NANO LETTERS
Biocatalytic Growth of Au Nanoparticles: From Mechanistic Aspects to Biosensors Design
2005 Vol. 5, No. 1 21-25
Maya Zayats,† Ronan Baron,† Inna Popov,‡ and Itamar Willner*,† Institute of Chemistry, and The Unit for Nanoscopic Characterization, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel. Received September 5, 2004
ABSTRACT The H2O2-mediated enlargement of Au nanoparticles (NPs) and the growth mechanism are described. In addition to the deposition of gold on the NP faces, the formation of nanocrystalline clusters at the intersection of the faces is observed. The detachment of the latter nanoclusters provides additional seeds for the deposition of gold. The biocatalyzed generation of H2O2 in the presence of O2/glucose and glucose oxidase enabled the development of an optical biosensor for glucose.
Au nanoparticles (Au NPs) are often used as optical labels for the detection of biorecognition events such as DNA hybridization1 or antigen-antibody complex formation2 or for the analysis of the catalytic functions of nucleic acids.3 Alternatively, Au NPs conjugated to biomaterials are used to amplify specific biomaterial binding events on surfaces by stimulating the electronic coupling between the localized plasmon of the NPs and the surface plasmon wave associated with the bulk Au surface.4 Also, Au NPs are used as “weight labels” for the detection of biorecognition events such as DNA hybridization using piezoelectric quartz crystals.5 The intrinsic property of metal nanoparticles to catalyze the reduction of metal ions on the NPs and thereby to enlarge the metallic nanoparticles is employed in different biosensing paths. The catalytic enlargement of Au NPs, acting as labels for DNA hybridization, is used for the amplified microgravimetric quartz-crystal-microbalance detection of nucleic acids.6 Similarly, the catalytic enlargement of Au NP conjugates associated with biorecognition complexes is used to yield conductive patterns that follow biosensing processes.7 Recently, the metallization of aptamer-functionalized Au NPs was used for the amplified optical detection of aptamerthrombin interactions.8 The use of enzymes as biocatalysts for the enlargement of metallic particles is, however, unexplored, and only recently the enlargement of Au NPs by NAD(P)H cofactors was demonstrated.9 The development of such systems may lead, however, to new sensitive enzyme assays and to novel biosensor configurations that employ * Corresponding author. Email:
[email protected]. Tel.: 972-26585272. Fax: 972-2-6527715. † Institute of Chemistry. ‡ Unit for Nanoscopic Characterization. 10.1021/nl048547p CCC: $30.25 Published on Web 12/03/2004
© 2005 American Chemical Society
optical, conductive, or microgravimetric stimuli as readout signals. Here we wish to report on the catalytic growth of Au NPs using AuCl4- and H2O2 and on the application of the process to develop a glucose sensor system based on the biocatalytic enlargement of Au NPs. The latter system may provide a model for numerous other oxidase-based biosensor assemblies. We also address the mechanism of growth of the Au NPs and reveal the catalytic deposition of nanocrystalline flakes on the NPs surfaces that eventually detach from the surface to form small crystalline flakes. The addition of H2O2 to a 0.01 M phosphate buffer solution that includes AuCl4-, 2 × 10-4 M, Au NP seeds (12 ( 1 nm stabilized by citrate), 3 × 10-10 M, cetyltrimethylammonium chloride (CTAC), 2 × 10-3 M, as surfactant, results in the immediate increase of the absorbance corresponding to the Au NP plasmon. Figure 1 shows the evolution of the absorbance spectra in the system in the presence of variable concentrations of H2O2 (spectra recorded after a fixed timeinterval of 5 min). As the concentration of H2O2 increases the absorbance is intensified. The calibration curve was derived (See Supporting Information, Figure 1S). Control experiments reveal that no Au NPs are formed in the absence of the Au NP seeds and that added H2O2 is essential to stimulate the absorbance changes. These results suggest that the Au NP seeds act as catalysts for the reduction of AuCl4by H2O2, resulting in the enlargement of the particles and the enhanced absorbance features, eq 1. Au NP
AuCl4- + 3/2H2O2 98 Au0 + 4Cl- + 3H+ + 3/2O2 A closer inspection of the spectral changes of the AuNP seeds upon treatment with H2O2, Figure 1, indicates that
Figure 1. Absorbance spectra of Au NP seeds (12 ( 1 nm), 3 × 10-10 M, upon growth in the presence of HAuCl4, 2 × 10-4 M, in 0.01 M phosphate buffer solution that included CTAC, 2 × 10-3 M, and using different concentrations of H2O2: (a) 0 M, (b) 2 × 10-5 M, (c) 5 × 10-5 M, (d) 1.1 × 10-4 M, (e) 1.8 × 10-4 M, (f) 2.4 × 10-4 M, g) 6.0 × 10-4 M. All spectra were recorded after a reaction time interval of 5 min.
