silver nano in biomedical

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C H A P T E R 3

Nanoparticles for Biomedical ApplicationsTianzhong Yang, Anca Mocofanescu, Chengmin Shen, Hongjun Gao, andCongwen Xiao

3.1 Introduction

Particles with diameters in the range of 2 to 100 nm, so-called nanocrystalline mate-rials, have become a major interdisciplinary area of research during recent decades.In fact, since the seventeenth century, noble metallic nanomaterials, though not

understood, have been obtained and used to give rise to a brilliant rose colorthroughout Europe in stained glass windows of cathedrals and by the Chinese incoloring vases and other ornaments [1, 2]. The scientific preparation of nano-particles dates back to the nineteenth century, with Faraday reporting the prepara-tion of colloids of relatively monodispersed gold nanoparticles. The scientists whomajor in nanoscience and nanotechnology should appreciate the inventors whodesigned transmission electron microscopy (TEM). The high-resolution TEM(HRTEM) and low-resolution (LRTEM) allow one to observe a substance at ananometer scale directly. The magic machines make it possible to investigate thenanomaterials with respect to size, size distribution, shape evolution, and shape uni-

formity and even the structure. The past couple of decades have witnessed an expo-nential growth of activities in this field worldwide, driven both by the excitement ofunderstanding new science and by the potential hope for applications and economicimpacts. Indeed, many efforts have been devoted to investigation into the synthesis,characterization, and application of nanomaterials. In general, nanomaterials canbe classified into three groups: zero-dimensional materials, so-called nanoparticles,with variations in shape and diameter, one-dimensional materials, includingnanorod and nanowire, and two-dimensional materials, including nanobelts,nanodisks, films, and nanosheets. Herein we focus on the nanoparticles (NPs), espe-cially the metallic ones.

The intense interest in the metallic NPs derives from their unique chemical andelectronic properties arising from the small volume to big surface area ratio and theseparation in the electronic energy level. The change in the properties at this lengthscale from their bulk counterparts is not a result of only scaling factors. It resultsfrom different causes as far as different materials are concerned. In semiconductors,it results from the further confinement of the electronic motion to a length scale thatis comparable to or smaller than the length scale characterizing the electronicmotion in bulk semiconducting material (called the electron Bohr radius, which is

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usually a few nanometers). As noble metals are reduced in size to tens ofnanometers, a new very strong absorption is observed, resulting from the collectiveoscillation of the electrons in the conduction band from one surface of the particle tothe other. This oscillation has a frequency that absorbs the visible light. This is calledthesurface plasmon absorption. In the case of transition metal nanoparticles, the

decrease in the particle size to the nanometer length scale increases the surface-to-volume ratio. This, together with our ability to make them in different sizes andshapes, makes them potentially useful in the field of catalysis.

The absorption spectra of many metallic nanoparticles are characterized by astrong broad absorption band that is absent in the bulk spectra. Classically, thisgiant dipole (or surface plasmon) band is ascribed to a collective oscillation of theconduction electrons in response to optical excitation [3]. The presence of this bandin the visible region of the spectrum is responsible for the striking colors of dilutecolloidal solutions of noble, alkali, alkaline Earth (Ca, Sr, Ba), and rare Earth (Eu,Yb) nanoparticles [4]. Mies theory predicts that below a certain size, less thanone-tenth of the optical wavelength, the position and width of this band shouldremain constant, independent of size [5]. Experimental evidence, however, togetherwith a dramatic increase in width with decreasing size [6], indicates a slight but sig-nificant shift to lower energy. Fragstein and Kreibig and others [79] have investi-gated the optical absorption spectra theoretically in the case of free-electron metalsand advanced the theory that when the particle diameter becomes small enough,with diameter less than the electronic mean-free path in the bulk metal (~20 nm forgold), the scattering of free electrons with the particle surface begins to have aninfluence on optical excitation. Such a simple and practical theory succeeds in terms

of spectra of relatively large particles (>3 nm for gold) but does not agree with theexperimental results when applied to the smaller sizes. It is anticipated that at onepoint the phenomenological description of free electrons, as well as inherent funda-mental assumptions of infinite lattice periodicity and a continuous energy level spec-trum, must fail, because it is widely known that the continuous energy level bandwill separate into individual levels since in the cluster the number of atoms is sosmall. Experimental measurements for the small nanoparticles are often degradedby a lack of size and shape uniformity that renders comparison with theory ques-tionable [10], and this is what makes it so difficult to unequivocally identify quan-tum size effects in the optical spectra of metal nanoparticles prepared in

macroscopic quantities, although such effects are well known from experiments onmetal-cluster beams and from conductance measurements on single-metalnanostructures [11].

Hence, it is a prerequisite to take a good grasp on the control of the size distribu-tion of the metallic nanoparticle. In later discussion, we see the same problem in thecase of magnetic metallic nanoparticles. So much effort has been devoted to findingan approach that can afford us nanoparticles with a narrow size distribution.

Besides noble metal nanoparticles, nanoparticles with novel magnetic propertiesare attracting more and more attention with regard to bioapplications. As far asmagnetism is concerned, magnetic particles with diameters smaller than some cer-tain critical value usually show properties different from their bulk counterparts. Atroom temperature, the measured M-H curve, namely the hysteresis loop, recordedon the magnetic nanoparticles shows saturation magnetization but no coercive field

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or magnetic remanence synchronously. In other words, from the viewpoint of thecoercive field or magnetic remanence, they behave similarly to paramagnetic mate-rials; on the other hand, the magnetization can reach the saturation state and islarger than paramagnetic ones. The scientists describe this behavior assuperparamagnetic. This indicates that magnetic moments in the particles are free

to align with the field during the measuring time at room temperature. This phe-nomenon is caused by the fact that in such small particles, because of the thermalfluctuation the magnetic moments can rotate freely despite the magnetic energy bar-rier. This characteristic allows a promising future in application of the magneticnanoparticles in biomedicine, particularly in magnetic resonance imaging (MRI),tissue engineering, and drug delivery.

Because of both the excitement of understanding a new science and its potentialfor applications having economic impact, a great deal of effort has been devoted toinvestigations into the metallic nanoparticles in terms of synthesis, size and shapecontrol, surface modification, and the primary applications. Significant break-throughs have been achieved in the synthesis of nanoparticles of varying diametersand in obtaining a narrow size distribution. Based on these, highly orderedself-assembly structures with large area of metallic nanoparticles have beenobtained. For the purpose of bioapplication, some kinds of organic molecules,including biomolecules of different lengths and functional groups, have been coatedor linked onto the surface of metallic nanoparticles.

In this review, we discuss the synthesis and properties of zero-dimensionalmetallic nanomaterials, including the noble metal nanoparticles and the magneticones. We start with a discussion of some effective methods aimed at the synthesis of

the nanoparticles. After that their novel properties are introduced. The applicationof the metal nanoparticles in biotechnology is presented in the final section. Wefocus on the synthesis of metallic nanoparticles by the wet chemistry method.Chemical vapor deposition (CVD) and the physical techniques such as using elec-tron, ion, or photon beams in lithography to make nanostructures are not discussed.In general, this chapter is constructed based on the remarkable work reported in thepast decades. Because of the limitation of our knowledge and experiences, theremust be some incorrect sayings or points in this overview. Due to the explosion ofpublications in this field, we do not claim that this review includes all of the pub-lished work, but rather an exposure to the methods. We apologize to other authors

who have contributed to the synthesis, characterization, and application of themetal NPs whom we have unintentionally left out.

3.2 Synthesis of Metallic Nanoparticles

3.2.1 Synthesis Approaches to Noble Metal Nanoparticles

In fact, as mentioned earlier, since the seventeenth century, noble metallicnanomaterials have been observed and used, but not understood, to give rise to a

brilliant rose color throughout Europe in stained glass windows of cathedrals andby the Chinese in coloring vases and other ornaments. And the scientific prepara-tion of nanoparticles dates back to the nineteenth century, with Faraday reportingthe preparation of colloids of relatively monodispersed gold nanoparticles. In the

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past decades, many approaches have been utilized to produce the noble nano-particles. Before moving into the idiographic synthesis methods, we discuss the par-ticles growth in kinetics and the mechanism.

3.2.1.1 IntroductionIn general, the process of generation of a solid phase from solution inevitablyinvolves in the chemical growth nanometer-sized materials. Two primary steps con-stitute the course of NPs growth: Step 1, the nucleation occur; step 2, by adsorbingthe substance separated out of the solution, the nucleoli grow into the particles ofdesired size and shape. A good understanding of the process and parameters demon-strating the precipitation helps to tailor the growth of nanoparticles to the desiredsize and shape. For a particular solvent, there is certain solubility for a solute,whereby addition of any excess solute will result in precipitation and formation ofnanocrystals. Thus, in the case of nanoparticle formation, for nucleation to occur,the solution must be supersaturated either by directly dissolving the solute at highertemperature and then cooling to low temperatures or by adding the necessary reac-tants to produce a supersaturated solution during the reaction [12, 13]. The precipi-tation process then basically consists of a nucleation step followed by particlegrowth stages [14, 15].

