly smaller masses of Pt and GOx for NP-GOx hybrid preparation and films assembly (ca. 5 mg of K2PtCl6 and only 1.8 mg of GOx /106 mL) in comparison with other enzyme-Pt biosensors discussed.3,4 There may be other advantages for nanostructuring the electrode as a random array of Pt NPs for biosensors based on H2O2 detection. In a recent related study we showed that random arrays of 2.5 nm polyacrylate (PAC)-capped Pt NP on PDDA featured a high current output for H2O2 oxidation at a low mass of Ptsestimated at ca. 190-870 ng/cm2s, a significant turnover rate per Pt site, a low detection limit (tens of nanomolar), and significant stability facing constant anodic polarization in a major improvement over bulk Pt (since deactivation by growth of a thick oxide film is a main drawback of bulk Pt detectors).19 This was attributed to the nanosize of the PAC-Pt active elements (2.5 nm) and their loosely packed surface distribution in the polyelectrolyte and to a more difficult surface oxidation of the NPs.19 The LOD of ca. 30 µM glucose at the PDDA/Pt NP-GOx film at low concentration of GOx in the assembly medium could also be due to the small Pt NPs size (4.1 nm) and their surface distribution improving the sensitivity and S/N ratio but also possibly to the spatial proximity between NP and enzyme. There exists supporting evidence in the literature that direct adsorption of GOx on Pt does not lead to enzyme denaturation, as inferred, for instance, from studies of glucose detection at GOx deposited while electrochemically growing Pt black,3b adsorbed in platinized Pt pores,3a or directly electrodeposited on Pt/Ir.20 The comparable NP size may be a factor assisting in preserving the enzyme native structure,5 but this requires further future study of the activity of free versus surface-bound GOx.

13850 J. Phys. Chem. C, Vol. 112, No. 36, 2008 Conclusions We showed that GOx can be coupled by a simple procedure to Pt NP and stabilizes in situ nanocrystal growth, creating a direct bond, and that the resulting NP and enzyme maintain catalytic activity. Amperometric glucose determination was possible by assembling the NP-enzyme structures in a polyelectrolyte matrix on electrodes. This method may prove to be of general nature to create miniaturized nanoscale biosensing elements, as we have evidence it also works for in situ modification of semiconductor nanoparticles with proteins, which will be the subject of another report. Acknowledgment. We thank the University Research Board at AUB for financial support of this work, and acknowledge the donors of the American Chemical Society Petroleum Research Fund (40548-B10) for partial financial support. We also thank NSF (DMR-9625692) and NHMFL under cooperative agreement DMR-0084173. Supporting Information Available: Photograph of Pt NP-GOx and control without GOx solutions; HRTEM; UV-visible absorption spectra of PDDA/Pt NP-GOx multilayers; continuous amperometry plot; amperometric response to glucose at a PDDA/Pt NP-GOx film in deoxygenated PBS compared to air-saturated solution; and amperometric response to glucose and to H2O2 at a PDDA/GOx film on ITO. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) (a) You, T.; Niwa, O.; Tomita, M; Hirono, S Anal. Chem. 2003, 75, 2080–2085. (b) Hrapovic, S.; Liu, Y.; Male, K. B.; Luong, J. H. T. Anal. Chem. 2004, 76, 1083–1088. (2) (a) Kim, C. S.; Oh, S. M. Electrochim. Acta 1996, 41, 2433–2439. (b) Niwa, O.; Horiuchi, T.; Morita, M. ; Huang, T.; Kissinger, P. T Anal. Chim. Acta 1996, 318, 167–173. (3) (a) Ikariyama, Y.; Yamauchi, S.; Aizawa, M.; Yukiashi, T.; Ushioda, H. Bull. Chem. Soc. Jpn. 1988, 61, 3525–3530. (b) Ikariyama, Y.; Yamauchi, S.; Yukiashi, T.; Ushioda, H. J. Electrochem. Soc. 1989, 136, 702–706. (4) Hrapovic, S.; Luong, J. H. T. Anal. Chem. 2003, 75, 3308–3315. (5) (a) Crumbliss, A. L.; Perine, S. C.; Stonehuerner, J.; Tubergen, K. R.; Zhao, J. G.; Henkens, R. W. Biotechnol. Bioeng. 1992, 40, 483–

