Copyright © 2010 American Scientific Publishers All rights reserved Printed in the United States of America

Journal of Nanoscience and Nanotechnology Vol. 10, 1–10, 2010

Solution Phase Interactions Controlled Ordered Arrangement of Gold Nanoparticles in Dried State Mandeep Singh Bakshi1
∗ , Vivek Sheel Jaswal4 , Fred Possmayer2 , and Nils O. Petersen3
∗ 2

1 Department of Chemistry, Acadia University, 6 University Avenue, Elliot Hall, Wolfville, NS B4P 2R6 Deparment of Biochemistry, University of Western Ontario, 339 Windermere Road, London, ON, Canada N6A 5A5 3 National Institute for Nanotechnology, Edmonton, Alberta, Canada 4 Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, Punjab, India

Gold (Au) nanoparticles (NPs) were synthesized in the presence of water soluble biomolecules such as DNA, chitosan, phospholipids, and BSA by using seed-mediated approach at room temperature. All reactions produced mostly spherical geometries with comparable size (≤20 nm). The NPs were arranged in a typical pearl-necklace type arrangement except in the presence of BSA. Different measurements such as UV-visible, TEM, XRD, and XPS were used to characterize the Au NPs. Fluorescence spectroscopy was used to identify the interactions between biomolecules and blank (uncapped) Au NPs in aqueous colloidal solutions. It was concluded that the favorable interactions between Au NPs and biomolecules in aqueous phase, in fact, drive them into pearl-necklace type arrangement in the dried state.

Keywords: Gold Nanoparticles, Biomolecules, Seed-Growth Method, Bioconjugate Materials.

Bionanomaterials are significantly important constituents of biocompatible devices which find applications in bioengineering, biomedical imaging, and molecular diagnostics.1 To synthesize bionanomaterials, one requires interfacial synergism between the nanoparticle surface and biomolecules. Gold (Au) nanoparticles (NPs) can easily provide the required synergism through their electroactive surfaces which attract electroactive partially charged biomolecules.2 This association provides both charge and steric stabilizations to colloidal Au and subsequently contributes to their biocompatible functionalities. In addition, biomolecules such as low molecular weight proteins, biocompatible polymers, and phospholipids can provide a soft template effect3 for an ordered arrangement of NPs. This is particularly true in those cases where the biomolecules exist in a self-assembled state. An ordered arrangement of NPs plays an important role in fabrication of nanoelectronic photonic devices. Deoxyribonucleic acid (DNA) is known to provide a soft-template effect4 due to its well defined polymeric sequence of sugar phosphate backbone which is well suited for electrostatic interactions with metal cations. ∗

Authors to whom correspondence should be addressed.

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Likewise serum albumin protein (BSA),5 chitosan,6 and phospholipids7 can drive NPs in an ordered arrangement. BSA5 is a single polypeptide chain composed of 583 amino acid residues. It is a well documented low molecular weight (66 kDa) globular protein which can provide good colloidal stability to Au NPs. Its secondary structure is highly
-helical and its tertiary structure is composed of three homologous domains with cystine residues forming disulfide bonds to produce a double loop bridging pattern. Chitosan,6 on the other hand, is nontoxic, biocompatible, and biodegradable naturally-derived polyelectrolyte. Chitosan possesses common features of most positively charged polyelectrolytes. Importantly, chitosan molecules are good vehicles for drug7a b or gene7c d delivery systems due to their strong electrostatic interactions with negatively charged molecules like DNA and RNA, and hence they are used for many applications in biomedicine8a–d and pharmaceuticals.8e f Phospholipids9 such as phosphatidylcholine (PC) and phosphatidylglycerol (PG) are well known micelle/vesicle forming molecules which possess strong affinity for Au NPs. Recently, we have demonstrated that phospholipid capped Au NPs can act as a model system for ultra-fine air-pollutants which inhibit pulmonary surfactant function.8g In the present study, we have used these biomolecules (DNA, BSA, chitosan, phospholipids) as capping agents

1533-4880/2010/10/001/010

doi:10.1166/jnn.2010.2050

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1. INTRODUCTION

Solution Phase Interactions Controlled Ordered Arrangement of Gold Nanoparticles in Dried State

for the synthesis of Au NPs so as to compare their individual capping ability in order to attain some organized morphologies by using a seed-growth method.10 We also expect that during the growth process, biomolecules may control the crystal growth of NPs by selectively adsorbing at some crystal planes of Au fcc geometry.7f Such a comprehensive study would help us to understand their soft-template properties as well as their capping abilities to achieve ordered morphologies.

