WO2003083480A1

WO2003083480A1 - Methods for manufacturing nanoparticle structures using hydrophobic or charged surfaces

Info

Publication number: WO2003083480A1
Application number: PCT/US2003/009655
Authority: WO
Inventors: David I. Kreimer; Thomas H. Nufert; Lev Ginzburg; Oleg A. Yevin
Original assignee: Array Bioscience Corporation
Priority date: 2002-03-29
Filing date: 2003-03-28
Publication date: 2003-10-09
Also published as: AU2003233458A1

Abstract

The invention provides methods for manufacturing nanoparticle structures using surfaces onto which the nonoparticles can aggregate to form nanoparticle structures. The surfaces suitable include gas-liquid interfaces (bubbles), beads, plastics, glasses, oil-water emulsions and films. The nanoparticle structures so obtained have improved Raman signal enhancement an improved radiation absorptivity and emmissivity.

Description

METHODS FOR MANUFACTURING NANOPARTICLE STRUCTURES USING HYDROPHOBIC OR CHARGED SURFACES

Related Applications This application claims priority to U.S. Provisional Patent Application

Serial No: 60/368,480 filed 29 March 2002 and to U.S. Provisional Patent Application Serial No: 60/368,932, filed 01 April 2002, each application incorporated herein fully by reference.

BACKGROUND

Field of the Invention

This invention relates to methods for manufacturing nanoparticle structures. Specifically, this invention relates to methods for manufacturing nanoparticle structures using surfaces. More specifically, this invention relates to the manufacture of nanoparticle structures using hydrophobic or charged surfaces.

Related Art

A. Manufacture of Nanoparticle Structures Nanoparticle structures are aggregates of nanoparticles containing two or more nanoparticles that are connected with each other either directly or via linking molecules. Nanoparticle structures can comprise particles with characteristic sizes within the range of a fraction of nanometer to hundreds of nanometers, or larger, having sizes up to hundreds micrometers. Nanoparticles can be spherical, ellipsoidal, rod-shape, branched, or disordered.

Metal nanoparticle structures can be manufactured using a number of approaches such as laser ablation, evaporation of metal, nanolitography, arranging nanoparticles of colloidal solutions using linkers, and the aggregation of colloidal solutions of metal nanoparticles. The aggregation of colloidal solutions does not require expensive instrumentation or generation of linkers that could be expensive. The aggregation is easy to implement: Metal nanoparticles are maintained in colloidal solution if they are charged and repel each other; addition of a salt to such a metal colloidal solution results in discharge of the particles and their aggregation. This approach is broadly used for generating aggregates capable of SERS and for other applications. Examples of this approach are included in United States Patent Application Serial Nos: 09/670,453; 09/815,909; 09/925,189; 09/669,369; 09/669,796; 09/815,828; "Enhancing Surfaces for Analyte Detection," David I. Kreimer and Thomas H. Nufert, filed March 15, 2002; 09/939,887. Each of the above applications is incorporated herein fully by reference.

Despite the ease of use and low cost, this approach suffers from poor reproducibility of the resulting nanoparticle structures. This poor reproducibility is the obstacle for the use of such nanoparticle structures.

SUMMARY OF THE INVENTION Thus, one object of this invention is to provide methods for the manufacture of nanoparticle structures having increased reproducibility.

Another object of this invention is to provide nanoparticle structures having increased enhancing properties for Raman spectroscopy and for thermal absorption and emission.

To address these and other objects, the instant invention provides for improved methods of manufacturing nanoparticle structures with increased reproducibility. In certain embodiments of this invention, nanoparticle structures can be made from colloidal solutions of nanoparticles on hydrophobic surfaces or the surfaces of bubbles. The use of such surfaces permits the production of better, more reproducible nanoparticle structures than were possible using prior art methods. Using these methods, nanoparticle structures having desirable characteristics of particle size, size of particle structures, means of association of particles and particle structures, and other properties. By providing methods for producing nanoparticle structures having desirable features permits more reproducible and controllable Raman enhancement, and radiation absorptivity and emissivity. Surfaces suitable for manufacturing nanoparticle structures of this invention include surfaces of bubbles, hydrophobic beads, emulsions, small charged beads and the like. Bubbles can be formed using solute ebullition, boiling, cavitation, ultrasound, alteration of temperature, pressure, and other methods known in the art. Once nanoparticle structures are manufactured, they can be used for a variety of purposes including Raman signal enhancement, improved radiation absorptivity and emissivity and other purposes for which nanoparticle structures are desirable.

BRIEF DESCRIPTION OF THE FIGURES

This invention will be described with reference to specific embodiments thereof. Other features and characteristics of embodiments of this invention can be found in the figures, in which:

Figure la depicts an embodiment of this invention in which nanoparticle structures are deposited on a substrate.

Figure lb depicts an embodiment of this invention in which nanoparticle structures are formed from a colloidal solution of nanoparticles in the presence ofbubbles.

Figure 2 depicts an optical micrograph of a surface of this invention as depicted in Figure la.

Figure 3 depicts a surface enhanced Raman spectrum of rhodamine 6G ("R6G") in the presence of an enhancing nanoparticle structures of this invention as in Figure la. Figure 4 depicts an optical micrograph of a surface of an embodiment of this invention as depicted in Figure lb.

Figure 5 depicts a surface enhanced Raman spectrum of rhodamine 6G ("R6G") as in Figure 3, except that the enhancing structures were manufactured using bubbles according to methods of this invention, as in Figure lb.

Figure 6a depicts an electron micrograph of nanoparticle structures of this invention as in Figure lb.

Figure 6b depicts an electron micrograph of the same nanoparticle structures as shown in Figure 6a, except at higher magnification. Figure 7 depicts a schematic representation of the manufacture of nanoparticle structures of this invention using bubbles.

Figures 8a-8h depicts schematically, a series of steps in the manufacture of nanoparticle structures of this invention using bubbles.

Figures 9a-9c depicts schematically, a series of steps in the manufacture of nanoparticle structures of this invention on the surface of a substrate.

Figure 10a depicts embodiments of this invention in which nanoparticle structures have receptors attached thereto.

