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 K 2 PtCl 6 (aq) reduced with H 2 (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 <d>) 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 -D-glucose by molecular oxygen to gluconic acid and H 2 O 2 , and H 2 O 2 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 apoen- zyme 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 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 PtCl 6 2- (aq) solution in the presence of GOx at a molar ratio of 889:1 of Pt(IV)/GOx under a reducing H 2 (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 H 2 O 2 oxidation, and the enzyme retains catalytic activity for glucose oxidation. A proof-of- concept 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 H 2 O 2 produced in the enzyme catalyzed reaction of glucose and oxygen. Experimental Methods Materials. Potassium hexachloroplatinate(IV), K 2 PtCl 6 (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 K 2 PtCl 6 (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 H 2 (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 * Corresponding author e-mail: Lara.Halaoui@aub.edu.lb. † American University of Beirut. ‡ Florida State University. J. Phys. Chem. C 2008, 112, 13846–13850 13846 10.1021/jp800779c CCC: $40.75  2008 American Chemical Society Published on Web 08/13/2008