Generally, there are three kinds of nucleation processes: homogeneous, hetero-geneous, and secondary. Homogeneous nucleation takes place in the absence of asolid interface by combining solute molecules to produce nuclei, and this procedureis involved in the synthesis of metallic NPs by the wet chemistry method. Homoge-

neous nucleation happens due to the driving force of the thermodynamics becausethe supersaturated solution is not stable in energy. The overall free energy change,G, which dominates the nucleation progress, is the sum of the free energy due tothe formation of a new volume and the free energy due to the new surface created.After the nuclei are formed from the solution, they grow via molecular addition,which relives the supersaturated step. On the other hand, when the reactants aredepleted due to particle growth, Ostwald ripening or defocusing will occur, wherethe larger particles continue to grow and the smaller ones get smaller and finally dis-solve. In addition to the growth by molecular addition, where soluble species depositon the solid surface, particles can grow by aggregating with other particles, and this

is called secondary growth. The rate of particle growth by aggregation is muchlarger than that by molecular addition. After the particles grow to a stable size, theywill grow by combining with smaller unstable nuclei and not by collisions with otherstable particles through such a mechanism of oriented attachment of nanocrystals[16, 17].

3.2.1.2 Synthesis of Gold Nanoparticles

The synthesis of colloidal gold has been studied intensively for a long time [18]. In1994, Brust et al. [19] developed an effective method for the generation oflong-chain alkanethiolate-stabilized gold nanoparticles. The product from this sys-tem is easy to disperse in organic solvent and to reisolate as purepowders. Due to thesuperb stability of the as-prepared particles, many groups have tried to follow or



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develop the approach. Much work has been done based on this smart design tomodify the properties, such as the reactivity and solubility, of the nanoparticlesthrough changing the molecular structures of the thiolates on the particle surface[2026]. Both long-chain thiolates containing an aromatic moiety [20], -substi-tuted (cyano, bromo, vinyl, ferrocenyl) [21] and poly-hetero--functionalized [22]

long-chain alkanethiolates and small molecular thiolates such as ( -mercapto-propyl) trimethyloxysilane [23] and the rigid aromatic thiols of 4-mercapto-biphenyl [24] and p-mercaptophenol [25] have been utilized. Kunitake andcoworkers also showed that sodium 3-mercaptopropionate could be used for thesynthesis gold nanoparticles using citrate as reductant [26]. Since the diluted aque-ous solution was needed in this method, it is difficult to separate the particles assolid powders; therefore, the detailed characterization of the surface structure witha variety of methods where solid powders are needed as the specimens is prevented.In addition, regarding the application of the gold nanoparticles in biomedicine,where the pH value is close to neutral, there still will be the challenge of developingthe approach from which the Au nanoparticles can be easily dispersed into aqueoussolution at a neutral pH value.

In 1999, Sihai Chen and Keisaku Kimura reported a new approach that canavoid the disadvantages mentioned above [27]. The shining points of their work arethe standout elucidation of the surface structure of these particles and that theas-synthesized nanoparticles are easily dispersible in water, a property that has notbeen achieved using other kinds of methods where the thiolates are employed[2026]. For biomedical purposes, the latter feature is a prerequisite because thenanoparticles should combine with macromolecules in living or fixed cells or tissues

in aqueous solution.Let us introduce the process briefly. In a typical preparation, aqueous solutionwas at first mixed with mercaptosuccinic acid (MSA) in methanol to give a trans-parent solution. Under vigorous stirring a freshly prepared aqueous sodiumborohydride solution was then added at a certain rate. The solution turned darkbrown immediately but remained transparent (which indicates that the nano-particles do not form) until enough reductant was added. The pH of the solutionincreased gradually with the addition of reactant. A flocculent dark brown precipi-tate would be generated by further addition of the reductant, and finally the pH ofthe solution was brought to 8.6. We can see that just as in the common synthesis in

aqueous solution, the pH must be adjusted. After being stirred for another duration,the solvent was removed by decantation after the centrifugation. The precipitatewas washed, suspended in ethanol, and dried by rotary evaporation withoutexceeding a temperature of 40C under vacuum and at last the product in powderstate was obtained.

They explained the whole procedure as follows:

( ) ( ) ( )

8 8 3 12

8 3 3

4 2

4

n m n n

nn m

HAuCL R SH NaBH H O

Au R S B OH

4 + + +

+ +

( )2 8 32 3n m n n+ + ++ +H Cl Na(3.1)

The initial molar ratio between MSA and chloroauric acid plays a dominate role intailoring the relative rates of particle nucleation and (then) growth, and hence itfinally determined the particle size. It was found that the particle sizes decreased



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implicating interactions between Au surface atoms and the nonbonding S orbitals ofan intact RSSR molecule was identified by Fenter et al. in 1994 [33].

In 2001, a report from Hongjun Gaos group presented outstanding work onthe Ag nanoparticles with narrow size distribution. And based on this characteriza-tion, the self-organized superstructure was obtained. From Figure 3.1 one can see

that the NPs have a diameter of about 4.18 nm and the standard deviation of thesize distribution is 0.23 [34].

3.2.2 Synthesis of Magnetic Metal Nanoparticles

Magnetic nanoparticles, especially those with diameters of less than 10 nm, are ofintense interest because theparticle size is smaller than the characteristic dimensionssuch as atomic or ionic diffusion length, electronic elastic and inelastic mean freepath length, and the magnetic domain size [3539]. Many new phenomena andproperties are expected to appear in the NP systems. The size and shape specificityof nanoparticles naturally acts as building blocks of the self-assembled passivatedNP superlattices (SLs) or NP arrays [40]. In this emerging field, monodisperse mag-netic NPs are a prerequisite to a broad range of applications, such as data storagedevices, biosensors, and drug delivery [4144]. The advances in preparing semicon-ductor and metallic NPs, specifically by the method of injecting molecular precur-sors into hot organic surfactant solution, have improved the NP samplesremarkably, with good size control, narrow size distribution, and good crystallinityof disperse nanoparticles [45, 46].

Now we introduce the general approach though the synthesis of cobalt

nanoparticles. The reason we chose to begin with the Co system is that Co nano-crystals possess a wealth of size-dependent structural, magnetic, electronic, and


90 nm

Diameter (nm)

Population(%)

3.0 3.5 4.0 4.5 5.0 5.505

1015

2530

20

Figure 3.1 TEM image of a two-dimensional silver nanoparticle superlattice and (inset) the histo-gram of the nanoparticles. (Source: [34].)


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presence of DiBAH have been reported by them [69]. In that work, they obtained Conanoparticles with small mean diameters of about 1.4 nm, by means of hydrogena-tion of Co(3C8H13)(

4C8H12) companied by poly(vinyl pyrrolidone). A comprehen-sive study of their magnetic properties has shown that they are similar to thoseexpected for free-standing cobalt NPs, which were discussed in 1998 [70]. Briefly, if

one assumed that the polymer matrix has few or no interactions with the NPs, thededuction is that surface metal atoms are undercoordinated and electronicallyunsaturated. The noteworthy point is that the formation of -cobalt has beenreported to be promoted by a careful choice of the ligands used during the synthesis[53, 54],which will be discussed latter. Hence, in the presence of trioctylphosphaneoxide (TOPO) or PR3ligands, which are known to damp the magnetic properties ofNPs, cobalt NPs adopt the -cobalt structure, whereas an fcc structure is observed inthe absence of such ligands [70]. In sample 1, the electron deficiency is also impor-tant. On the contrary, NPs in sample 2 (when [CoN(SiMe3)2]2is utilized as precur-sor; for detail, see [71]), the surfaces of which are surrounded by donating ligands,adopt a compact structure displaying features of both fcc and hcp phases. It is worthnoting that the hcp structure is not often reported for such small sizes, especially forNPs obtained at low temperature. It is suggested that coordination of a -donorligand at their surfaces favors this structure. The synthesis conditions also play animportant role on the structure of the product. In fact, sample 1 is obtained at roomtemperature by a slow reaction process (48 hours), whereas the formation of sample2 is fast at 50C. This is in contradiction with the expected kinetic quenching of ametastable structure and further attests the influence of the coordinated ligand onthe structure adopted by the NPs.

Now let us come to the identification of the new phase of cobalt in detail. As wasmentioned, besides the well known structures including hcp and fcc, another phaseof cobalt has been found in cobalt nanomaterials. Though the insight was reportedby Hull [72] in 1921 and then Krainer [73] and Kajiwara et al. [50], it is from 1976that the evidence for a new structure of cobalt present in small cobalt clusters pro-duced by the decomposition of organometallic precursors is provided by Respaud etal. [74]. However, the structure was not identified until Dinega and Bawendireported their work on this new structural cobalt in 1999 [53]. In the report, thermaldecomposition of octacarbonyldicobalt in solution in the presence of TOPO as acoordinating ligand was employed to synthesize cobalt nanoparticles. This method

provides a clean route for the preparation of the material since elemental cobalt isthe only nonvolatile product of the reaction, which can be depicted as follows:

( )[ ]Co CO Co CO2 8 2 18 + (3.3)

They claimed that the as-synthesized powder was highly susceptible to magneticfields generated by a small permanent magnet, suggesting that it consisted of mag-netic materials without or with thimbleful of cobalt oxides. TEM showed that thepowder consisted of roughly spherical particles with an average diameter of 20 nm

and a 15% size distribution. Later experiments showed that the size of the crystals isnot limited to this range. In fact, injection at higher temperature yields regularpolyhedron-shaped faceted crystals as large as 0.3 m and possibly even larger(Figure 3.3). These results are reasonable since we know that the temperature will



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influence the nucleation and the growth process, as discussed previously. Theyalso found the presence of oxygen, which indicates surface oxidation of the cobaltparticles, corresponding to coverage of one to two monolayers of cobalt oxide forparticle sizes in the 20-nm range. The other elements, in their opinion, come fromresidual organic solvents adsorbed on the particle surfaces. The absence of phos-phorus in the elemental analysis indicates that TOPO was completely removed dur-ing washing of the particles. It is noted in this work that freshly synthesized cobalt

nanoparticles are extremely reactive toward oxidation. In fact, rapid exposure to airresults in immediate oxidation accompanied by a red glow. Therefore, even simplywashing the particles in organic solvents inevitably leads to surface oxidation byresidual moisture and dissolved air. The consideration is reasonable that the result-ing oxide layer appears to passivate the surface of the particles and considerablyreduce the speed of further oxidation. It should be mentioned that the new -cobaltphase appears to be metastable under normal conditions. Although stable at roomtemperature for at least several months, heating the sample at 500C completelytransforms it to the known fcc phase. Furthermore, subsequent cooling does notreturn the sample to its original -cobalt structure.