Figure 10b depicts an embodiment of this invention in which nanoparticle structures are manufactured using chemical linkers to attach nanoparticles to one another.

DETAILED DESCRIPTION In general, the methods of this invention can utilize surfaces suitable for the aggregation of nanoparticles to each other. It can be readily appreciated that according to prior art methods in solution, formation of aggregates of nanoparticles can occur in 3-dimensions. It is well known that movement of particles in three dimensions leads to interactions between particles that obeys principles elucidated by Albert Einstein in the early 1900s. Unfortunately, the types of interactions that occur in "3d-space" are somewhat variable and the time required to achieve a certain degree of aggregation can be variable.

However, using 2-dimensional surfaces ("2d"), the predictability of interactions can be increased substantially compared to those which occur in 3d- space. Therefore, according to one possible theory, the improved reproducibility of the nanoparticle structures of this invention is due to the fact that the nanoparticle structures form on surfaces.

The surfaces on which nanoparticle structures can be desirably formed include bubbles (e.g., pockets of gas phase in a liquid), hydrophobic surfaces such as neutral plastics, Teflon ^R, and on the surfaces of oils (e.g., emulsions). Additionally, surfaces that are suitable include charged surfaces, such as charged plastics, glasses, foams, detergent foams and the like.

In particular, for metal nanoparticle structures, the spatial distribution of density of particles within an aggregate can determine electromagnetic and other characteristics of these aggregates and of the media containing such nanoparticle structures. Optical characteristics of such aggregates and their capacity to emit or absorb electromagnetic radiation in near-infrared and infrared ranges of electromagnetic energy (heat absorption and radiation) can be dependent on the density. When nanoparticles within such an aggregate are distributed randomly, such aggregates allow for propagation of electromagnetic wave through the aggregate. When nanoparticles are ordered within the aggregate, such a nanoparticle structures can also propagate electromagnetic waves; this propagation is dependent upon orientation of the wave relative to the preferred directions within the nanoparticle structure. When nanoparticles are distributed in a fractal arrangement, such an aggregate propagates electromagnetic waves poorly. Due to this property, fractal nanoparticle structures are of practical interest because of their ability to concentrate and re-emit electromagnetic energy from tiny areas herein called "hot spots." Fractal and fractal-like arrangements of nanoparticles are desirable, in particular, for generation of nanoparticle structures displaying high enhancement of Raman signals in Surface Enhanced Raman Spectroscopy ("SERS") and other kinds of optical responses. These arrangements can also be used for manufacturing of materials with superior heat absorption and emission characteristics.

I. Aggregation of Nanoparticles in Colloidal Solutions

One approach for manufacturing of fractal nanoparticle structures is based on assumption that fractal aggregates can form spontaneously when nanoparticles engage in a complex with each other upon very first encounter(s). Depending on the rate of diffusion of particles and aggregates and on probability of particles adhering or "sticking" to each other, the resulting aggregates can either form via formation of clusters of comparable size that then aggregate with each other, or alternatively via individual aggregates growing due to addition of single particles to the aggregates. Correspondingly, certain aggregates have fractal dimensionality from about 1.8 (high openness of aggregates) to about 2.5 (somewhat more dense structures). If nanoparticles encounter each other several times prior to sticking, the resulting structures are dense and have dimensionality close to 3.0. These mechanisms are disclosed by Ramsay J.D.F. in: Sol-gel processing, D.J. Wedlock (Ed.), Controlled Particle, Droplet And Bubble Formation, Butterworth-Heineman, Oxford, UK, (1994), Chapter 1, p. 1-38, incorporated herein fully by reference. While this manufacturing method can be useful for many applications, it does not permit controlling the resulting fractal nanoparticle structures. II. Mechanisms of Bubble Formation

Bubbles in bulk liquid can be a gas either surrounded entirely by a liquid or, if a surfactant is present, gas can be surrounded by a layer of such a surfactant. Bubbles can be also associated with surfaces of the vessel or particles present in the liquid. The size of bubbles can vary from several nanometers to up to 1 cm, beyond which hydrodynamic forces are likely to cause the bubbles to degrade. Bubble formation can be spontaneous or induced. Spontaneous formation of bubbles in a liquid that does not contain other components can occur by cavitation or boiling. In addition to these mechanisms, in a liquid saturated with gas, bubble formation may occur as the result of the presence of greater than equilibrium concentration of a dissolved gas. Various aspects of formation and growth of bubbles are disclosed in the prior art by S.D. Lubetkin, in:D.J. Wedlock (Ed.), Controlled Particle, Droplet And Bubble Formation, Butterworth-Heineman, Oxford, UK, (1994), Chapter 6, p. 159- 190, herein incorporated fully by reference.

Spontaneous bubble formation can occur via one or more of the following: (a) homogenous nucleation; (b) heterogeneous nucleation; (c) cavitation; (d) electrolysis; (e) chemical reactions; and (f) Harvey nuclei. Additionally, bubbles can be present in a solution as pre-existing and/or colloidally stable free bubbles. Homogenous nucleation refers to the case when, due to alteration of pressure or temperature, a vapor forms initial bubbles of a critical size in the bulk of liquid away from surfaces of a vessel, surface of the liquid and in the absence of dust particles. If there is a detergent present in the liquid, the rate of nucleation of bubbles and the rate of their growth can be altered.

When bubbles are formed at a surface (walls of a vessel, dust or other inhomogenieties), such bubbles are nucleated heterogeneously. The formation of bubbles via this mechanism can occur more easily than in the bulk of liquid, and can depend upon the contact angle of gas/solution/solid interface and the geometry of nucleation side. While the dependence on the angle is well understood - the larger the angle, the easier formation of the bubble, the geometry of nucleation sites are understood only for spherical and conical depressions and protrusions.

Cavitation is the process of bubble formation by reduction in pressure. The reduction can be achieved by mechanical means, placing the liquid under tension, or by acoustic means, where waves introduce negative pressures. In particular, in certain embodiments of this invention, ultrasound can be desirable to form bubbles

Electrolytic bubble generation can occur on the surface of an electrode. Remarkably, in this process, bubbles are charged. The size of bubbles after detachment from the electrode can depend upon the composition of the electrode surface and of electrostatic interactions between the charged bubbles and the electrode. Generation of bubbles using electrolytic method allows for effective control over the process of bubble nucleation and growth.