2. EXPERIMENTAL DETAILS 2.1. Materials

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Tetrachloroauric acid (HAuCl4 , sodium borohydride (NaBH4 , and trisodium citrate (Na3 Cit) were obtained from Aldrich. Deoxyribonucleic acid (DNA) from Herring Sperm, Chitosan (poly(D-glucosamine)), 75–85% deacetylated, and bovine serum albumin (BSA) 99%, were purchased from Sigma. 1,2-Diheptanoyl-sn-Glycero3-phosphocholine (DHPC) and 1-palmitoyl-2-oleoylsn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (POPG) were products of Avanti Polar Lipids, Inc. Ultra pure water (18 M cm) was used for all aqueous preparations. 2.2. Synthesis of Biomolecule-Capped Au NPs by the Seed-Growth (S-G) Method The S-G method was used for the synthesis of Au NPs in the presence of different biomolecules. A preliminary solution consisting of 25 ml of HAuCl4 aqueous solution ([HAuCl4 ] = 0.5 mM) was mixed with 0.5 mM of sodium citrate (Na3 Cit) in a screw capped glass bottle. Addition of 0.6 ml of aqueous NaBH4 ([NaBH4 ] = 0.1 mol dm−3  solution led to a ruby red color in the final solution at room temperature. The seed solution (total 5 ml) was composed of aqueous biomolecules (DNA/Chitosan/BSA/POPG = 120 g;/[DHPC] = 1.75 mM), [HAuCl4 ] = 0.5 mM, and 0.2 ml of freshly prepared ascorbic acid (AA) aqueous solution ([AA] = 0.1 M). Finally, 0.5 ml of the preliminary solution was added and the seed solution, and total solution was mixed a couple of times. A similar procedure was adopted to synthesize Au-Ag bimetallic NPs. Here, the addition of AgNO3 was carried out immediately after the addition of HAuCl4 to the seed solution while the rest of the reaction sequence was the same as mentioned in the previous paragraph. Three concentrations of AgNO3 , namely, 0.1, 0.2, and 0.4 mM, were used. Purification of each sample was carried out by repeated washing (at least 3 to 4 times) with double distilled water and centrifuging at 10000 rpm for 10 minutes so as to remove most of the remaining soluble biomolecules from the aqueous phase. 2