at low H2O2 concentrations a red shift (ca. 15 nm) in the absorbance maximum is observed, curve (a), while at higher H2O2 concentrations, concomitant to the absorbance growth, a blue shift in the absorbance maxima occurs (ca. 10 nm as compared to the seeds). While the red shift observed at low H2O2 concentrations may support the enlargement of the particles,10 the blue shift at higher H2O2 concentrations with
the concomitant absorbance growth may imply the formation of more Au NPs of lower dimensions or the formation of a mixture of very small crystalline Au NPs together with enlarged Au NPs. To understand the mechanism of growth of the Au NPs we have performed a detailed HR-TEM analysis that follows the enlargement process: Figure 2A shows the HR-TEM image of the Au NP seeds. We find that the Au NPs exist in several morphologies (spheres, rhombs, triangles, and polygons) with a very narrow size distribution, 12 ( 1 nm. Figures 2B-D show the NPs formed upon treatment of the Au NP seeds with a low (5 × 10-5 M) and a high (2.4 × 10-4 M) concentration of H2O2 in the presence of AuCl4-/CTAC (for 5 min), respectively. For the low concentration of H2O2, Figure 2B, we observe NPs with a strong dark contrast of dimensions corresponding to ca. 13 nm × 13 nm that are coated by numerous nanocrystallites of lighter contrast that yield Au NP clusters of dimensions up to ca. 18 nm × 27 nm. Interestingly, for the higher concentration of H2O2, Figure 2C and 2D, the Au NPs coated with the Au nanocrystallites reached larger dimensions, up to ca. 32 nm × 28 nm, but in addition to these enlarged particles numerous small separated Au nanocrystal flakes of variable sizes, 2.5 to 7 nm (with maximum distribution of 3.5 nm), are observed. Figure 2C shows separated clusters marked with arrows. Figure 3 shows the Fourier filtered image of a 3.5 nm sized separated flake in which lattice fringes corresponding to the (111) plane of Au0 are clearly resolved. The majority of the observed flakes contain dislocations, and part of them were found to be folded and to appear as two-dimensional Au crystallites. Figure 4 shows the HR-TEM image of the Au NP that includes the attached, catalytically grown Au nanocrystals on the core Au NP seed. We observe that the Au crystallites are catalytically grown at the intersection of the faces of the parent seed Au NPs. The HR-TEM analysis indicates that the initial Au NP seeds
Figure 2. HR-TEM images of (A) Au NP seeds, (B) after 5 min of reaction, with HAuCl4, 2 × 10-4 M, CTAC, 2 × 10-3 M, and 5 × 10-5 M H2O2, (C and D) after reaction with HAuCl4, 2 × 10-4 M, CTAC, 2 × 10-3 M, and 2.4 × 10-4 M H2O2. 22
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Figure 3. Fourier filtered HR-TEM image of a 3.5 nm detached crystalline cluster. Lattice fringe of the 0.235 nm of {111} Au crystal plane is indicated.