Chemical bubble generation is somewhat similar to electrolytic generation in that control of bubble size and density can be readily achieved. In this approach, gas is produced as the result of a chemical reaction such as thermal decomposition, for example decomposition of carbonates, nitrates, hydrogen peroxide, and diazo derivatives. An acid-base reaction is another example for chemical bubble generation.

When pre-existing sources of bubbles exist, macroscopic bubbles can be formed without the need for nucleation step. Three known sources of bubbles are: Harvey nuclei; pre-existing bubbles, and entrained or sparged bubbles. Harvey nuclei are bubbles entrapped at a solid-liquid interface in cavities of such geometry that the entrapped gas is stable to displacement by the surrounding liquid phase. An example is the use of antidumping granules during distillation, where the aim is to introduce as many Harvey nuclei as possible. Pre-existing bubbles are obtained by shaking or stirring a liquid in a vessel. These bubbles can be stabilized by surfactants.

A sudden change in conditions for a liquid containing pre-existing bubbles can result in surge of bubbles as these pre-existing, microscopic bubbles rapidly grow to a visible size. The changes can include reduction of pressure, increase in temperature, etc.

To get rid of bubbles, sufficiently high pressure can be applied to the liquid. This pressure will collapse essentially all bubbles. Often a pressure as high as 1000 atmospheres is applied for such purposes, although lower pressures may also be suitable. It can be appreciated that persons of ordinary skill can determine desirable pressures.

Non-spontaneous bubble formation include approaches such as sparging, entrainmant, and attrition. These approaches can be useful for controlled insertion of gas bubbles into a liquid phase. In sparging, bubbles are inserted directly into the liquid phase by pumping gas through a frit, filter or bubble column. Sparging is a controllable and reliable method of producing bubbles having consistent sizes. Entrainment occurs when gas is enveloped by liquid at an interphase between two phases. Such a process can occur where liquid is in motion (for example, waves in the ocean). Mechanical agitators can be used to produce bubbles using this process. Another way for gas entrapment is mechanical disruption of gas-liquid interface that can be also achieved with agitators. This process called attrition extensively disclosed in chemical and engineering literature. For example, Perry, R.H., Green, D.W., Maloney J.O. (ed) (1984) Liquid- gas systems. In: Chemical Engineers Handbook, 6th edn, McGraw-Hill, New York, Section 18, incorporated herein fully by reference. A. Stability of Bubbles

Bubbles in general, can reach a free liquid surface where they may burst rapidly or may persist for a long time, they may adhere to a solid surface, they may coalesce with each other and they may dissolve. These ways of bubble removal are understood and can be controlled. Some means for the control include the use of surfactants and the use of gases that chemically interact with liquid, such as CO₂/water, SO₂/water and NH₃/water. Other methods are well known in the art.

B. Bubbles on Hydrophobic and Hydrophilic Surfaces

Bubbles have an affinity for surfaces and are often attracted to and retained on such surfaces. In particular, hydrophobic surfaces are attractive for retention of hydrophobic (not charged) gas bubbles. The following papers describing the affinity of gas bubbles to hydrophobic and hydrophilic surfaces are incorporated herein fully by reference: Carambassis et al., Forces Measured between Hydrophobic Surfaces due to a Submicroscopic Bridging Bubble. Physical Review Letters 80(24}: 5357-5367 (15 June 1998); Ishida et al., Nano Bubbles on Hydrophobic Surface in Water Observed by Tapping-Mode Atomic Force Microscopy, Langmuir 16: 6377-6380 (2000); Suresh, L. and Walz, J.Y., Effect of Surface Roughness on the Interaction Energy between a Colloidal

Sphere and a Flat Plate, J. Colloid and Interface Science 183:199-213 (Article # 0535) (1996) ; M. Preuss and H.-J. Butt, Direct Measurement of Particle-Bubble Interaction in Aqueous Electrolyte: Dependence on Surfactant, Langmuir 14 3164-3174 (1998); Semal et al., influence of Surface Roughness on Wetting Dynamics, Langmuir 15: 8765-8770 (1999). C. Bubble Formation by Release of Dissolved Gases with Solutes

Addition of salts to a liquid containing dissolved gas can result in ebulliation of the gas from the liquid and the formation of a bubble. L.A. Bol'shov, A.L.Bol'shov, The Sechenov Law of Salting out and the Nagner, Russian J. of Physical Chemistry, 71(2): 244 (1997), incorporated herein fully by reference. Furthermore, ebullition of gas can occur near hydrophobic or hydrophilic surfaces upon addition of salts. Bunkin et al., Effect of Salts and Dissolved Gas on Optical Cavitation near Hydrophobic and Hydrophilic Surfaces, Langmuir 13: 3024-3028 (1997), incorporated herein fully by reference. The effect can be increased by application of optical stimulation (e.g., exposure to laser light to produce cavitation).

Similarly, ebullition of gas can be induced by the dissolving of other, non-salt solutes in a liquid containing dissolved gas. Such solutes include polar materials such as sugars including glucose, fructose, galactose, fucose, Ν- acetylarabinoside and the like, peptides, nucleic acids, proteins and the like. One can readily apreciate that any solute that does not interact in undesirable ways with nanoparticles, nanoparticle structures or other elements in the medium can be used. In embodiments in which the nanoparticle structures can be made in non-polar medium, one can use non-polar solutes to elicit ebullition of gas. In embodiments in which detergents or other surfactants are desirably used to stabilize bubbles, such surfactants can also elicit ebullition of gas.

HI. Methods for Manufacturing Nanoparticle Structures Using Surfaces

In general, methods of this invention comprise forming a colloidal solution of nanoparticles in liquid medium. In some embodiments of this invention, the Raman active structures desirable for use according to this invention can include any structure in which Raman signals can be amplified. The following discussion regarding metal fractal structures is not intended to be limiting to the scope of the invention, but is for purposes of illustration only.