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2.3. Methods UV-visible spectra of prepared NP solutions were obtained using a UV spectrophotometer (Multiskan Spectrum, model no. 1500) in the wavelength range of 200–900 nm to determine the absorbance due to surface plasmon resonance (SPR). The formation of Au NPs was monitored around 520 nm in the visible absorption range. The shape and size of the NPs were characterized by transmission electron microscopy (TEM). Samples were prepared by mounting a drop of NP solution on a carbon coated Cu grid and allowing it to dry in air. They were observed with the help of a Philips CM10 TEM operating at 100 kV. The X-ray diffraction (XRD) patterns were characterized by using Bruker-AXS D8-GADDS with Tsec = 480. The chemical composition of some samples was confirmed with the help of X-ray photoelectron spectroscopic (XPS) measurements. A portion of an aqueous NP solution was placed onto a clean silicon wafer and then it was introduced into the sample chamber of the XPS instrument. The liquid was then pumped away. The XPS analyses were carried out with a Kratos Axis Ultra Spectrometer using a monochromatic Al K
source (15 mA, 14 kV). The instrument work function was calibrated to give a binding energy (BE) of 83.96 eV for the Au 4f7/2 line for metallic gold and the spectrometer dispersion was adjusted to give a BE of 932.62 eV for the Cu 2p3/2 line of metallic copper. A Kratos charge neutralizer system was used with all samples. Survey and high-resolution analyses were carried out with an analysis area of ∼300 × 700 microns using pass energies of 160 eV and 20 eV, respectively. Interactions between Au NPs and biomolecules in aqueous phase were studied with the help of fluorescence spectroscopy using a Hitachi fluorescence spectrophotometer F2500. Here, we took seed solution containing Au NPs of 2–3 nm as blank (uncapped) NPs to study their interactions with biomolecules in aqueous phase. Aliquots of 0.1 ml pipette out from a stock solution of Au NPs of concentration equal to 0.5 mM (assuming whole gold salt is reduced to NPs) were added in aqueous BSA solution to determine their effect on the quenching efficiency of tryptophan emission. Tryptophan residue emission in the case of BSA provides an opportunity to evaluate its interactions with NP surface, while pyrene fluorescence was used to explore such interactions between chitosan or DHPC with Au NPs. The ratio of the intensities of first (I1 ) to third (I3 ) vibronic bands of the pyrene emission spectrum reflects the polarity of the medium in which it is solubilized.11 The emission spectra were recorded employing an excitation wavelength of 334 nm, and the intensities I1 and I3 were measured at wavelengths corresponding to the first and third vibronic bands located at ca. 373 and 384 nm. J. Nanosci. Nanotechnol. 10, 1–10, 2010

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Solution Phase Interactions Controlled Ordered Arrangement of Gold Nanoparticles in Dried State

3. RESULTS AND DISCUSSION

(a)

(b)

3.1. TEM Measurements 3.1.1. Pearl-Necklace Arrangement in the Presence of DNA, Chitosan, and Phospholipids

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20 nm 20 nm

13.3±5.8 nm

(d)

(c)

12.2±3.9 nm

20 nm 20 nm

(f)

(e)

23.3±4.7 nm

20 nm 20 nm

Fig. 1. (a) Pearl-necklace arrangement of Au NPs generated on DNA examined in the dried state on Cu-grid; (b) arrangement of Au-Ag bimetallic NPs on DNA; (c) arrangement of Au NPs in the presence of chitosan; (d) arrangement of Au-Ag bimetallic NPs in the presence of chitosan; and (e) similar arrangement demonstrated by Au NPs in the presence of DHPC. (f) In the presence of Ag, the pearl-necklace arrangement of Au NPs converts into a continuous Au-Ag bimetallic nanowires in the presence of DHPC.

salts results in further reaction at the bead-bead interfaces (presumably {111} crystal planes), leading to nanowires (Fig. 1(f)). POPG molecules, on the other hand are approximately cylindrical in shape and cannot shield their fatty acyl groups by forming micelles. So they generate extended bilayers which form closed vesicles.13 The anionic POPG vesicles attract gold cations which act as counter ions adsorbed at vesicle-solution interfaces (Stern layer). Thus vesicles bearing gold cations may act as vehicles for delivery of Au ions to the seed surface for reduction 3