Figure 6. Absorbance spectra of the Au NP-functionalized glass surfaces after 10 min of reaction with 2 × 10-4 M HAuCl4 in 0.01 M phosphate buffer solution that includes CTAC upon reaction with different concentrations of H2O2: (a) 0 M, (b) 2 × 10-5 M, (c) 5 × 10-5 M, (d) 8 × 10-5 M, (e) 2.4 × 10-4 M, (f) 6.0 × 10-4 M, (g) 7.2 × 10-3 M.
Figure 4. Au NP and 3.5 nm height triangular shape flake (marked with arrow) grown on its surface. Inset: fast Fourier transformed image of the flake area confirming its orientation along 〈011〉 direction.
Figure 5. HR-TEM image of the enlarged Au NP with nanocrystalline clusters at faces intersections.
are in the distincts decahedral or icosahedral structures.11 Upon the enlargement of the particles, Figure 5, gold is deposited on the seeds, and the nanocrystalline flakes are deposited and catalytically enlarged at the sharp intersections of (111)/(111), (100)/(110), and (110)/(210) faces. This mechanism may smooth out the sharp edges of the NPs and decrease their surface energy. This detailed HR-TEM analysis reveals important features involved in the catalytic H2O2-mediated growth of the Au NPs. At low H2O2 concentrations the flakes are mainly associated with the parent Au NPs, leading to structurally enlarged particles. This is consistent with the red shift observed for the enlarged Nano Lett., Vol. 5, No. 1, 2005
particles at low H2O2 concentrations. At high H2O2 concentrations a mixture of small Au nanocrystallites (2.5-7 nm) together with enlarged Au NPs (32 × 28 nm) is generated. The increased content of Au NPs yields the high absorbance features, yet the mixture of different sized particles leads to the observed blue shift of the absorbance spectra. The enlargement of the Au NP seeds by H2O2 was also examined on surfaces. The citrate-capped Au NPs were assembled on an aminopropylsiloxane film that was assembled on glass plates.12 The resulting plates were then reacted with a solution of AuCl4-/CTAC in the presence of different concentrations of H2O2. The absorbance of the surfaces was checked in water after 10 min of reaction. Figure 6 shows the absorbance spectra of the Au-NPmodified surfaces upon treatment with different concentrations of H2O2. (The derived calibration curve is provided as Supporting Information, Figure 2S). The absorbance of the Au-NP-modified interface increases as the concentration of H2O2 is elevated. Interestingly, however, and in contrast to the spectra in solution, the absorbance bands characteristic to the Au NPs are constantly red shifted. This observation is reasonable and supports the catalytic Au NP growth mechanism that we discussed for the system in solution. As the H2O2 concentration increases the Au NPs are enlarged, but the small flakes separated from the parent Au NP cores are washed off into the growing solution. The absorbance spectra of the slides correspond then to the net surface enlarged Au NPs. HR-SEM measurements support the enlargement process of the Au NPs on the surface. The HRSEM images of the Au NP seeds deposited on aminopropylsiloxane-functionalized ITO plates prior to reaction with 23
Scheme 1 Assembly of Au-NPs on a Glass Support and the Biocatalytic Au NPs Growth in the Presence of Glucose
H2O2 and after reaction with H2O2, 1.8 × 10-4 M, for 10 min, confirm the enlargement of the Au NPs (the images
Figure 7. Absorbance spectra of solutions containing Au NP seeds, 3 × 10-10 M, 2 × 10-4 M HAuCl4, 47 µg mL-1 GOx, in 0.01 M phosphate buffer solution and CTAC, 2 × 10-3 M, upon reaction for 10 min at 30 °C, with different concentrations of β-D(+) glucose: (a) 0 M, (b) 2 × 10-6 M, (c) 1 × 10-5 M, (d) 2 × 10-5 M, (e) 5 × 10-5 M, (f) 1.1 × 10-4 M, (g) 2.4 × 10-4 M. 24