A. Manufacture of Metal Particles

To make metal particles for nanoscale arrays of receptors according to some embodiments of this invention, we can generally use methods known in the art. Tarcha et al., U.S. Patent No: 5,567,628, incorporated herein fully by reference. Metal colloids can be composed of noble metals, specifically, elemental gold or silver, copper, platinum, palladium and other metals known to provide surface enhancement. In general, to make a metal colloid, a dilute solution containing the metal salt is chemically reacted with a reducing agent. Reducing agents can include ascorbate, citrate, borohydride, hydrogen gas, and the like. Chemical reduction of the metal salt can produce elemental metal in solution, which combine to form a colloidal solution containing metal particles that are relatively spherical in shape.

Example 1 Manufacture of Gold Colloid and Fractal Structures In one embodiment of this invention, a solution of gold nuclei is made by preparing a 0.01% solution of NaAuCl₄ in water under vigorous stirring. One milliliter ("mL") of a solution of 1% sodium citrate is added. After 1 minute of mixing, 1 mL of a solution containing 0.075 % NaBH₄ and 1% sodium citrate is added under vigorous stirring. The reaction is permitted to proceed for 5 minutes to prepare the gold nuclei having an average diameter of about 2 nm). The solution containing the gold nuclei can be refrigerated at 4° C until needed. This solution can be used as is, or can be used to produce particles of larger size (e.g., up to about 50 nm diameter), by rapidly adding 30 μl of the solution containing gold nuclei and 0.4 mL of a 1% sodium citrate solution to the solution of 1% HAuCl₄3H₂O diluted in 100 mL H₂O, under vigorous stirring. The mixture is boiled for 15 minutes and is then cooled to room temperature. During cooling, the particles in the solution can form fractal structures. The resulting colloid and/or fractal particle structures can be stored in a dark bottle.

Deposition of enhancing particles on dielectric surfaces including glass can generate films that can enhance electromagnetic signals. Such films can be as thin as about 10 nm. In particular, the distribution of electric field enhancement on the surface of such a film can be uneven. Such enhancing areas are resonance domains. Such areas can be particular useful for positioning receptors for analyte binding and detection. For films or particle structures embedded in dielectric materials, one way to manufacture enhancing structures is to treat the surface until "percolation points" appear. Methods for measuring sheet resistance and bulk resistance are well known in the art.

Example 2

Manufacture of Metal Particles and

Nanoparticle Structures Using Laser Ablation In addition to liquid phase synthesis described above, laser ablation is used to make metal particles. A piece of metal foil is placed in a chamber containing a low concentration of a noble gas such as helium, neon, argon, xenon, or krypton. Exposure to the foil to laser light or other heat source causes evaporation of the metal atoms, which, in suspension in the chamber, can spontaneously aggregate to form fractal or other particle structures as a result of random diffusion. These methods are well known in the art. B. Manufacture of Films Containing Particles

To manufacture substrates containing metal colloidal particles of one embodiment of this invention, the colloidal metal particles can be deposited onto quartz slides as described in Examples 1 or 2. Other films can be made that incorporate random structures or non-fractal ordered structures in similar fashions.

Example 3 Manufacture of Quartz Slides Containing Gold Fractal Structures Quartz slides (2.5 cm x 0.8 cm x 0.1 cm) are cleaned in a mixture of

HCl:HNO₃ (3:1) for several hours. The slides are then rinsed with deionized H₂O (Millipore Corporation) to a resistance of about 18 MΩ and then with CH₃OH. Slides are then immersed for 18 hours in a solution of aminopropyltrimethoxysilane diluted 1:5 in CH₃OH. The slides are then rinsed extensively with CH₃OH (spectrophotometric grade) and deionized H₂O prior to immersion into colloidal gold solution described above. The slides are then immersed in the gold colloid solution above. During this time, the gold colloid particles can deposit and can become attached to the surface of the quartz slide. After 24 hours, colloid derivatization is complete. Once attached, the binding of colloidal gold nanocomposites to the quartz surfaces is strong and is essentially irreversible. During the procedure, ultraviolet and/or visual light absorbance spectra of such derivatized slides are used to assess the quality and reproducibility of the derivatization procedure. The manufacturing process is monitored using electron microscopy to assess the density of the colloidal coating, the distribution of gold colloid particles on the surface, and the size of the gold colloid particles.

In other embodiments, fractal aggregates can be attached to substrates having a layer of gold metal thereon. The types of substrates are not limited, and can be quartz, conventional glass, plastic or any other substrate upon which a layer of gold metal can adhere. Because gold metal is relatively chemically inert, once prepared, the gold-coated slides can be cleaned using conventional methods prior to attachment of fractal aggregates thereto. Gold or silver fractal aggregates can be prepared using methods described herein or using methods from the prior art. Colloidal of fractal aggregates can then be applied to the surface of the gold-coated substrate and the fractal aggregates tend to adhere to the gold surface, forming a fractal-derivatized or colloidal derivatized substrate. After attachment of fractal aggregates to the gold surface of the substrate, the fractal-derivatized substrate can be washed to remove unbound colloids. For example, for gold fractal aggregates, it can be desirable to wash the substrate with a solution containing an acid. In certain embodiments, it can be desirable to use nitric acid, and in other embodiments, it can be desirable to use concentrated nitric acid for a period of several hours at a temperature above the freezing point of the acid solution, up to the melting temperature of the fractal aggregates. In other embodiments, other acids can be used, such as HC1, sulfuric acid, acetic acid or other acid. Furthermore the conditions of the washing can be determined by methods known in the art. In certain embodiments, it can be desirable to use a mixture of an acid and an organic solvent, such as acetone to remove materials soluble in such solvents. The types of acids and organic solvents can be selected depending on the types of reagents or contaminants present in the solutions used to prepare the fractal aggregates.