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TEM images of Au NPs synthesized in the presence of DNA, chitosan, and DHPC are shown in Figure 1. All images show systematic pearl-necklace type arrangement of NPs. The cross-linked structure of DNA macromolecules allows the sugar-phosphate backbone to interact electrostatically with gold cations. Gold cations are slowly reduced into gold atoms while adsorbed on the DNA backbone by a weak reducing agent like AA. The gold atoms thus obtained simultaneously nucleate with small seeds following an autocatalytic process, thus allowing them to grow into Au NPs (133 ± 58 nm). Simultaneous hybridization between the long DNA strands and the NP surface facilitates DNA-NP conjugate pearlnecklace arrangement formation (Figs. 1(a, b)) as observed in the dried state.4a Furthermore, such arrangements provide good plasmon coupling efficiency when the size of NPs is close to 20 nm and inter-particle distance is less than or equal to the diameter of the NP.4e In the case of chitosan (Figs. 1(c, d)), the protonated amine groups in the presence of acidic medium undergo electrostatic interactions with anionic tetrachloroauric ions. Further addition of AA simply reduces the gold ions to atoms and thereby starts a simultaneous nucleation of small seeds to form Au NPs6b (122 ± 39 nm). In the dried state, NPs are arranged in a well defined pearl-necklace arrangement most probably due to their association (because of citrate stabilized seeds) with protonated amines which are exposed to aqueous phase because of the intermolecular hydrophobic aggregation.12 In contrast to the long strand-like DNA and chitosan, DHPC/POPC are small compact amphipathic molecules which spontaneously form self-associated assemblies. Short-chained PCs such as DHPC form micelles where the polar zwitterionic groups extend outward and thereby shield the hydrophobic fatty acyl groups from water. However, DHPC possesses a relatively high critical micellar concentration = 1.8 mM.11e As a result, in the studies described here soluble DHPC molecules can react with the surfaces of the seeds, likely generating an adsorbed bilayer (Stern layer). Previous studies by our group9e–g using long-chained PCs suggested that such a zwitterionic bilayer would not impede growth of the Au seeds via slow reduction by AA. In those studies, long-chained (bilayer-forming) PCs tended to produce poor capping and anisotropic growth. It appears that employing the shortchained, relatively soluble micelle-forming DHPC promotes superior capping effects, leading to approximately round Au NPs and the formation of beads on a necklace arrangement (Fig. 1(e), 233 ± 47 nm). Including silver

Solution Phase Interactions Controlled Ordered Arrangement of Gold Nanoparticles in Dried State

by AA. Furthermore, due to the surface active properties of POPG molecules, they will adsorb at solutionNP interface of freshly prepared NPs with preferences for {100} or {110} crystal planes rather than {111}, and hence will provide NPs with appropriate charge and steric stabilizations14 required for colloidal stability. Both DHPC zwitterionic and POPG ionic head groups will have electrostatic interactions with charged Au surface though magnitude of latter interactions should be much higher. Such an association eventually results in a bilayer capping arrangement on the NP surface as we9f g and other groups15 have already reported. In the dried state, lipid capped NPs show a fine pearl-necklace arrangement and tend to produce one dimensional (1D) NPs chains, presumably continuously capped by POPG due to bilayer fusion16 9e or coalescence via active edge effects15f as indicated by block arrows in Figure 2(a). Similar overall mechanisms are thought to operate with other NP morphologies (polyhedral NP, 376 ± 55 nm, Fig. 2(b); dendrite NPs, 615 ± 166 nm NPs, Fig. 2(c); Sea-Urchin type NPs, 144 ± 212 nm, Fig. 2(d)) arranged as one dimensional

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(a)

(b)

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super-structures5 prepared under slightly different S-G conditions (see their figure captions). The organization of Au NPs in 1D chains17 provides an opportunity to tune their optical properties due to collective surface plasmon resonance of conduction band electrons along the 1D chains of Au NPs. Such an arrangement of NPs can act as a waveguide which guides electromagnetic waves. Here, the electromagnetic energy can be propagated over a distance of a few hundred nanometers to create a plasmon based waveguide.18 This organization of NPs in 1D chains with controlled inter-particle spacing provides a potential method for the development of photonic devices19 for biomedical applications. Addition of small amounts of Ag usually converts the pearl-necklace type arrangement into a more or less continuous Au-Ag bimetallic nanowire (Fig. 1(f)). Such nanowires are not observed with DNA and chitosan in the presence of Ag (Figs. 1(b) and (c)), respectively). A polymeric nature with relatively poor capping ability due to weak interfacial adsorption of DNA and chitosan molecules in comparison to DHPC/POPG could be the reason for the lack of nanowire formation. On the other hand, the presence of highly surface active DHPC/POPG, drives interconnected NPs into a more or less continuous nanowire formation in the presence of Ag as has already been discussed in detail in our previous work.9ef Therefore, TEM images of Figures 1 and 2 demonstrate that the presence of DNA, chitosan, and DHPC/POPG drives the pearl-necklace arrangement of Au NPs. 3.1.2. No Pearl-Necklace Arrangement in the Presence of BSA