Figure 3.2 shows the X-ray powder diffraction patterns of the as-synthesizedproduct, which present the evolution of the nanoparticle structure with variation ofthe molar ratio of TOPO/precursor, and one can see that this product takes a new


21

0

21

1

11

0

32

1

311

3102

21

33

0

42

05

10

511 5

20

52

1

531

44

2

610

611

20 30 40 50 60 70 80 90 100

2 /

(b)

(c)

(a)

Figure 3.2 (a) Experimental and (b) calculated X-ray powder diffraction patterns of the -cobaltstructure and (c) the sample shown in (a) after being heated to 500C. Peaks corresponding to fcccobalt are denoted by X. (From: [53]. 1999 Wiley-VCH Verlag GmbH, Weinheim, Germany.

Reprinted with permission.)


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phase that does not correspond to either of the two known structures of cobalt, fccand hcp. In other words, neither the positions of the peaks nor their relative intensi-ties correspond to any known cobalt phases. Based on the detailed analysis, the con-clusion is that this pattern corresponds to a new structure of cobalt, so-called-cobalt. This structure is cubic (space group P4132) with a unit cell parameter=

6.097 0.001 . The unit cell structure is similar to that of -manganese (ahigh-temperature phase of manganese) [75]. Twenty cobalt atoms constitute oneunit cell, and they are divided into two types: 12 atoms of type I and 8 atoms of typeII. These two types of atoms differ in their local coordination. Unlike an idealclose-packed structure, which has 12 nearest neighbors, -cobalt has only threenearest neighbors for type I and two nearest neighbors for type II atoms. This phe-nomenonthe same atoms being divided into several types based on the local coor-dinationoften happens in compounds such as magnetite. This results in a structurefor-cobalt that is less dense than both the hcp and fcc structures. The calculated

density of-cobalt is 8.635 g(cm)

3

, compared to 8.788 g(cm)

3

and 8.836 g(cm)

3

for the fcc and hcp structures, respectively [53].Hence, the structure of-cobalt was identified for a certainty. Figure 3.3 shows

the TEM images of the as-obtained nanoparticles. However, as far as the uniformityof the size and shape is concerned, which we continue to emphasize, the systemneeds to be developed for the uniformity of the morphology.

Figure 3.4 shows models of the unit cell for -cobalt with respect to different lat-tice direction projections. In addition, for the purpose of applications, it should bementioned that the magnetic moment per atom in-cobalt, measured as 1.70B, issimilar, within experimental error, to those of the two known structures (1.75 and

1.72Bfor bulk fcc and hcp cobalt) [53, 76].It was also claimed that the use of TOPO seized the key role here because of the

fact that the same preparation method but without the presence of TOPO yieldsnanocrystals with a pure fcc phase. Hence, the influence of TOPO on the structureof the as-obtained nanoparticles should be clarified, and it was indeed done. Tightcoordination of ligand molecules (TOPO) around the growing crystal and aroundsolubilized cobalt atoms is responsible for changing the energetics of growth in favorof the new, less dense phase. However, the particular synthesis of this paper is notunique in producing-cobalt. Sun and Murray [54] have confirmed this assignment


0.1 m0.1 m

Figure 3.3 TEM image of cobalt crystals with the new -cobalt structure. (From: [53]. 1999Wiley-VCH Verlag GmbH, Weinheim, Germany. Reprinted with permission.)


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of the structure of-cobalt in nanocrystals obtained by reducing cobalt salts in solu-tion in the presence of alkylphosphanes serving as the coordinating ligands. Due toits metastability, -cobalt may be accessible only by solution-phase approaches,rather than more common techniques, such as electrochemical deposition or chemi-cal vapor deposition and so on. As far as the uniformity of nanoparticle morphol-ogy is concerned, remarkable achievements have been obtained by Sun andMurrays work. In their work, briefly, the particle generation began with the injec-tion of dioctylether superhydride solution into a hot (200C) CoCl2dioctylethersolution in the presence of oleic acid (octadec-9-ene-1-carboxylic acid,CH3(CH2)7CH=CH(CH2)7COOH) and trialkylphosphine (PR3, R=n-C4H9, or

n-C8H17). Reduction occurred instantly upon injection, leading to the simultaneousformation of many small metal clusters serving as nuclei. Continued heating at200C allowed steady growth of these clusters into nanometer-sized, single crystalsof cobalt. The steric bulk of the alkylphosphine controlled the rate of particlegrowth and consequently the morphology. Short-chain alkylphosphines (e.g.,tributylphosphine) allowed faster growth, leading to the generation of large parti-cles, while bulkier species (e.g., trioctylphosphine) reduced particle growth andfavored production of smaller nanocrystals (NCs). However, it is worth mentioningthat the influence of the organic coatings steric hindrance on the particles size, sizedistribution, and shape must play some essential role in such cases. These organi-

cally stabilized cobalt NCs were readily dispersed in aliphatic, aromatic, and chlori-nated solvents and could be precipitated with the addition of short-chain alcohols,facilitating a size-sorting procedure that isolated nearly monodisperse samples [77].

In Sun and Murrays work, average particle size was coarsely controlled byvarying the type of alkylphosphine used in combination with oleic acid during thegrowth. For instance, bulky P(C8H17)3limited growth to producing particles withdiameters of 2 to 6 nm, and less bulky P(C4H9)3led to larger particles (7 to 11 nm indiameter). A size selection procedure is needed for the purpose of fine tuning theparticle size. It should be pointed out that the size sorting procedure is the disadvan-

tage of such a system in view of the cost for application in industry. And somegroups developed approaches that lead to nanoparticles with tunable size and nar-row size distribution without the size selection procedure. Based on the fact thattrialkylphosphine reversibly coordinates neutral metal surface sites, slowing but not


(a) (b)

Figure 3.4 The unit cell of-cobalt: (a) unit cell cube filled with 8 atoms of type I (dark) and 12atoms of type II (light) and (b) 111 projection of the same cube showing threefold symmetry alongits main diagonal. (From: [53]. 1999 Wiley-VCH Verlag GmbH, Weinheim, Germany. Reprintedwith permission.)


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stopping the particles growth, it cannot prevent the particles from eventually grow-ing to undispersible aggregates at high temperature when used alone. If oleic acid isemployed alone, though it is an excellent stabilizing agent, it binds so tightly to theparticle surface during synthesis that it greatly impedes the particle growth. Thecombination of trialkylphosphine and oleic acid produced a tight ligand shell that

allowed the particles to grow steadily while it protected them from aggregation andoxidation.

It is well known that a temporal separation of the nucleation and growth stagesis needed to produce a monodisperse colloid. Ideally, a large number of criticalnuclei should be formed in a short interval of time followed by the simultaneous andsteady growth of those nuclei. In Sun and Murrays approach [54], the reducingagent (superhydride) was injected to induce rapid reduction of cobalt (II) chloride inhot (200C) media. This procedure provided the temporally discrete homogeneousnucleation desired. Growth of the nuclei was continued by addition of cobalt-con-

taining species to the surface of the particles and was halted by cooling to below100C naturally. From the TEM characterization (Figure 3.5), one can see the stand-out results obtained by this means. However, the size selection procedure is needed.This may lead to a high cost in terms of economics when it is realized in industry.

In 2003, a report from Gaos group presented the advancement in the control ofsize, size distribution, and the monolayer and multilayer self-assembly based on thenarrow size distribution [78, 79]. In their work, they employed the approach inwhich thermal decomposition of octacarbonyldicobalt was utilized. This methodprovides a clean route for the preparation of the material since the cobalt is theonly nonvolatileproduct during the whole and the reaction can be described by (3.3)

[53, 80]:A combination of surfactants in the presence of stabilizing ligands triphenyl-

phosphine and oleic acid was employed for controlling the particle growth, stabiliz-ing the particles, and preventing oxidation. Because the phenyl can provide greatersteric hindrance than that of the straight-chain alkyl, they adopted the triphenyl-phosphine, instead of the tributylphosphine or trioctylphosphine, to synthesizecobalt NPs with smaller size.