C. Aggregation of Particles to Form Nanoparticle Structures According to other embodiments of this invention, several methods can be used to form particle structures. It is known that metal colloids can be deposited onto surfaces, and when aggregated can form fractal structures having a fractal dimension of about 1.8. Safonov et al., Spectral Dependence of Selective Photomodification in Fractal Aggregates of Colloidal Particles, Physical Review Letters 80(5V 1102-1105 (1998) incorporated herein fully by reference. Figure 1 depicts a particle structure suitable for use with the methods of this invention. The particles are arranged in a scale-invariant fashion, which promotes the formation of resonance domains upon illumination by laser light. In addition to fractal structures, ordered non-fractal structures and random structures can be generated. These different types of structures can have desirable properties for enhancing signals associated with detection of analytes using electromagnetic radiation. To make ordered non- fractal structures, one can use, for example, chemical linkers having different lengths sequentially as described in more detail below. In addition, using linkers of the same size, one can generate ordered structures, which can be useful for certain applications.

In certain embodiments of this invention, particles can be attached together to form structures having resonance properties. In general, it can be desirable to have the particles being spheres, ellipsoids, or rods. For ellipsoidal particles, it can be desirable for the particles to have a long axis (x), another axis (y) and a third axis (z). In general, it can be desirable to have x be from about 0.05 to about 1 times the wavelength (λ) of the incident electromagnetic radiation to be used. For rods, it can be desirable for x to be less than about 4 λ, alternatively, less than about 3 λ, alternatively less than about 2 λ, in other embodiments, less than about lλ, and in yet other embodiments, less than about ^lΛ λ. The ends of the rods can be either flat, tapered, oblong, or have other shape that can promote resonance. For two particle structures, it can be desirable for the particle pair to have an x dimension to be less than about 4 λ, alternatively, less than about 3 λ, alternatively less than about 2 λ, in other embodiments, less than about lλ, and in yet other embodiments, less than about Vi λ. For two-dimensional structures, pairs of particles, rods, rods plus particles together can be used. The arrangement of these elements can be randomly distributed, or can have a distribution density that is dependent upon the scale of observation in a non-linear fashion. In other embodiments, rods can be linked together end-to end to form long structures that can provide enhanced resonance properties.

For three-dimensional structures, one can use regular nested particles, or chemical arrays of particles, associated either by chemical linkers in a fractal structure or in ordered, nested arrays. In yet other embodiments, of third-order structures, a suspension of particles can be desirable. In certain of these embodiments, the suspended particles can have dimensions in the range of about Vi λ to about 1 millimeter (mm).

Using the strategies of this invention, a researcher or developer can satisfy many needs, including, but not limited to selecting the absorbance of electromagnetic radiation by particle elements, the nature of the surface selected, the number of resonance domains, the resonance properties, the wavelengths of electromagnetic radiation showing resonance enhancement, the porosity of the particle structures, and the overall structure of the particle structures, including, but not limited to the fractal dimensions of the structure(s).

1. Photoaggregation

Photoaggregation can be used to generate particle structures that have properties which can be desirable for use in Raman spectroscopy.

Irradiation of fractal metal nanocomposites by a laser pulse with an energy above a certain threshold leads to selective photomodification, a process that can result in the formation of "dichroic holes" in the absorption spectrum near the laser wavelength (Safonov et al., Physical Review Letters 80(5): 1102- 1105 (1998), incorporated herein fully by reference). Selective photomodification of the geometrical structure can be observed for both silver and gold colloids, polymers doped with metal aggregates, and films produced by laser evaporation of metal targets.

One theory for the formation of selective photomodification is that the localization of optical excitations in fractal structures are prevalent in random nanocomposites. According to this theory, the localization of selective photomodification in fractals can arise because of the scale-invariant distribution of highly polarizable particles (monomers). As a result, small groups of particles having different local configurations can interact with the incident light independently of one another, and can resonate at different frequencies, generating different domains, called herein "optical modes." According to the same theory, optical modes formed by the interactions between monomers in fractal are localized in domains that can be smaller than the optical wavelength of the incident light and smaller than the size of the clusters of particles in the colloid. The frequencies of the optical modes can span a spectral range broader than the absorption bandwidth of the monomers associated with plasmon resonance at the surface. However, other theories may account for the effects of photomodification of fractal structures, and this invention is not limited to any particular theory for operability.

Photomodification of silver fractal aggregates can occur within domains as small as about 24 x 24 x 48 nm³ (Safonov et al., Physical Review Letters 80(5):1102-1105 (1998), incorporated herein fully by reference). The energy absorbed by the fractal medium can be localized in a progressively smaller number of monomers as the laser wavelength is increased. As the energy absorbed into the resonant domains increases, the temperature at those locations can increase. At a power of 11 mJ/cm², light having a wavelength of 550 nm can produce a temperature of about 600 K (Safonov et al., Physical Review Letters 80T5 1102-1105 (1998), incorporated herein fully by reference). At this temperature, which is about one-half the melting temperature of silver, sintering of the colloids can occur (Safonov et al., Id.) incorporated herein fully by reference), thereby forming stable fractal nanocomposites.

As used in this invention, photoaggregation can be accomplished by exposing a metal colloid on a surface to pulses of incident light having a wavelengths in the range of about 400 nm to about 2000 nm. In alternative embodiments, the wavelength can be in the range of about 450 nm to about 1079 nm. The intensity of the incident light can be in the range of about 5 mJ/cm² to about 20 mJ/cm². In an alternative embodiment, the incident light can have a wavelength of 1079 nm at an intensity of 11 mJ/cm².

Fractal aggregates that are especially useful for the present invention can be made from metal particles having dimensions in the range of about 10 nm to about 100 nm in diameter, and in alternative embodiments, about 50 nm in diameter. A typical fractal structure of this invention is composed of up to about 1000 particles, and an area of the aggregate typically used for large-scale arrays can have a size of about 100 μm x 100 μm.

Figure 2 depicts a particle structure that have been photoaggregated and that are suitable for use with the methods of this invention. Local areas of fusion of the metal particles can be observed (circles).

2. Chemically Directed Synthesis of Particle Structures

In certain embodiments of this invention, particle structures can be made using chemical methods. First, metal particles can be either made according to methods described above, or alternatively can be purchased from commercial suppliers (NanoGram Inc., Fremont, California). Second, the particles can be joined together to form first-order structures, for example, pairs of particles. Then, the first-order structures can be joined together to form second-order structures, for example, pairs of particle pairs. Finally, third-order fractal structures can be made by joining second-order structures together.