Karam et al. 490. (b) Stonehuerner, J. G.; Zhao, J.; O’Daly, J. P.; Crumbliss, A. L.; Henkens, R. W. Biosens. Bioelectron. 1992, 7, 421–428. (6) Lin, Y.; Lu, F.; Tu, Y.; Ren, Z. Nano Lett. 2004, 4, 191–195. (7) (a) Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. J. Am. Chem. Soc. 2001, 123, 3838–3839. (b) Bestman, K.; Lee, J.; Wiertz, F. G. M.; Heering, H. A.; Dekker, C. Nano Lett. 2003, 3, 727–730. (8) (a) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003, 299, 1877–1881. (b) Zayats, M.; Katz, E.; Baron, R.; Willner, I. J. Am. Chem. Soc. 2005, 127, 1877–1881. (9) (a) Zayats, M.; Baron, R.; Popov, I.; Willner, I. Nano Lett. 2005, 5, 21–25. (b) Baron, R.; Zayats, M.; Willner, I. Anal. Chem. 2005, 77, 1566– 1571. (10) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Anal. Chem. 2006, 78, 2266–2271. (11) (a) Hecht, H. J.; Schomburg, D.; Kalisz, H.; Schmid, R. D. Biosens. Bioelectron. 1993, 8, 197–203. (b) Hecht, H. J.; Kalisz, H. M.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153–172. (12) Voet, J. G.; Coe, J.; Epstein, J.; Matossian, V.; Shipley, T. Biochemistry 1981, 20, 7182–7185. (13) (a) Losic, D.; Shapter, J. G.; Gooding, J. J. Langmuir 2002, 18, 5422–5428. (b) Chi, Q.; Zhang, J.; Dong, S.; Wang, E. J. Chem. Soc. Faraday Trans. 1994, 90, 2057–2060. (14) (a) Ahmadi, T. S.; Wang, Z. L.; Green, T. C.; Henglein, A.; ElSayed, M. Science 1996, 272, 1924–1926. (b) Ghannoum, S.; Xin, Y.; Jaber, J.; Halaoui, L. I. Langmuir 2003, 19, 4804–4811. (c) Yu, W.; Liu, M.; Liu, H.; Zheng, J. J. Colloid Interface Sci. 1999, 210, 218–221. (d) Narayanan, R.; El-Sayed, M. A. J. Phys. Chem. B 2003, 107, 12416–12424. (e) Zhao, M.; Crooks, R. M. AdV. Mater. 1999, 11, 217–220. (15) Pocard, N. L.; Alsmeyer, D. C.; McCreery, R. L.; Neenan, T. X.; Callstrom, M. R. J. Am. Chem. Soc. 1992, 114, 769–771. (16) Markarian, M.; El Harakeh, M.; Halaoui, L. I J. Phys. Chem. B 2005, 109, 11616–11621, and references therein. (17) This was thought to have resulted from a change in surface modification, i.e., a possible loss in the coupled heterostructure between NP and enzyme during ultracentrifugation. After being ultracentrifuged at 15 000 rpm, the Pt NPs still exhibited the average nanocrystal size of ca. 4.0 nm (Figures 1 and 2), but they were no longer readily solvated in aqueous medium and resuspension required sonication; possibly indicating some NP agglomeration. By comparison, prior to this procedure, the Pt NP-GOx as-prepared structures were fully solvated forming a golden-brown solution as shown in Supporting Information Figure S1. This apparent change in solvation properties led us to believe that there may have been a change in surface modification by this procedure which prevented NP assembly on PDDA, explaining the lack of measurable electrocatalytic activity of the film thus assembled. (18) Zhang, X.; Wang, J.; Ogorevc, B.; Spichiger, U. E. Electroanalysis 1999, 11, 945–949. (19) Karam, P.; Halaoui, L. Anal. Chem. 2008, 80, 5441–5448. (20) Matsumoto, N.; Chen, X.; Wilson, G. S. Anal. Chem. 2002, 74, 362–367.

JP800779C