48nm

Figure 3.5 TEM image of a two-dimensional assembly of 9-nm cobalt nanocrystals. Inset:High-resolution TEM image of a single particle. (From: [54]. 1999 American Institute of Physics.Reprinted with permission.)


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Briefly, a three-neck flask with dichlorobenzene is heated to ~220C under N2and stirred for a certain time to exclude ambient air, and then equivalent amounts inmole of oleic acid and triphenylphosphine are added. In another vessel, Co2(CO)8iscombined with dry dichlorobenzene, warmed until fully dissolved, and then after-ward rapidly injected into the flask. The solution turns black quickly and foams,

indicating the precursor decomposition and the nucleation. The black solution isrefluxed and then the particles are obtained by the classical separation procedure.Cobalt nanoparticles with 7-nm average diameter are obtained by the size-sortingprocedure with a narrow size distribution. When the concentration of the oleic acidand triphenylphosphine was increased by a factor of 2, cobalt NPs of about 5 nmwere obtained.

Compared to Dinega and Bawendis work [53], at first the employment of oleicacid played a role in leading to the differences. As we have discussed, oleic acid,when employed alone, is an excellent stabilizing agent. However, it binds so tightly

to the particle surface during synthesis that it greatly impedes the particle growth.The combination of triphenylphosphine and oleic acid produced a tight ligand shellthat allowed the particles to grow steadily while protecting them from aggregationand oxidation. Second, by using the triphenylphosphine instead of the tributyl-phosphine or trioctylphosphine, based on the fact that the phenyl can providegreater steric hindrance than that of the straight-chain alkyl, the nanoparticles withsmaller diameter (7 nm) were obtained. In addition, since it is generally acceptedthat a temporal separation of the nucleation and the growth stages is required forthe production of a monodisperse colloid of nanoparticles [54], the injection of thedichlorobenzene solution of Co2(Co)8into a hot system (~220C) forms a large

number of critical nuclei. The growth of the nuclei continues by the addition ofcobalt-containing species to the surface of the particles with a decreasing reactionrate (temperature tuned to 185C). This process can narrow the size distribution ofcobalt NP diameters. Figure 3.6 exhibits the highly ordered superlattice structure ofthe cobalt nanoparticles.

3.3 Novel Properties of Metal Nanoparticles

As we all know, the composition and the structure determine the properties, and theproperties determine where and how the materials can be used. So it is necessary toknow about the properties before moving into the applications.

3.3.1 Unique Properties of Noble Metal Nanoparticles

In the following we will locate space to discuss the properties of metal nano-particles, especially those with organic coating, because these underlie the applica-tion of the nanoparticles in all kinds of purposes, and in most cases the ligandsinfluence the properties of the nanoparticles, more or less. According to the back-

bone of this issue, let us begin with those of noble metal nanoparticles.At a fundamental level, optical absorption spectra provide information on theelectronic structure of small metallic particles. The absorption spectra of manymetallic nanoparticles are characterized by a strong broad absorption band that is

3.3 Novel Properties of Metal Nanoparticles 57


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In Whettens groups investigation, they measured the optical absorption spectraof a series of nanocrystal gold molecules such as larger, crystalline Au clusters witheffective core diameters ranging from 1.4 to 3.2 nm (constituted of ~70 to ~800 Auatoms), passivated by a compact monolayer ofn-alkylthiol(ate)s, across the elec-tronic range (1.1 to 4.0 eV) in dilute solution at ordinary temperature. After a series

of designed experiments, the conclusions can be summarized as follows [84]: Withdecreasingcore mass (crystallite size), the spectra uniformly show a systematic evo-lution. Concretely, (1) the so-called surface plasmon bands are broadened until theeffective diameter of crystallites is less than 2.0 nm, (2) the emergence of a distinctonset for strong absorption near the energy (~1.7 eV) of the interband gap (5d 6sp), and (3) the appearance in the smallest crystallites of a weak steplike structureabove this onset, which can be interpreted as arising from a series of transitions fromthe continuum d band to the discrete level structure of the conduction band justabove theFermi level.Theclassical electrodynamic (Mie) theory, basedon bulk opti-cal properties, can reproduce this spectral evolutionand thereby yield a consistent

core sizingonlyby making a strong assumption about the surface chemical interac-tion. Quantitative agreement with the spectral line shape requires a size-dependentoffset of the frequency-dependent dielectric function, which may be explained by atransition in electronic structure just below 2.0 nm (~200 atoms).

After the discovery of methods for preparing gold nanoparticles by Dubois andNuzzo in 1992 [32], which has overcome the obstacle of a too broad size distribu-tion limiting the theoretical and successful experimental investigation into the opti-cal properties of the gold clusters, the subsequent discovery and increasingly wideexploration of the highly stable Au:SR nanocrystal systems over the years has estab-lished the stable existence and many properties of these systems, including thepoints presented below [84].

They are charge-neutral entities comprising a compact crystalline core ofclose-packed metal atoms and a dense mantle of straight-chain groups, which can


1.6

0.0300 400 500

Wavelength600 700 800

0.4

Ab

sorbance

0.8

1.2

abcde

Figure 3.7 UV-visible spectra of Au particles of different sizes: (a) 10.2, (b) 10.8, (c) 12.8, (d)19.4, and (e) 33.6 . (From: [27]. 1999 American Chemical Society. Reprinted with permission.)


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be prepared quantitatively by at least two distinct methods [19, 32], effective acrossthe ~1.4- to 3.5-nm core diameter range (~70 to 1,000 atoms). Their surface proper-ties are determined by the surfactant tail group (usually methyl, CH3), and hencethe interactions with external agents (e.g., solvents, strong acids, or bases) and witheach other is weakened; the surfactant groups (RS) initially adsorbed can later be

exchanged with excess thiol (RSH) in solution [8587]. A small metal particle sur-rounded by a condensed dielectric medium [88] governed the electronic properties.It is notable that the raw, as-synthesized samples have been shown by mass spec-trometry to contain enhanced abundances at certain sizes, which is probably attrib-utable to the filled structural shells of Au atoms. The components of these mixturescan be fractionated from one another according to size and then handled as purifiedmolecular substances in various manners [87, 89]. Realistic simulations [90] andX-ray diffraction patterns obtained on selected samples provided considerableinsight into the structure of these assemblies and their interactions, which is consis-

tent with patterns calculated from theoretically generated structures. Whetten et al.[91] in 1997 found methods to produce and isolate smaller nanocrystals (1.4 to 1.7nm) in large quantities, which allows one to obtain a rather complete picture of theevolution of the optical properties of gold nanoparticles.

Based on the achievement mentioned above, the scientists are able to investi-gate the properties, especially the optical ones, at will. The spectra shown in Figure3.8 are arbitrarily normalized at the high-energy end and are presented superim-posed, without risk of confusion, based on the fact that the relative spectral intensityat 2.5 eV decreases monotonically with mass (core size) for each series shown. Fur-thermore, Figure 3.9 suggests that chain lengths for a given core mass have nearly no

influence on the spectra, while at smaller sizes (~1.7 nm) they can be very sensitive tosample purity [Figure 3.9(b)].

Cursorily, the evolution of the optical spectrum of Au particles with diametersranging from 1.4 to 3.2 nm is so simple that it could even be described as that whichis already known about Au from spectroscopic investigations of unfractionatedsamples of nanometer-scale particles in glasses, in solution, and from the smallercluster compounds [92, 93]. However, the use of well-fractionated samples of goldnanocrystals, grown in a strongly etching environment [89], allows one to distin-guish certain features that had not been noticed previously:

There is anonsetof strong absorption located near 1.7 eV, where it behavesincreasingly distinctly at smaller nanocrystal sizes. These show a clean breakfrom the preonset behavior occurring at the energy of ~1.6 eV, which can beattributed to the interband (d sp) absorption edge, as shown in the semi-logarithmical scale in Figure 3.10.

A weak,steplike structureoccurs, beginning with the onset mentioned earlier,in the spectra of all the smallest clusters studied. It is clearly evidenced in thedifferential (or log-differential) spectral intensities plotted in Figure 3.11 (andits inset), which transforms the weak steps into a series of distinct peaks and

shoulders. These also bring out the onset structure (1.7 eV) as a first peak.

For these effects to be mastered and understood, one should acquaint oneself withthe elementary facts of the electronic structure of close-packed Au, as are confirmed



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also to apply to Au clusters by cluster-beam experiments [94]. Now we discuss themin general. In fact, there are two mechanism that originate the optical absorptionintensity (in the 0- to 8-eV range) in Au. First, common to all metals are theintraband transitionsoccurring within the broad conduction band; as far as Au isconcerned, it comes mainly from the 6s1p hybridized atomic orbitals, whose onset

is at zero frequency (or above the Kubo gap in small particles [95]). This leads to aquite weak absorbance, increasing as the square of the frequency until it approachesthat of the surface plasmon frequency. For conduction electron densities of Au(~60/nm3), it should appear near 5 eV when the particles have a spherical geometry.The secondinterband transitionsoccur between the 5d10 band and the unoccupiedstates of the conduction band, which have an onset at the energy difference betweenthe highest point in the narrow, flat d band and the lowest unoccupied level of theconduction band (i.e., at the Fermi level). In bulk Au, this first interband excitationtakes place at the X point of the first Brillouin zone, with energy of 1.7 eV. The sub-

stantial d p character of these transitions gives rise to very strong absorption andsequentially to the colors of Au surfaces, thin films, and nanoparticles. Theresearchers consider it to be a truth that the high polarizability of the 5d10 cores


1.5 2.0 2.5 3.0 3.5 4.0

C18

C6

0

0

0

(c)

Energy (eV)

(b)

(a)

C12

Figure 3.8 Optical absorption spectra for dilute solutions of several purified gold molecules passi-vated by (a) hexyl-, (b) dodecyl-, and (c) octadecylthiolates. The spectra are scaled to unity at 4 eVand are compared to an aqueous solution of commercial colloidal Au particles (dotted lines) of9-nm mean size. The peak amplitude (near 2.5 eV) descends with the metallic core diameters: (a)3.2, 2.5, 2.4, 2.2, 2.0, and 1.7 nm (SC6 passivant); (b) 2.5, 2.4, 2.2, 2.1, 2.0, and 1.7 nm (SC12passivant); and (c) 1.7 and 1.4 nm (SC18 passivant). (From: [84]. 1997 American Chemical Soci-ety. Reprinted with permission.)