In alternative embodiments of this invention, the formation of a fractal array of metal particles can be carried out using chemical methods. Once metal colloid particles have been manufactured, each particle can be attached to a linker molecule via a thiol or other type of suitable chemical bond. The linker molecules then can be attached to one another to link adjacent colloid particles together. The distance between the particles is a function of the total lengths of the linker molecules. It can be desired to select a stoichiometric ratio of particles to linker molecules. If too few linker molecules are used, then the array of particles will be too loose or may not form at all. Conversely, if the ratio of linker molecules to particles is too high, the array may become too tight, and may even tend to form crystalline structures, which are not random, and therefore will not tend to promote surface enhanced Raman scattering. In general, it can be desirable to perform the linking procedure sequentially, wherein the first step comprises adding linker molecules to individual particles under conditions that do not permit cross-linking of particles together. By way of example only, such a linker can comprise an oligonucleotide having a reactive group at one end only. During this first step, the reactive end of the oligonucleotide can bind with a metal particle, thereby forming a first particle- linker species, and having a free end of the linker. The ratio of linker molecules to particles can be selected, depending on the number of linker molecules are to be attached to the particle. A second linker can be attached to another group of particles in a different reaction chamber, thereby resulting in a second linker-particle species, again with the linker having a free end. After those reactions have progressed, the different linker-particle species can be mixed together and the linkers can attach together to form "particle pairs" joined by the linker molecules.

In other embodiments, nucleic acids can be used as linkers, based upon the ability of DNA to form hybrids with nucleic acids comprising complementary sequences. DNA ligases or other mechanisms can be used to join the linkers together to form a complete linker between metal particles.

After the pairs of particles are formed, additional linkers can be attached to the particle pairs, and the process can be repeated to form "pairs of particle pairs." Subsequently, the process can be repeated until 3 or more orders of particle structures are formed. Under these conditions, one can manufacture structures having any desired porosity. In general, the size of the nanoscale structures should have average dimensions in the range of about 20 nm to about 500 nm. In alternative embodiments, the dimensions can be in the range of about 50 nm to about 300 nm, and in other embodiments, in the range of about 100 to about 200 nm, and in yet other embodiments, about 150 nm.

In other embodiments of this invention, the linking can be carried out using an aryl di-thiol or di-isonitrile molecules. Alternatively one can use any active moiety that can be used to attach the linker to the metal particle. It can be desirable to use the above types of aryl linkers with nucleic acid or other types of linker molecules. The linker can have a central area having ethylbenzene moieties, where n is a number between 1 and about 10,000.

In general, the ratio of length for each subsequent pairs of linkers can be in the range of about 2 to about 20. Alternatively, the ratios of lengths of subsequent pairs of linkers can be in the range of about 3 to about 10, and in other embodiments, about 5. In certain other embodiments, the ratio of linker lengths in successive orders can be non-constant, thus resulting in the manufacture of an ordered, non- fractal structure. For example, for a three-order manufacturing process, it can be desirable for the ration of LI :L2:L3 to be in the range of about 1 :2:4. Alternatively, the ratio can be about 1:5:25, and in yet other embodiments, the ratio can be about 1 :20:400. In other embodiments, the ratio between LI and L2 and from L2 to L3 need not be the same. Thus, in certain embodiments the ration of LI :L2:L3 can be 1:3:20, or alternatively, 1:20:40.

D. Manufacture of Suspensions of Nanoparticle Structures

In certain other embodiments of this invention, suspensions of fractal particle associates (fractal associates) can be used, for example, to provide a structure in solution that can bind or retain analytes for detection using methods of this invention. The size of fractal particle associates can be in the range of from hundreds of nanometers to mm dimensions. The fractal associates can comprise a number of particles arranged by means of chemical linkers. The number of particles per fractal associate can be as few as about 100 particles, or alternatively, thousands can be used to form a fractal associate. By increasing the number of particles in a fractal associate, the increase in the void size increases by a greater proportion.

In another series of embodiments of this invention, nested fractal structures are provided. Nested fractal structure, for example, comprises a core of a large particle, surrounded by a "halo" of smaller particles, and each of the smaller particles is surrounded by a "halo" of even smaller particles. Nested fractal structures can be especially useful for generation of essentially uniform fractal surfaces for enhanced analyte detection. It can be desirable to include large excesses of smaller particles compared to larger particles for each successive step. For example, it can be desirable to have excess of smaller particles in the range of about 10 to about 1000 times the number of larger particles. Alternatively, it can be desirable to have an excess of smaller particles of between 10 and 100 times the number of larger particles, and in other embodiments, it can be desirable to have smaller particles in excess of about 10 times the number of larger particles.

E. Manufacture of Nanoparticle Structures Using Surfaces

Nanoparticle structures of this invention can be made in general, by first forming a colloidal suspension of nanoparticles. Regardless of how a suspension of nanoparticles is provided, the following methods for manufacturing nanoparticle structures using surfaces can be advantageous. To such a colloidal suspension containing dissolved gas, a solute can be added. Adding a solute can cause bubbles of the gas to form in the suspension. The nanoparticles can partition into the bubbles on the gas/liquid interface of the bubble. If the nanoparticles are hydrophobic, they tend to associate with the interior surface of the bubble. The composition of gas in a suspension can be controlled using many methods known in the art. For example, the suspension can be degassed and then a controlled composition of gas can be introduced into the suspension by a frit, bubbler, exposure of a thin layer of suspension in a gaseous environment, or other means known in the art. By way of example, suitable gases include Helium (He), oxygen (O₂), CO₂, air, N₂, Ar or any other gas that does not adversely react with the components in the solution.