(d)

(c)

Fig. 2. (a) A chain like arrangement of POPG capped Au NPs synthesized by using 0.125 ml of Au seed solution. Block arrows indicate the junction between the NPs presumably created by the fused POPG bilayers of adjoining NPs. (b) Chain like arrangement of Au NPs synthesized in the presence of POPG by using 0.25 ml of Au seed solution; and (c) and (d) chains of dendrite shaped large Au NPs synthesized in the presence of POPG by allowing 1 hour time interval between the two steps of the seed-growth method.

4

On the contrary, no such arrangement of Au NPs is observed in the presence of BSA (Fig. 3). Although BSA is a single polypeptide chain with high
-helical secondary and tertiary structures, the dried state assembly observed on the Cu grid seems to consist of a number of nanosized polyhedral domains (Fig. 3(a)). Such domains have already been reported by Gunning et al.20 for BSA + Tween 20 films created at the air-water interface and observed by a scanning near optical microscope. The onset of such domains might occur due to its self-aggregation behavior in macromolecular assemblies.21 Such assemblies might act as soft templates22 23 for the growing NPs (Fig. 3(b), 117 ± 34 nm). Addition of Ag further facilitates the anisotropic growth of the NPs (Fig. 3(c)) as observed for other biomolecules. The formation of interconnected networks of NPs is promoted at higher levels of Ag (Fig. 3(d)). The absence of pearl-necklace arrangement is presumed to be caused by poor capping and low surface activity in comparison to other biomolecules used in this study. BSA is a globular protein and hence its liquid-solid interfacial adsorption does not cause any extensive unfolding as a result the secondary structure still retains even after the adsorption.5g J. Nanosci. Nanotechnol. 10, 1–10, 2010

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Solution Phase Interactions Controlled Ordered Arrangement of Gold Nanoparticles in Dried State (a)

(c) Au NPs

Polyhedral domains of BSA

50 nm

20 nm

(b)

20 nm

(d)

Fig. 3. (a) Shows a dried film of macromolecular aggregated aqueous BSA on Cu-grid containing several polyhedral light-shaded domains and dark shaded Au NPs occupy some of these domains. (b) A close up of same polyhedral Au NPs synthesized in the presence of BSA. (c) and (d) show the Au-Ag bimetallic NPs appear to be interconnected in the presence of 0.1 and 0.2 mM of AgNO3 , respectively.

XRD analysis suggests a fcc geometry for Au or Au-Ag bimetallic NPs in each case. Some representative examples are given in Figure 4. The XRD patterns with peaks at 38.1 , 44.4 , 64.5 , 77.5 , and 81.6 are indexed as {111}, {200}, {220}, {311}, and {222} facets which are consistent with fcc geometry of Au or Ag bulk.

XPS is an excellent tool to evaluate the adsorption of biomolecules on metal surface. Many studies24 have been devoted to this direction. High resolution data of four principle atoms i.e., N, P, C, and O, present in the immobilized DNA film on Au surface are shown in Figure 5. The binding energies and percent areas occupied by different elements have been listed in Table I. A clear XPS emission of both N 1s and P 2p provides direct evidence of adsorbed DNA on the Au surface.24cd N 1s electrons emission shows a doublet with binding energies at 400.66 eV and J. Nanosci. Nanotechnol. 10, 1–10, 2010

Intensity/a.u.

3.2. XPS Measurements

I(111)

Au NPs (DNA) Au NPs (DNA, AgNO3 = 0.4 mM)

I(200) I(220)

I(311) I(222)

30

40

50

60

70

80

90

2θ/degree Fig. 4. XRD patterns of Au NPs synthesized in the presence of DNA and DNA + AgNO3 .

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20 nm

Solution Phase Interactions Controlled Ordered Arrangement of Gold Nanoparticles in Dried State

N 1s

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C 1s

P 2p

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O 1s

Fig. 5.

High resolution XPS spectra of N 1s, C 1s, P 2p, and O 1s emissions from dried DNA capped Au NPs.