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(equivalent to a dielectric constant near 10) results in a second effect, which is thelarge redshift of the collective resonance to its observed location (2.4 eV), with theunusual results that its intensity is dominated by the interband transitions, its exis-tence by the intraband transitions, and its location by the latter but modifiedstrongly by the polarizable Au+ ion cores. It seems that the breadth and proximity of

this resonance compared to the interband transition onset will make it more difficultto distinguish the interband absorption edge. Fortunately, Taylor et al. [94] gainedthe photoemission spectra on mass-selected cold AuN- beams, thereby demonstrat-ing that this interband energy, which is located at just below 2 eV, was definedclearly and exactly for clusters ofN=21 to more than 200 atoms. As a result, itslocation is probably constant for all close-packed Au clusters, in spite of the fractionof atoms at the surface. Based on these facts, the onset feature observed near 1.7 eVcan be ascertained without ambiguity to the first interband transitions, that is, totransitions from the highest occupied d orbitals to the lowest unoccupied level(s) of

the conduction band.Because no such structure has previously been ascertained in condensed Au, itis not easy to propose an interpretation of the discrete steplike structure (the peaks inFigure 3.11). However, by elimination methods, the evidence points toward the rea-son that it results from the pattern of unoccupied energy levels located just above theFermi level, that is, from the quantum size effect in the conduction band. Each stepwould then represent a newly accessible channel in the sparse conduction bandlevelstructure for transitions from the quasicontinuum of d-band levels.

Scientists prefer to address the applicability of various size-dependent correc-tions to the optical properties derived from bulk Au in a quantitative analysis of the

optical spectra of passivated gold nanocrystals. The effects of surface scattering(mean free path correction), surfactant effects on the core electron density, and


1.5 2.0

(b)

Energy (eV)

(a)

2.5 3.0 3.5

00

Figure 3.9 Optical spectra comparing nanocrystals of like metallic cores. (a) Spectra (normalizedat 4 eV) from 2.5-nm-diameter clusters passivated with hexyl (solid line) and dodecyl (dashed). (b)Spectra (offset for ease of viewing) of 1.7-nm clusters passivated by (top to bottom) SC6, SC12,and SC18. (From: [84]. 1997 American Chemical Society. Reprinted with permission.)


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quantum size effects are taken into account. Now it is time to discuss the basic Mietheory.

The essential assumption of Mies theory of (linear) optical absorption bysmall particles is that the particle and its surrounding medium are each homoge-neous and describable by their bulk optical dielectric functions. Briefly, by analyz-

ing Maxwells equations for this geometry one can obtain an expression for theabsorption cross section, which is a sum over electric and magnetic multipoles(spherical vector harmonics and Legendre polynomials). In the case that the size of aparticle is much smaller than the wavelength of the exciting radiation, the absorp-tion is dominated by the dipole term, with a cross section() given by [96]

( ) ( )

( )[ ] ( )

=

+ +9

2

3 20

2

1

2

22

m Vc

/

m

(3.4)

In this expression,is the frequency andcis the speed of light, mis the dielectricconstant of the embedding medium (assumed to be frequency independent over thespectral range of interest),V0is the volume of the absorbing particle, and1() and2() are the real and imaginary parts of the frequency-dependent dielectric con-stant of the absorbing solid.

According to the bulk optical constants [97] extracted by cubic spline fit,Whetten calculated the bulk dielectric function for Au and the correspondingabsorption spectrum based on (3.4). The spectrum (Figure 3.12), calculated assum-ingm= 1, shows a giant dipole resonance peaking near 2.3 eV, which is consistentwith the vanishing of 1()+2m. From (3.4) one can see that the spectral features


1.0 1.5 2.0 2.5 3.0 3.5 4.0

Energy (eV)

0.01

0.10

1.00

10.00

Figure 3.10 Optical spectra (offset for ease of viewing) of diverse selected fractions replotted on asemilogarithmic scale (top to bottom): 9-nm colloidal gold and 3.2-, 2.5-, 1.7-, and 1.4-nmnanocrystal gold molecules. The vertical dashed line marks the onset of the interband absorptionjust above 1.6 eV. (From: [84]. 1997 American Chemical Society. Reprinted with permission.)


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are size independent and the particle dimension acts only as a volumetric scaling fac-

tor. To obtain a size dependence for metal particles, one must decompose the dielec-tric function into two terms: an interband (IB) contribution, accounting for theresponse of 5d electrons, and a free-electron contribution (Drude,D) [98] from theelectrodynamics of the nearly free conduction electrons [99]:

( ) ( ) ( ) ( ) ( ) ( ) 1 11B 1D 2 21B 2D= + = +, (3.5)

( ) ( )( )

1

2

202 2D

2

202

= +

=+

1 0p p

, (3.6)

Here, pis the frequency of the plasma oscillation of free electrons expressed interms of the free-electron density N, the electron charge e, and the effective mass m:

p m2 Ne= 2 / (3.7)

which corresponds energetically to 8.89 eV for gold and 9.08 eV for silver, respec-tively [98]. The term 0, which usually takes a magnitude on the order of hun-dredths of an electron volt, acts as the deputy of the frequency of inelastic collisions(electron-phonon coupling, defects, impurities) of free electrons within the metal.From Figure 3.12 we can see the intuitive separation of susceptibilities of bulk fccAu. The variation in the 1() is free electron in nature below 2.4 eV, although offsetby the large, positive, and near-constant interband contribution. A significant inflec-tion appears near 2.4 eV. The 2() function is dominated by the free-electron term


dA

d/

1.5 2.0 2.5 3.0 3.5

01.5 2.0 2.5

Energy (eV)

Figure 3.11 Derivative (with respect to wavelength , dA/d ) of the optical absorption of diverseselected fractions (top to bottom): 2.6-, 2.4-, 2.2-, 2.0-, 1.7-, and 1.4-nm-diameter nanocrystalgold molecules. The inset shows the logarithmic derivative (A1 dA/d ) for the smallest of these.(From: [84]. 1997 American Chemical Society. Reprinted with permission.)


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below 1.7 eV and by the interband contribution above 2.4 eV. To introduce sizeeffects, one should assume that as the size of the particle diminishes, the rate of scat-tering from the particle surface (s) begins to greatly exceed the bulk scattering rate0 [99]. The surface scattering rate is described in terms of the Fermi velocity (F=1.4108 cm/s for gold and silver) and particle radius [98]:

s F=A R/ (3.8)

This expression can be explained as a limitation on the mean free pathof the freeelectrons by the particle sizes. The coefficientA is of the order of unity, and itsmeaning is not sufficiently identified [100]. It takes the value unity if the scattering isassumed to be isotropic, three-fourths if diffusive, or zero if elastic. When other fac-tors such as electron density at the surface, the effect of the interface, the anisotropyof particle, and quantum mechanical computations are incorporated into the the-ory, values from 0.1 to above 2 can be deduced and theoretically justified [6]. To

identify dielectric functions of the particles, one assumes that the interband contri-bution (Figure 3.12) is the same as that of the bulk, but that free-electron contribu-tions for small particles use sin place of0in (3.6).


0

1

23

4

5

6

50

40

30

20

10

0

10

1 1.5 2 2.5 3

Energy (eV)

3.5 4 4.5

2

21B

2D

ID

11B

2

1

1

(a)

(b)

Figure 3.12 Decomposition of the experimental dielectric functions (solid lines) of bulk fcc Auinto free-electron (Drude) and interband (5d f 6sp) contributions: (a) real and (b) imaginary parts.The onset of significant interband absorption [dotted curve in (b)] can be seen near 1.8 eV. (From:[84]. 1997 American Chemical Society. Reprinted with permission.)


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Assuming that the scattering rate in nanomaterials is the same as that in thebulk, the researchers calculated the size evolution of the optical absorption spectrumand compared the results with those obtained using this mean-free path correction(A= 1). The conclusion can be described as follows.

The damping and redshift of the giant dipole band are clearly evident. The

redshift in the position of the plasmon band is the result of the correction to 1,which is not negligible at smallest sizes [99].

Whetten and coworkers attempt to fit the measured absorption coefficientsrecorded on SC12surfactant with Mie theory and simple MFP correction (3.4) to theDrude terms in the dielectric function. The fits failed to describe the broadening ofthe surface plasmons and their positions. Nor did they observe a relative increase inlow-energy absorption, accompanying the quenching of the surface plasmon, whichis demanded by the functional behavior in the mean-free path model (Figure 3.13).