Once the suspension has a desired type and amount of gas, bubbles can be formed using a variety of means, including solute ebullition, cavitation, or other methods known. It can be convenient to use salt ebullition because salts are used to cause aggregation of nanoparticles. Alternatively, salts can be added in sufficient amount to cause aggregation, and then another solute can be added to form bubbles. In those embodiments using salt as a solute to form bubbles, NaCl, KC1, Ca^, Al⁺⁺⁺, Fe^, Fe^, or other anion along with any anion, such as SO₄ ^2", CO₃ ^2", HCO₃ ^"citrate or other organic anions. The concentrations of solutes can vary depending upon the charge. Thus, trivalent cations can be present in concentrations in the range of about 0.1 μM to about 5 M (saturating concentration). Di-valent cations can be present in concentrations of about 1 μm to saturating concentration, and monovalent cations can be present at a concentration of about 2 μm to saturation. In certain embodiments, NaCl in a concentration of from about 0.5 M to about 1M can be used. The salt can be added either in solid form as crystals, or it can be added as a solution of pre- dissolved salts in medium. It can be readily appreciated that routine experimentation can be performed to determine optimal concentrations of solutes.

Non-salt solutes can be used, and include by way of example, sugars, amino acids, peptides, complex carbohydrates, or other non-reactive substances, so long as a sufficient osmotic pressure is provided by the solute to cause bubble formation.

Temperature changes can also lead to bubble formation. It is well known that increasing temperature lowers the partial pressure of dissolved gases in solutions. Thus, a suspension of nanoparticles equilibrated at room temperature (about 22°C) can generate bubbles upon heating. The exact temperatures desired can be determined using routine experimentation.

The diameters of bubbles suitable for manufacture of nanoparticle structures of this invention can be from 50 nanometers (nm) to about 1 mm, alternatively from about 100 nm to about 1 micrometer (μm), about 200 nm to about 800nm, about 300 to about 400 nm, and in other embodiments, about 500 nm in diameter. It can also be appreciated that the use of a variety of different bubble sizes can lead to formation of different sized nanoparticle structures, which can be especially useful for broad band absorption, emission and enhancing effects on Raman spectroscopy. Bubble size can be controlled by regulating the surface tension of the liquid in the suspension. For example, the use of surfactants (e.g., non-ionic detergents, such as Tween 80), ionic detergents can be used to alter the surface tension of aqueous solutions, and other liquids, such as alcohols (ethanol, methanol, butanol, propanol, and the like, as well as ketones (e.g., acetone, butanone, and the like) can be used. It can be appreciated that a variety of single solvents or mixtures of solvents can be used.

Likewise, the life-time of bubbles can be varied. In general, desirable lifetimes are from about 1 millisecond (msec) to about 10 hours, alternatively from about 1 sec to about 1 hour, and in other embodiments, about 30 minutes. Bubble lifetimes should be sufficiently long to permit a desired number of nanoparticles to associate with the bubble. It can be appreciated that if one wishes to make nanoparticle structures having higher nanoparticle density, one can use the following variables among others, bubbles with longer lifetimes, higher nanoparticle concentrations, or higher partition coefficients of nanoparticle for the bubbles.

Likewise, bubble density (expressed as a ratio of bubbles to nanoparticles) can be in the range of about 1:20,000, alternatively about 1000:1, or about 100:1, 10:1 or about 1 :1. In some embodiments of this invention, depicted in Figure la, a surface

100 is prepared in which a substrate 104 has nanoparticle structures 108 thereon. This structure of relatively evenly distributed nanoparticle structures can be obtained by placing the substrate 104 in a flask containing a colloidal suspension. In Figure la, no salt was added and few bubbles formed. The resulting nanoparticle structures were small, as shown in Figure 2 below (Example 4).

Alternatively, salt is added to the suspension and bubbles can be formed, resulting in the formation of nanoparticle aggregates on the surface of the bubbles, which then precipitated to the bottom of the flask and onto the surface of the substrate.

Alternatively, the colloidal solution can be initially degassed using vacuum or boiling to remove gas. Gas can then be introduced into the solution using a variety of means known in the art. These include the use of a frit, bubbler, or through the formation of gas by chemical reactions as described above.

Unless otherwise indicated, like reference numerals refer to the same elements, regardless of the figure in which they are described. Figure lb depicts an alternative embodiment 102 in which a colloidal solution of nanoparticles is placed on a surface of substrate 104 and then bubbles are formed, and the nanoparticle structures form and are deposited in a characteristic "X-shaped" pattern on the surface of the substrate. The exact mechanism for the formation of this characteristic shape are not well understood, but according to one theory, the shape of the precipitated nanoparticle structures depends upon the geometry of the surface of the colloidal suspension on the substrate, which may alter the ways in which bubbles in the solution move up and down within the suspension.

The presence of such an X-shaped region, which is visible to the unaided eye, can be useful to visualize the nanoparticles precipitated on the surface, and therefore can be an indicator of the progress of reactions.

Example 4 Nanoparticle Structures Made Without Bubbles To manufacture nanoparticle structures in the absence of bubbles, we prepared a colloidal solution nanoparticles as described above, boiled the solution for 2 hours, and then added sufficient salt to cause aggregation only. No bubbles formed. Figure 2 depicts an optical micrograph of the surface depicted in Figure la prepared as described. Nanoparticle structures are present in a relatively random pattern and the nanoparticle structures were small.

Figure 3 depicts a Raman spectrum of R-6G on a surface containing nanoparticle structures prepared without bubbles as shown in Figures la and 2. Note the presence of two Raman spectral features A and B.

Example 5 Nanoparticle Structures Made With Bubbles To determine the effects of bubble formation on nanoparticle structure formation, we prepared a colloidal suspension of nanoparticles as described above. We then boiled the suspension for 2 hours, and then cooled the suspension on ice (0°C) for 12 hours, to permit air equilibration to occur. Then, to one part colloidal suspension saturated with air, we added 3 parts water that had been saturated with air, and then added 1 part of a solution of NaCl (4M). Under these conditions, numerous bubbles formed, and nanoparticle structures formed within the bubbles. The solution was allowed to stand for a period of time, and as nanoparticles partitioned into the bubbles, the bubbles, with nanoparticle structures therein sank to the bottom of the suspension. Subsequently, the bubbles broke free of the nanoparticle structures, resulting in nanoparticle structures on the surface of the substrate and free bubbles rising to the surface of the suspension.