399.12 eV. The first peak can be attributed to thymine multilayers (binding energy between 401.1 to 402.1 eV),25c 26 whereas a second peak at relatively lower binding energy (i.e., 399.12 eV) may be due to free amine groups.10h 26 The P 2p core level emission peak arises due to the phosphate backbone of the adsorbed DNA molecule, which can be split into a single spin-orbit pair i.e., 134.57 eV and 133.73 eV.24c The P 2p3/2 binding energy of 133.73 agrees fairly well with other studies related to DNA monolayers.27 The high resolution C 1s spectrum gives the following three contributions.10h 24b–d 26 The first peak with lowest binding energy at 285.56 eV can be assigned to the C only bound to C or H, the second peak at 287.06 eV can be attributed to the C available in C–N and C–O single bonds, while the third peak belongs to the highest binding energy of 289.06 eV and may be due to C present in CONH and COOH groups. It should be noted that the maximum contribution to the electron emission (i.e., 75%) with a binding energy of 285.56 eV is contributed by C–C or C–H functional groups which are available in the sugar or base moieties of DNA macromolecules.24c d 27 O 1s spectra can be decomposed into two main components24a c d with binding energies at 532.1 eV and 530.45 eV. The peak at 530.45 eV is mainly attributed to the oxygens of phosphate groups24a while the one at 532.15 eV could be from sugar moieties. 6

Thus elemental analysis of N 1s, P 2p, C 1s, and O 1s suggests the presence of a DNA film on the surface of Au NPs. Likewise similar information can be drawn from adsorbed BSA as well as DHPC molecules on Au or AuAg biometallic NPs surface with binding energies of C 1s, N 1s, P 2p, and O 1s listed in Table I. 3.3. Solution Behavior TEM and XPS analysis gives clear indication about the adsorbed biomolecules on the Au or Au-Ag bimetallic surface in the dried state. We believe that the ordered arrangement is driven by self aggregation of biomolecules capped Au NPs in aqueus phase. UV-visible and fluorescence measurements have been employed to explore such interactions in aqueous phase. 3.4. UV-Visible Measurements Surface plasmon resonance of colloidal Au NPs produces absorbance in the visible region due to collective resonance of the conduction band electrons with light photons.28 Monodisperse Au NPs smaller than 20 nm produce absorbance close to 520 nm, while Ag NPs of similar size show absorbance around 400 nm. J. Nanosci. Nanotechnol. 10, 1–10, 2010

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Solution Phase Interactions Controlled Ordered Arrangement of Gold Nanoparticles in Dried State

Table I. Binding energies/eV (I) and area in percent (II) of some species constituting the surface composition of Au NPs in the presence DNA, DHPC, and BSA.

Sample with DNA DNA + Ag* DHPC DHPC + Ag* BSA BSA + Ag*

Au 4f 5/2

Au 4f 7/2

Ag 3d

C 1s (C–C, C–H)

N 1s

P 2p1/2

P 2p3/2

(I)

(II)

(I)

(II)

(I)

(II)

(I)

(II)

(I)

(II)

(I)

(II)

(I)

(II)

(I)

(II)

87.73 87.78

42.8 42.8

84.06 84.11

57.2 57.2

368.16

100.0

285.60 285.58

75.3 67.7

400.66 400.75

44.8 43.8

399.12 399.21

55.2 56.2

134.57 134.63

33.3 33.3

133.73 133.79

66.7 66.7

87.62 86.38 87.83

34.4 8.4 42.8

83.95 82.71 84.16

46.0 11.2 57.2

285.47

56.5

402.10

15.4

399.71

84.6

368.28

100.0

284.42

57.4

402.31

19.9

400.70

80.1

87.66 87.78

42.8 42.8

83.99 84.11

57.2 57.2

400.27 400.17

80.7 91.9

398.24 398.4

19.3 8.1

133.56 181.73 133.44 131.68

47.2 19.5 22.6 44.1

100.0

45.3 64.4

23.6 9.7 11.3 22.0

368.17

285.29 285.30

134.40 132.57 134.28 132.52

Note: Ag* → [AgNO3  = 04.