Enlightened by Hengleins work [101], Whetten and coworkers take thatadsorbates can significantly affect the metals electronic properties, both for bulksurfaces and in small particles as the second correction. The dependence in (3.7) ofthe plasmon frequency (p) on the free-electron density Nallows for a simplephenomenological description of the adsorbate effect. It is possible that they provideor withdraw additional electron density at the interface based on the facts thatthiol(ate)s are intimately bound to the particle surface. In the case of flat gold sur-faces with self-assembled thiol monolayers on them, approximately 0.2 nm2 of sur-face supports one thiol [102], and the coverage density is increased in small particlesbecause of curvature introduced by edges and vertices [90]. When correction is


4.0

Energy (eV)

1.0 1.5

0

2.0 2.5 3.0 3.5

Figure 3.13 Absorption spectra predicted by Mie theory for particles of decreasing diameter (topto bottom): 40 (bulk), 3, 2, and 1 nm. All curves were normalized to unity at 4.1 eV. The sizedependence results entirely from mean-free path corrections to both real and imaginary dielectricfunctions. (From: [84]. 1997 American Chemical Society. Reprinted with permission.)


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employed, taking as adjustable parameters the number of electrons donated (orwithdrawn), the core size, and the thiol surface coverage density, one can obtain thebetter fit only by adding the assumption that the surfactant groups donate (or with-draw) electrons. In fact, based on the assumption that one electron is donated perRS chain adsorbed, researchers indeed got reasonable values for the extracted core

dimensions and surfactant packing densities.Unfortunately, when these modifications are utilized to explain the abnor-

mally wide or depressed collective oscillation band, which resists being fitted withsimple corrections even after our introducing hypothetical cluster-size distributions,incorporating a Lorentz term for outer core electrons, or invoking Kubos quantummechanical correction to the relaxation time [43], the loss is the only thing that isobtained. Huffman and Henglein together with their coworkers suggested that thepassivating layer provided a dielectric coating on the surface of the sphere, amount-ing to a change in the dielectric constant of the medium [3, 103]. Another proposedmodificationsupposing that the outer surface gold atoms bound to the thiol(ate)lose their metallic character and are effectively removed from the metallic core,leaving its electron density unchangeddoes no better. In fact, both of these modifi-cations make no improvement to the fit of the patterns from calculations andexperiments.

However, regardless of the flaw of the specific theoretical foundation as dis-cussed above, it is possible to reproduce the spectra mathematically by modifyingthe dielectric functions, although this must be done in a way that satisfies certainconstraints (sum rule and Kramers-Kronig relations). For example, a much better fitis obtained simply by adding an energy-independent (offset) correction to the opti-

cal constants, and this suggests a transition in the electronic structure of Au at a sizejust below 2.0 nm. In 1996, Kreibig et al. [104] suggested that an abrupt change inthe experimentally determined dielectric constants for small gold particles tookplace at approximately 3-nm diameter. Whetten et al reported that their resultsplaced this change in the electronic structure of thiol-passivated gold particles pre-cisely where a transition in the atom-packing structure had recently been found[27], suggesting that these may be interrelated [84].

3.3.2 Magnetic Properties of Metallic Nanoparticles

When the magnetic properties are talked about in terms of magnetic metallicnanoparticles, the most noteworthy point of the origin is that the volume is smalland the surface/bulk ratio is big. Because of the small volume, the thermal fluctua-tion energy of the magnetic moment can compete with the magnetic barrier energyand even overcome it. Hence, the magnetic moment can rotate freely despite themagnetic barrier energy. In the most cases, the saturation magnetization is smallerthan the bulk counterpart, and it is attributed to the surface/volume ratio and thesituation of the surface.

In general, there are two basically points in the nanoparticles that are differentfrom those of their bulk counterpart. First, and also most important, when the parti-cle size is smaller than a certain critical value, they present very different behaviorfrom their bulk counterpart. Concretely, at room temperature, they exhibit zerocoercive field and no remanence, but they do saturate and the slope of the M-H



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curve varies continually before it reaches the saturation state. Second, the measuredaverage moment per molecule (atom, in the case of a metallic nanoparticle) usuallyis smaller than the one in bulk materials. The loss in magnetization corresponds to apartial quenching of the magnetic contribution of the surface. The fact that the mag-netization of the surface layer is not completely quenched is explained by a partial

coverage of the surface, in good agreement with the bulkiness of the ligands. Beforethe discussion of the magnetic properties, we introduce some terms that are neededwhen we talk about the magnetic properties of nanoparticles.

Superparamagnetism.At room temperature, the nanoparticles usually exhibitzero coercive fields and no remanence, but they do saturate and the slope ofthe M-H curve varies continuously before it reaches the saturation state. Sothey are considered to be super paramagnetic and the magnetic properties arecalledsuperparamagnetism.

Blocking temperature.As mentioned above, the nanoparticles are superpara-magnetic at room temperature. However, when the measurement is taken atlow enough temperature, either the coercive field or the remanence is not zeroany more. The behavior can be fully attributed to ferromagnetism. So thereexists some temperature at which the ferromagnetism begins to take the placeof superparamagnetism. That is theblocking temperature.

Following are the achievements obtained to date. As always, the statements are con-structed based on the outstanding work from different groups.

In the work reported by Gaos group [79], magnetic properties of the ~7-nm

cobalt nanoparticles deposited on highly oriented pyrolytic graphite (HOPG) sub-strate are measured by SQUID using a standard airless procedure. The magnetiza-tion as a function of the temperature in a 10-Oe field between 5K and 300Kdetermines the blocking temperature using a zero-field cooling (ZFC) procedure.Figure 3.14(a) shows the typical result for magnetic NPs. One can see that below thecritical size at which a particle becomes a single-domain magnet and is smallenough, the nanoparticles display superparamagnetism [106] at high temperature.

From the concrete measurement data, the blocking temperature (Tb) is 92K. Thebroad transition from superparamagnetism to ferromagnetism shown in Figure3.14(a) around 92K is probably due to the magnetostatic particle interactions in the

close-packed arrays. The blocking temperature should roughly satisfy therelationship

T KV kb B= / 30 (3.9)

whereKis the anisotropy constant,kBis Boltzmanns constant, andVis the averagevolume of the particles. With the knowledge of the blocking temperature and theparticle size, the anisotropy constant is 2.1 104 erg/cm3 for the Co particles, whichis smaller than that of the bulk fcc cobalt (2.7 106 erg/cm3).

Figures 3.14(b) and 3.14(c) show the hysteresis loop of the cobalt NPs powderrecorded at 250K and 10K, respectively. Cobalt NPs show no remanent magnetiza-tion in their magnetization field data at 250K. As discussed above, the superpara-magnetism behavior was observed at room temperature. While at 10K, the remnant



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magnetizationMris about 1.5 emu/g, the coercive fieldHcis 163 Oe, and the mag-netization at saturation (Ms) is estimated to be only 14.0 emu/g (the estimation isbased on an extrapolation of curves ofH/MversusH).

The noteworthy point of this work is the measurement conducted in differentstates of the cobalt nanoparticles. Yang et al. diluted the Co NPs in wax with a massratio of cobalt nanoparticles:wax equal to 1:4. Figure 3.14(d) shows the hysteresisloop of diluted particle powder, and a clear change in the shape of the hysteresisloop can be found. The Mrreaches 7.3 emu/g, Msreaches 59.6 emu/g, andHcincreases from 163 to 600 Oe in comparison with the cobalt nanoparticles in pow-der states. The hysteresis loop of ordered arrays of cobalt nanoparticles on HOPGsubstrate is shown in Figure 3.14(e). TheMr reaches 12.6 emu/g andHcincreases to790 Oe. However, the improvement ofMsis not obvious compared to that of theparticles diluted with wax, which is 61.6 emu/g. These values are lower than those


Temperature (K)0 50

M

(emu

,n

orma

lize

d)

100 150 200 250

H(kOe) H(kOe)

20

60

40

20

20

10 0 10

10K

10 0 10 20

10K

10K250K

(b)

(a)

(c)

(d) (e)

M

(emu

/g)

M

(emu

/g)

0

40

60

20

10

10

0

20

Figure 3.14 (a) ZFC magnetization versus temperature of cobalt NPs showing a blocking temper-ature (T

b) of 92K. Hysteresis loop of powder of cobalt NPs compacted into a capsule obtained at (b)

250K and (c) 10K. Hysteresis loop obtained at 10K: (d) diluting cobalt NPs with wax and (e) cobaltNPs deposited on HOPG and dried under N2 to prevent oxidation. (Source: [79].)


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obtained from the bulk phase. Because TEM images taken over large areas of thesample show no evidence of the coalescence, the observed changes cannot be attrib-uted to the coalescence of the cobalt NPs. They suggested that the exchange cou-pling between adjacent particles should account for explanations for the change inmagnetic properties. The dipole coupling enhancements are attributed to the long-

range order of the two-dimensional lattice and collective flips of the magneticdipoles.