Figure 4 depicts an optical micrograph of a surface of a substrate having nanoparticle structures made with a large excess of bubbles. Nanoparticle structures (dark areas) are numerous, and distributed randomly over the surface, but the nanoparticle structures themselves are fractal in nature as further depicted below in Figures 6a and 6b.

Figure 5 is a Raman spectrum of R-6G obtained on a surface having nanoparticle structures deposited using an excess of bubbles as described in Figure 4. Note that peaks A and B are located in the same wavenumber as in Figure 3, but the amplitude of the peaks are substantially higher than the corresponding peaks in Figure 3, indicating that the nanoparticle structures formed using an excess of bubbles provide higher degrees of enhancement that those formed with fewer bubbles.

Figure 6a depicts an electron micrograph of a surface of a substrate having nanoparticle structures deposited using an excess of bubbles, as described in Figures 4 and 5. The nanoparticle structures are the light areas in the picture.

Figure 6b is a higher-power view of a portion of the nanoparticle structures shown in Figure 6a, and depict the fractal nature of the nanoparticle structure.

Figure 7 depicts schematically, the formation of a bubble with nanoparticles of this invention 700. Vessel 704 contains solution 708 having surface 712, and which has nanoparticles 716 suspended therein. Bubble 720 is shown with nanoparticles 724 within the bubble associated with the inner surface. Because nanoparticles 716 are metal and are hydrophobic, they tend to partition to the interior of bubble 720. When so concentrated within bubble 720, the nanoparticles can associate with each other easily to form clusters of nanoparticles, or nanoparticle structures.

Figures 8a - 8h depict a series of steps in the manufacture of nanoparticle structures of this invention. Figure 8a depicts an embodiment 800 within a flask 804 containing medium 808 having nanoparticles 816 suspended therein.

Figure 8b depicts an embodiment of this invention 800-b in which the same vessel 804 as for Figure 8a, but also containing bubble 820.

Figure 8c depicts a situation 800-c in which nanoparticles 816 partition to the interior of bubble 820 thereby forming particles 824 associated with the bubble. The bubble having nanoparticles therein is shown as 821.

Figure 8d depicts an embodiment of this invention 800-d in which two bubbles with nanoparticles coalesce into a bubble pair 822. Figure 8e depicts an embodiment 800-e in which bubbles 821 have precipitated to the bottom of the flask 804, and in which some nanoparticle structures 828 have been released from bubbles 820 which float toward the top of the solution 808.

Figure 8f depicts an embodiment of this invention 800-f in which a plurality of bubbles containing nanoparticles 821 have precipitated to the bottom of the flask, forming a coalesced bubble structure 822. Note that bubbles can be of varying sizes.

Figure 8g depicts an embodiment of this invention 800-g in which two bubbles 823, each having nanoparticles had precipitated to the bottom of the bubble. These bubbles 823 can release nanoparticles at the bottom of the flask, producing nanoparticle structures.

Figure 8h depicts an embodiment of this invention 802 in which a cluster of nanoparticle structures 828 has been deposited on the surface 832 of the substrate. Figures 9a to 9c depict steps in the formation of an embodiment 900 of this invention, in which a flask contains a substrate 904 with a layer of gold metal 908 thereon. Solution 914 having suspended nanoparticles 916 is placed on the surface 912 of substrate 904. The surface 915 of the solution is indicated. Figure 9b depicts the step of formation of bubbles 920 in the solution

914. Nanoparticles 916 partition into bubbles 920 forming partitioned nanoparticles 924 within bubbles 921.

Figure 9c depicts the situation in which clusters of nanoparticle structures 928 have been deposited on the upper surface 912 of gold layer 908. Bubbles 932 released from the nanoparticle structures 928 float toward the surface 915 of solution 914.

Centrifugation may be used to facilitate the process. Figure 10a depicts embodiment 1000 of this invention in which solution

1004 has a free nanoparticles 1016, free receptors, 1018, nanoparticles with attached receptors 1017 and bubble 1020. Some of the nanoparticles 1017 have partitioned to the interior of bubble 1020.

Figure 10b depicts an alternative embodiment of this invention in which solution 1004 contains linked nanoparticle pairs 1019 and larger linked aggregates 1021. Some of the nanoparticle pairs 1019 and the larger aggregates 1021 are shown partitioned into the bubble 1020.

INDUSTRIAL APPLICABILITY The methods of this invention produce nanoparticle structures with improved reproducibility and improved enhancing properties, making them suitable for use in detection of analytes by Raman spectroscopy and for use in radiation absorption and emission devices.

Claims

We Claim:

1. A method for manufacturing a nanoparticle structure, comprising the steps of: (a) providing a solution containing at least two nanoparticles;

(b) providing at least one bubble in said solution;

(c) permitting said nanoparticles to aggregate.

2. The method of claim 1, further comprising the step of permitting the aggregated nanoparticles to deposit on a substrate.

3. The method of claim 1, wherein the step of providing at least one bubble comprises adding a solute to said solution.

4. The method of claim 3, wherein said solute is a salt.

5. The method of claim 3, wherein said solute is a polar solute.

6. The methods of claim 3, wherein said solute is a non-polar solute.

7. A method for manufacturing a nanoparticle structure, comprising the steps of:

(a) providing a solution containing at least two nanoparticles;

(b) providing at least one surface in said solution; (c) permitting said nanoparticles to aggregate on said surface

8. The method of claim 7, wherein said surface is selected from the group consisting of emulsions, beads and films.

9. The method of claim 7, wherein said surface is hydrophobic.

10. The method of claim 7, wherein said surface is hydrophilic.

11. The method of claim 7, wherein said surface is charged.

12. A method for detecting the presence of an analyte, comprising:

(a) providing a substrate having at least one nanoparticle structure thereon; said nanoparticle structure made according to the steps of: (i) providing a solution containing at least two nanoparticles;

(ii) providing at least one surface in said solution; and (iii) permitting said nanoparticles to aggregate, forming said nanoparticle structure;

(b) applying said analyte on said substrate near said at least one nanoparticle structure; and

(c) detecting the presence of an enhanced spectral feature of said analyte.

13. The method of claim 12, wherein said enhanced spectral feature is a Raman spectral feature.

14. The method of claim 12, wherein said surface is a surface of a bubble.