are provided as Supporting Information, Figure 3S). The Au NPs are enlarged from 12 ( 1 nm to 18 ( 1 nm, respectively. The size of the enlarged particles elucidated by the HR-SEM measurements is in very good agreement with the size of the particles deduced from the respective absorbance spectrum10 and is in good agreement with the theoretically calculated value using the Mie theory. Numerous oxidases generate H2O2 upon the oxidation of the respective substrates by molecular O2. This suggests that the catalytic growth of Au NPs could be employed as a process for the optical sensing of the respective substrates. We have applied the method for the optical detection of glucose using glucose oxidase, GOx, and O2/glucose as the H2O2 generating system. Figure 7 shows the spectral changes of the Au NP seed solution, 3 × 10-10 M, that included AuCl4-/ CTAC and GOx, 47 µg ml-1, upon the addition of different concentrations of glucose (the experiments were performed under O2 at 30 °C and the spectra were recorded after a fixed time interval corresponding to 10 min). The absorbance values of the particles increase as the concentration of glucose is elevated (the derived calibration curve is provided as Supporting Information, Figure 4S). Control experiments indicate that no growth of Au NPs occurs upon exclusion of glucose, GOx, or in the absence of O2. These results indicate that the biocatalyzed formation of H2O2 is essential to induce the growth of the particles. As the concentration of glucose increases, the concentration of the generated H2O2 is higher and the growth of the Au NPs is enhanced. Nano Lett., Vol. 5, No. 1, 2005
whereas Figure 8B shows the derived calibration curve corresponding to the optical detection of glucose by the catalytically enlarged Au NPs. The increase of the absorbance values as the concentration of glucose is higher implies the glucose-controlled growth of Au NPs. On modified glass slides β-D(+) glucose is detected with a sensitivity limit that corresponds to 2 × 10-6 M. In conclusion, the present study has demonstrated the H2O2-mediated growth of Au NPs. The microscopic studies have revealed important mechanistic aspects involved in the NP growth process. We find that in addition to the deposition of gold on the nanocrystalline faces accelerated growth of nanocrystals occurs at the intersection of faces, and these eventually detach to form new seeds. The process enabled us to develop a glucose biosensor system based on the H2O2mediated growth of the Au NPs. Since numerous oxidases generate H2O2, the extension of this method to other oxidases to sense substrates such as cholesterol, lactate, choline, or xanthine seems feasible. Acknowledgment. This research is supported by the German-Israeli Program (DIP). M.Z. acknowledges the Israel Ministry of Science for the allowance of a Levi Eshkol PhD fellowship. Supporting Information Available: Supplementary figures and description of experimental procedures. This material is available free of charge via the Internet at http:// pubs.acs.org. References
Figure 8. (A) Absorbance spectra of the Au-NP-functionalized glass supports upon reaction with 2 × 10-4 M HAuCl4, 47 µg mL-1 GOx in 0.01 M phosphate buffer that includes CTAC, 2 × 10-3 M, and different concentrations of β-D(+) glucose: a) 0 M, b) 5 × 10-6 M, c) 2 × 10-5 M, d) 5 × 10-5 M, e) 1.1 × 10-4 M, f) 1.8 × 10-4 M, g) 3.0 × 10-4 M. For all experiments the reaction time interval was 10 min, 30 °C. (B) Calibration curve corresponding to the absorbance at λ ) 542 nm of the Au NP functionalized glass supports upon analyzing variable concentrations of β-D(+) glucose. (Experimental conditions as in (A).)
The analysis of glucose by this method was also performed on glass supports. The glass surface consisting of Au NP seeds bound to the aminopropylsiloxane film associated with the solid support, Scheme 1, was interacted with solution of AuCl4-/CTAC/GOx in the presence of variable concentrations of glucose for 10 min at 30 °C. Figure 8A shows the spectral changes of the glass slides upon the growth of Au NPs in the presence of variable concentrations of glucose,
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