In the work of Catherine Amiens [6467], she and coworkers discussed the mag-netic properties of the cobalt NPs they synthesized. The most striking result is thehigh differential susceptibility observed in high fields and leading to a very low mag-netization at 5T in a certain sample (namely, sample 1). The fact that the NPs adoptstructures very different from the bulk ones, even if it may lead to strong changes inmagnetic anisotropy, should not account for this strong decrease in magnetization.Indeed, body-centered-cubic (bcc), fcc, and hcp phases present very close values ofmagnetic moment per atom (1.6 to 1.7

B/Co). It is also the same in -Co, where the

value determined is close to 1.7B/Co, or in polytetrahedral arrangements, where nomagnetization reduction could be evidenced, whereas the atom packing is very dif-ferent from the bulk one [52, 53]. Either partial oxidation of the NP surface or astrong damping of the magnetic moment of the surface atoms introduced by coordi-nation of the ligands or other chemical species at their surface could then result inthis magnetization damping. Either through a strong alteration of surface aniso-tropy or via the formation of a diamagnetic surface layer, a strong coordinationeffect would also be responsible for the fact that the magnetization is difficult to sat-urate. Oxidation can be ruled out by the given XANES and EXAFS spectra. This

sample was synthesized under a di-hydrogen pressure, whereas another sample(namely, sample 2) was not. In fact, surface hydrides are expected to be present atthe surface of the particles, as recently demonstrated for Ru NPs [104]. One can thusquestion the effect of hydrogen adsorption on the magnetic properties. However, inSelwoods book one can find experiments showing that hydrogen adsorptioninduces only a small decrease of magnetization in the case of cobalt [107]. Further-more, as Amiens et al. demonstrated in 1998, NPs prepared in the same conditionsof hydrogen pressure but stabilized by poly-(vinylpyrrolidone), displayed magneticproperties similar to those of free-standing Co NPs [52]; hence, the possible role ofcoordinated hydrides in the lowMsvalue found for sample 1 was ruled out. How-

ever, the ligand used during the synthesis of sample 1 can strongly interact withhydrides coordinated to transition metals [24]. In this particular case, the formationof hydrido aluminate species at the NP surface should take place, which could thentransform into tricoordinated surface alkyl aluminum species. Formation of surfacealkyl radicals may also be envisaged, in agreement with the short distance observedby EXAFS. Hence, the surface of the NPs should then be regarded as electronicallydepleted. As is well known, electron-withdrawing ligands such as carbon monoxidedeprive the surface atoms of their magnetic properties [108]. In the assumption ofAmiens et al., a similar phenomenon takes place in sample 1 and accounts for theobserved low magnetization.

The loss in magnetization corresponds to a partial quenching of the magneticcontribution of the surface. The fact that the magnetization of the surface layer is



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not completely quenched is best explained by a partial coverage of the surface, ingood agreement with the bulkiness of the ligands.

Now, we have a grasp on the primary properties of the metal nanoparticles andcan move into the bioapplications of them.

3.4 Application of Metal Nanoparticles in Biomedicine

3.4.1 Biomedical Detection Using Novel Metal Nanoparticles

3.4.1.1 Au Nanoparticles

Functional nanomaterials that are designed to perform a reaction are of intenseinterest because of their potential uses in medical diagnostics [109], drug delivery[110], and catalysis [111, 112]. To be useful and biocompatible for biomedicalapplication, the nanoparticles must be coated by and/or linked with suitable mole-

cules, and the most well-understood method is the utilization of place-exchangereactions, which are based on displacement of surface coatings. Regarding the real-ization of the bioapplication of the NPs, the technique that allows us to modify thesurface of the NPs must play the determinative role. So we introduce this even morethan the idiographic bioapplication. The employment and generality of these reac-tions for the modification of Au nanoparticles was first described by Ingram et al.[113], who produced chemically useful particles by replacement of the monolayerligands with-functionalized alkanethiols. A significant drawback to this methodis the need to synthesize individual thiolated ligands for the aim at insertion into themonolayer. The inclusion and tailoring of functional moieties has been an aim insmall-molecule organic chemistry for decades, and as a result, a huge number ofreactions have been developed to convert, for instance, alcohols into carboxylicacids and halides into alkenes [114] or to combine them with molecular speciesusing amide or ester condensation chemistry [115]. These reactions have all beenemployed for the modification of Au nanoparticles surface structure and chemistry[116]. However, these chemistries are not compatible with every desired applica-tion, and the result is that the development of additional routes in general towardnanoparticle functionalization is still necessary for their use in emergingnanotechnologies. Molecular species can be conjoined by 1,3-dipolar cycloaddition

reactions [117], which were first described by Huisgen [118] and have recentlygained a revisit with the advent of click chemistry.In the latter method, azide-containing based on the discussion from triazole

coupling reactions could analogously be utilized as a general method for thefunctionalization of metal nanoparticle surfaces. Elizabeth Williams et al. [119]designed a system as a general route for functionalization of Au nanoparticles.Based on the facts that only mild reaction conditions are required and that theextreme selectivity toward molecules bearing azides and alkynes prevents unwantedside products, this reaction scheme is a particularly effective design. Figure 3.15shows thegeneral synthetic strategy for triazole functionalization of nanoparticles.

Let us tell about the synthesis of a series of redox-active, fluorescent, andsolubilizing species and the use of click chemistry as a facile route towardfunctionalization of monolayer-protected Au nanoparticles.

3.4 Application of Metal Nanoparticles in Biomedicine 71


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In the case of synthesis of 1-Pyren-1-yl-propyn-1-one (Pyr), an amount ofpyrene-1-carboxaldehyde was set in a round-bottomed flask together with anhy-drous tetrahydrofuran (THF) with ethynylmagnesium bromide as solute. From thebeginning to the end of the process, the reaction system was protected under nitro-gen. The reaction was quenched with saturated aqueous ammonium chloride afterthe solution was allowed to be stirred overnight. Finally, the product was extractedwith diethyl ether. The combined organic layers were dried over sodium sulfate, andthe ether was removed. After the product was dissolved in 50 mL of anhydrous ace-tone, Jones reagent was added dropwise under stirring until a red color occurred.After the reaction was quenched with isopropyl alcohol, the solution color turned

green. An excess of saturated aqueous sodium metabisulfite was added in, then theacetone was removed by rotary evaporation. After the extraction and purification,an intense yellow solid was obtained. The synthesis of 1-Anthracen-9-yl-propyn-1-one (An), Propynoic Acid 2-[2-(2-Methoxy-ethoxy) ethoxy]ethyl Ester(PEG) and Propynoic Acid Phenylamide (Ani) is similar except for a little change ormodification and the use of some new reactant instead [119].

They prepared the modified Au particles by employing the two-phase synthesismethod of Brust et al. [120] with slight modifications. Briefly, HAuCl4in distilledwater was transferred into toluene using tetraoctylammonium bromide and theorganic phase was isolated. After decanethiol was added, the solution was cooled to

0C and stirred for 10 minutes, after which an aqueous solution containing 380 mgof NaBH4 was added in. The mixture was stirred for an additional 3 hours before theorganic layer was separated and evaporated; a black waxy solid was produced andwas washed with copious amounts of ethanol.

Imitating the approach employed by Murray and coworkers, Williams et al. dis-solved the as-synthesized Au nanoparticles and an amount of BrC11H22SH in DCMand kept the mixture under stirring at room temperature for 60 hours to performligand exchange with BrC11H22SH. Following standard characterization methods[121], the NMR and Fourier transform infrared (FTIR) spectra reveal that protec-tive monolayers contain both CH

3- and Br-terminated ligands.

To the synthesis of azide-functionalized Au nanoparticles, they dissolved theAu--Brfunctionalized particles in DCM and then added them into an equal vol-ume of NaN3in DMSO. After the solution was allowed to remain under stirring for


(a) (b)

(c)

Br

NN

N

O

O

R

R

= = NN

N= =

NN

N= =

=

=

=

=

Figure 3.15 (a) Br(CH2)11SH in DCM, 60 hours at room temperature; (b) 0.25 M NaN 3 inDCM/DMSO solution, 48 hours; and (c) R = propyn-1-one derived compounds as in scheme 2, 24to 96 hours in dioxane or 1:1 hexane/dioxane. (From: [119]. 2006 American Chemical Society.Reprinted with permission.)


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48 hours, they added water and the black organic layer was isolated. They per-formed reaction of azide-functionalized Au nanoparticles with alkynyl derivativesusing a procedure that can be described as follows: N3-functionalized particles andthe alkynyl-modified compound (Figure 3.16) were codissolved in dioxane or hex-ane/dioxane and stirred for 24 to 96 hours. Under vacuum, the solvent was removed

and ethanol was utilized to remove any unreacted alkynyl derivative. Finally, theparticles were dried and then redissolved in DCM. Additionally, Au nanoparticlesamples could be decomposed using standard disulfide-forming reactions. For test-ing the functionalization approach, reactions involving triazole ring formation wereconducted using small Au particles.

Because its length sufficiently stabilizes the particles from aggregation but is notso sterically hindered as to prevent ligand exchange, decanethiol served as the sur-face ligand in the synthesis of monolayer-protected Au clusters.