Figure 6 illustrates the UV-visible spectra of Au and Au-Ag bimetallic NP suspensions. Absorbance close to 520 nm belongs to Au NPs without DNA (Fig. 6(a)). It is 20 nm red-shifted in the presence of DNA. A red shift of 20 nm provides a clear evidence of aggregation29 among Au NPs due to pearl-necklace arrangement as demonstrated by TEM images. Addition of Ag produces (a) 2

Absorbance

1.5

1

0.5

3.5. Fluorescence Measurements 0

320

400

480

560

720

640

800

880

Wavelength/nm Au NPs (BSA) Au NPs (BSA, 0.1 mM AgNO3) Au NPs (BSA, 0.2 mM AgNO3) Au NPs (BSA, 0.4 mM AgNO3)

(b) 2

Absorbance

1.5

1

0.5

0

300

400

500

600

700

800

900

Wavelength/nm Fig. 6. (a) UV-visible spectra of Au and Au-Ag bimetallic NP suspensions in the presence of fixed amounts of DNA. Note a blue shift in the absorbance with the increase in the amount of AgNO3 from 0.1–0.4 mM. (b) UV-visible spectra of Au and Au-Ag bimetallic NP suspensions in the presence of BSA with identical amounts of AgNO3 .

J. Nanosci. Nanotechnol. 10, 1–10, 2010

Tryptophan residues available in domain I and domain II of BSA show fluorescence emission at 340 nm when excited at 295 nm.31 The top curve in Figure 7(a) belongs to tryptophan emission of aqueous BSA in the absence of Au NPs. In the presence of Au NPs, the emission of tryptophan at 340 nm is significantly quenched. This is presumably due to the formation of a bioconjugate complex of BSA at the metal surface. On the other hand, chitosan, DHPC, and DNA lack such fluorescence emission by themselves. Therefore, these molecules cannot be used as an effective fluorophore like BSA. For these molecules, we need an indirect method (such as pyrene fluorescence) to investigate their complexation behavior with Au NPs. As the amount of Au NPs increases in aqueous BSA solution (see experimental for details), the fluorescence emission of BSA due to tryptophan at 340 nm decreases (Fig. 7(a)). The quenching of the tryptophan emission by Au NPs can be analysed in term of Stern-Volmer equation in the following form: I0 /I = 1 + Kq 0 Q = 1 + Ksv Q 7

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Au NPs (DNA) Au NPs (no DNA) Au NPs (DNA, 0.1 mM AgNO3) Au NPs (DNA, 0.2 mM AgNO3) Au NPs (DNA, 0.4 mM Ag NO3)

blue shift30 due to Au-Ag bimetallic NPs formation. TEM images also show that even bimetallic NPs are arranged in pearl-necklace arrangement. Similar behavior is observed with chitosan (not shown), and POPG.9e f On the contrary, in the presence of BSA (Fig. 6(b)), a broad and distorted absorbance around 540 nm can be attributed to the presence of polyhedral geometries as demonstrated by TEM images in Figure 3(b). Generally, poor capping ability of a capping/stabilizing agent leaves some of the crystal planes vulnerable for further crystal growth and results in the formation of anisotropic geometries. In addition, BSA also acts as a mild reducing agent30c and hence, it may further facilitate anisotropic growth due to its reducing ability. In such a situation, BSA may not be as good capping/stabilizing agent as other biomolecules are. Hence, this might result in the inhibition of pearl-necklace arrangement formation.

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(b)

1.8 1.7 1.6

Io/I

1.5 1.4

Intensity/a.u.

Intensity/a.u.

1.3 1.2 1.1 1

0

5

10 15 20 25 30 35 40

[Au NPs]/µM

300

350

400

450

500

350

400

450

Wavelength/nm (c) 0.932

0.65

600

(d) 1.05

1

0.45 0.924

I1/Iexc

0.5 0.926

I1/I3

0.55

0.928

I1/I3

550

0.6

0.93

0.95

0.4

0.922 0.92

500

Wavelength/nm

0.35 0

5

10

15

20

25

30

35

0.3 40

[Au NPs]/µM

0.9

0.85

0

10

20

30

40

50

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[Au NPs]/µM Fig. 7. (a) Plot of intensity versus wavelength of tryptophan emission of BSA around 340 nm in the presence of different amounts of Au NPs. Top curve represents aqueous BSA solution without NPs. Inset shows a linear variation between I0 /I and the amount of Au NPs in order to calculate Stern Volmer Constant. (b) Pyrene emission spectrum of solubilized pyrene in the hydrophobic domains of chitosan suspensions in the absence and presence of different amounts of NPs. (c) Plots of I1 /I3 and I1 /Iex ratio versus the concentration of Au NPs in the presence of chitosan. (d) Plot of I1 /I3 ratio of pyrene solubilized in the micelles of DHPC with respect to the amount of Au NPs.

Where I0 and I are the maximum fluorescence intensities in the absence and presence of Au NPs, respectively. The I0 /I ratio varies linearly within certain concentration limits of the quencher (inset, Fig. 7(a)), in our case Au NPs. Deviations from linearity at high concentrations can be attributed to the “sphere of action mechanism.”32 33 The slope of this plot gives the value of Stern-Volmer fluorescence quenching constant (Ksv ) which reflects the quenching efficiency. This corresponds to the product of the biomolecules’ quenching constant (Kq ) and the life time of fluorophore (0 ). Ksv thus calculated is equal to 30 × 104 M−1 consistent with a strong quenching ability of Au NPs. Though, BSA has been found to be poor capping agent as compare to other biomolecules, still Au NPs show a significant fluorescence quenching of tryptophan emission. A close affinity between Au NPs and BSA macromolecule can also be understood from a substantial reducing ability of BSA macromolecules of gold ions to nucleating centres. Similar studies have been carried out under identical conditions by using pyrene fluorescence in the presence of chitosan. It has been reported that 1 g/L of chitosan 8

produces hydrophobic domains which are quite stable.12 Although, the exact nature in terms of molecular arrangement of such hydrophobic domains is unknown, the protonated amine groups in the presence of acidic medium enclosing hydrophobic domains should be in contact with aqueous phase in order to attain proper stability. As soon as citrate stabilized Au NPs are added to the solution, the protonated amine groups undergo strong electrostatic interactions with negatively charged regions of the Au surface. Such an association would influence the hydrophobic environment of hydrophobic domains due to a structural rearrangement6d of chitosan molecules on the Au surface. Figure 7(b) shows typical pyrene emission spectra of pyrene molecules in the presence of chitosan with increasing amounts of Au NPs. As the amount of Au NPs is increased (see experimental for details), the I1 /I3 value increases while I1 /Iexc (excimer formation) decreases before tending to become constant (Fig. 7(c)). The former can be interpreted on the basis of a decrease in the polarity of the medium in which pyrene is solubilized, while the latter can be attributed to a decrease in the formation of excited state dimers.11 Both factors indicate a loosening of J. Nanosci. Nanotechnol. 10, 1–10, 2010

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Solution Phase Interactions Controlled Ordered Arrangement of Gold Nanoparticles in Dried State

4. CONCLUSIONS The present study demonstrates a fine compatibility between the results in the dried state and in aqueous phase. The self aggregation of Au NPs in a typical pearl-necklace arrangement in the dried state is the consequence of favorable interactions between the capping biomolecules and the Au NP surface in aqueous phase. Such interactions act as driving force to have an ordered arrangement of Au NPs on solid surfaces. Thus, a careful selection of biomolecules can provide us a simple and inexpensive method to fabricate wave guides or photonic devices for applications in bio-medical imaging.

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Acknowledgments: These studies were supported by Grants MOP 66406 and FRN 15462 from the Canadian Institutes of Health Research.

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Solution Phase Interactions Controlled Ordered Arrangement of Gold Nanoparticles in Dried State

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Received: 8 October 2008. Accepted: 14 March 2009.

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