One can see in the representative TEM image in Figure 3.17 that the Au nano-particles are spherical and have an average diameter of about 1.8 nm. For the pur-pose of replacing a fraction of these ligands with Br-terminated undecanethiolligands, the decanethiol-stabilized Au particles were then stirred in a solution con-taining BrC11H22-SH. By means of NMR and FTIR spectra, the replacement wasconfirmed. In the reaction of the Br termini, in the way that nucleophilic substitu-tion with NaN3 was used to append azide functionalities to the Au nanoparticles, asshown in Figure 3.17(b), the size and shape of the resultant particles were notaffected. According to the FTIR and NMR spectra, the ligand replacement withBrC11H22-SH and subsequent reaction with NaN3 and the resultant Aunanoparticles containing mixed monolayers that are 44% CH3-and52%N3-termi-

nated alkanethiol ligands were confirmed.In Williamss work, they also conducted the triazole functionalization of Aunanoparticles, and further particle functionalization through 1,3-dipolar cyclo-addition reactions (i.e., click chemistry), by fusing ethynyl- and azide-bearing


3

Fe

Fc NB Pyr

HN

An PEG Ani

NO2

CH3OO

O

O

O

O

OO

Figure 3.16 Scheme 2. Propyn-1-one compounds for attachment via triazole ring formation.(From: [119]. 2006 American Chemical Society. Reprinted with permission.)


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molecules, can be allowed by decorating the nanoparticle surface with N3functionalities. They utilized six different alkynyl compounds including the alkynederivatives of ferrocene (Fc), nitrobenzene (NB), pyrene (Pyr), anthracene (An),poly(ethylene glycol) (PEG), and aniline (Ani) to demonstrate the utility and gener-ality of this method [119]. After the reactants were connected to the Au nano-

particles, a range of physical, electronic, and spectroscopic properties areintroduced or improved. Aiming at the enhancement of the rate of triazole forma-tion, each was synthesized to include a carbonyl group adjacent to the terminalalkyne to provide a more electron-withdrawing environment [117, 123].

According to the NMR and FTIR results recorded for the as-synthesized func-tionalized Au nanoparticles (for the purpose of this chapter, we prefer to ignore theconcrete procedure for preparing samples and the patterns measured).

Other than the ring formation, the surface attachment does not affect the struc-tures of the compounds drastically. In addition, the alkyl stretching vibrations of thealkanethiolate remain unchanged by cycloaddition, indicating that the protecting

surface monolayer is largely unaffected by triazole ring formation. The completereaction that the triple bonds with the terminal N3 groups on the Au nanoparticles toform a triazole ring rules out binding by intercalation of the ligand into the particlesurface monolayer or the possibility of unreacted alkynyl compounds present asimpurities.

In this approach they also utilized cycloaddition reactions to preparemultifunc-tional Au nanoparticles by simultaneously stirring the N3-terminated nanoparticleswith several acetylenic small molecules to obtain N3-containing Au nanoparticles,which was reacted with a solution containing both the Fc and the NB propyn-1-onespecies. Analysis based on FTIR results indicates that the multifunctional Aunanoparticles containing both Fc and NB species linked through the fact that atriazole ring formed.


(a) (b)

5040302010

0#ofParticles

04080

120160

#

ofParticles

0 1 2 3 4 5 6Size (nm)

0 4 62Size (nm)

Figure 3.17 (a) Representative TEM images of synthesized C10H21SH-modified Au particles and (b)azide-functionalized Au nanoparticles. Insets contain particle size distributions. The scale bar is 50nm. (From: [119]. 2006 American Chemical Society. Reprinted with permission.)


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Quantitative assessment of extent of this functionalization approach has beencarried out following a method that has been previously described [119]. An estima-tion of the percentages of surface-bound ligands per Au nanoparticle was per-formed by assuming the following:

1. No ligands are destroyed though side reactions.

2. All ligands are completely cleaved from the surface by means of reactionwith I2. Analysis of peak integrations in the NMR result for theN3-terminated particles suggests that there are about 1.2 N3-terminatedthiols for every CH3-terminated thiol and an average of ~4% ligands perparticle that are not converted to N3upon reaction with NaN3 [119].

However, this may result from the fact that a difference in the degree of solubil-ity of the reactants exists and there is no apparent tendency with respect to size orreactivity for Pyr- or PEG-coupled Au nanoparticles. As far as the role of solvent onthe extent of functionalization is concerned, by repeating these reactions in a rangeof polar and nonpolar solvents, it was found that the latter ones are more effectiveand have a larger yield.

Since solubility played a primary role when determining reactivity, the research-ers have observed that solutions containing both hexane and dioxane wouldsolubilize both the Au nanoparticles and click compounds. In the click compoundstudied, an average threefold increase in the efficiency of the azide conversion totriazole is the result from addition of the more nonpolar (such as hexane) solvent.

These facts are in good agreement with prior reports, where hydrophobic solventsbetter stabilized the nonpolar transition state [124, 125].The better solubilization of the hydrophobic monolayer on the Au nanoparticle

surfaces by addition of the nonpolar hexane to solution should also possiblyaccount for this effect.

Sharpless et al. have demonstrated that Cu catalysts can greatly enhance therate of triazole formation, particularly in aqueous solutions, which is a prerequisite[126, 127], so the researchers have also sought a proper catalyst system to increasethe conversion. Because of the hydrophobic habit of the monolayer-protected parti-cles, which made them insoluble in aqueous solutions and prevented the use of the

most commonly employed CuSO4-ascorbic acid system, the use of several organicsoluble catalysts, including bromotris(triphenylphosphinato) copper(I) [128], CuI,[129], and CuBr/2,6-lutidine [130], seems reasonable. However, in all cases imme-diate and extensive particle aggregation or decomposition was observed. This phe-nomenon may be caused by the attractive force between the particle surface and theorganic coating, which may have different charges.

By the use of triazole ring formation as a general method, chemical, spectro-scopic, or electrochemical functionality may be appended to metal nanoparticles.This procedure can be named clicked functionality on Au nanoparticles. Hence,we take an overall look at the modification of the surface. And the idiographicapplication of Au nanoparticles will be introduced, together with Ag NPs, in thenext section.



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3.4.1.2 Ag Nanoparticles

First, we introduce the useful technology that utilizes localized surface plasmon res-onance (LSPR) nanosensors in detecting biological molecules. This novel, nanoscaledevelopment is a refractive indexbased sensing device that relies on the extraordi-nary optical properties of noble (Ag, Au, Cu) metal nanoparticles [131135].Briefly, it is based on the nanoscale limit of surface plasmon resonance sensors. Inother words, LSPR, which refers to the ability of the conduction electrons in thenanoparticle to oscillate collectively [136], induces electromagnetic fields surround-ing the nanoparticle, which determine the sensing volume in which refractiveindexbased sensing can occur [135, 137]. Nanoparticles exhibit selective photonabsorption, which can easily be monitored using ultraviolet-visible, since it is wellknown that the conduction electrons oscillate collectively to only specific wave-lengths of light (UV-visible) spectroscopy. It is well established that the maximumextinction wavelength maxof the LSPR is dependent upon the composition, size,

shape, and interparticle spacing of the nanoparticles. And the dielectric properties oftheir local environment (i.e., substrate, solvent, and surface-confined molecules)[135, 136] also play an important role.

The first nonmodel application of the LSPR nanosensor was reported by Haes etal. in 2004 [135]. In their work, the LSPR nanosensor underlain by the optical prop-erties of Ag nanotriangles was shown to aid in the understanding of the interactionbetween amyloidderived diffusible ligands (ADDL) and the anti-ADDL antibody,molecules possibly involved in the development of Alzheimers disease. By varyingthe concentration of anti-ADDL antibody, a surface-confined binding constant of3.0107 M1 for the interaction of ADDLs and anti-ADDLs was measured. Influ-

ences of Cr, the nanoparticle adhesion layer, will be shown to be the limiting factorin the sensitivity of this assay.

In fact, in 2002, they presented the work on the employment of Ag nanoparticlestogether with Au nanoparticles in the LSPR procedure. Briefly, it is based on the factthat triangular silver nanoparticles (~100 nm wide and 50 nm high) have remark-able optical properties and the peak extinction wavelengthmaxof their LSPR spec-trum is unexpectedly sensitive to nanoparticle size, shape, and local (~10 to 30 nm)external dielectric environment. This sensitivity of the LSPR max to thenanoenvironment has provided the opportunity for developing a new class ofnanoscale affinity biosensors. Utilizing the well-studied biotin-streptavidin system,the essential characteristics and operational principles of these LSPR nanobio-sensors were demonstrated. In this work, a 27.0-nm redshift in the LSPR maxoccurred, after the exposure of biotin-functionalized Ag nanotriangles to 100-nMstreptavidin (SA). They measured the LSPR maxshift, R/Rmax, versus the [SA]response curve over the concentration range 1015 < [S] < 106 M. By comparingthe data with the theoretical normalized response expected for 1:1 binding of aligand to a multivalent receptor with different sites but invariant affinities, one gotapproximate values for the saturation response. The values are Rmax= 26.5 nmtogether with the surface-confined thermodynamic binding constant Ka,surf= 10

11

M1

for the actual limit of detection (LOD) for the LSPR nanobiosensor, which is inthe low-picomolar to high-femtomolar region. Hence, a strategy for amplifying theresponse of the LSPR nanobiosensor with employment of biotinylated Au colloidsand thereby further improving the LOD was carried out. Several control



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experiments were conducted to define the LSPR nanobiosensors response to non-specific binding as well as to demonstrate its response to the specif

silver nano in biomedical

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