J. Phys. Chem. B 2000, 104, 9795-9810
9795
Preparation, Microscopy, and Flow Cytometry with Excitation into Surface Plasmon Resonance Bands of Gold or Silver Nanoparticles on Aminodextran-Coated Polystyrene Beads Olavi Siiman*,† and Alexander Burshteyn‡ AdVanced Technology, Beckman Coulter, Inc., 11800 SW 147th AVenue, Miami, Florida 33196-2500, and Reagents Applications & DeVelopment, Beckman Coulter, Inc., 11800 SW 147th AVenue, Miami, Florida 33196-2500 ReceiVed: January 20, 2000; In Final Form: July 11, 2000
Spherical polystyrene latex beads of about 2.0 µm diameter were coated with islands of silver or gold metal, about 5-200 nm in diameter, by reduction of aqueous silver or gold ions in the presence of sugar-coated polystyrene latex beads. The metal islands are held on the bead surface by a polymeric sugar derivative, aminodextran, covalently bound to the polystyrene aldehyde/sulfate bead. Images of the gold or silver nanoparticle-coated polystyrene beads, obtained with an optical microscope, show that gold, silver, and uncoated polystyrene beads can be distinguished by their different colors, red-purple, black, and colorless, respectively. Also, scanning electron micrographs of coated versus uncoated polystyrene beads show a distinct granular bead surface when metal nanostructures are present versus a smooth bead surface when they are absent. Nanoparticles of gold greater than about 50 nm in diameter on polystyrene beads showed enhanced 90° or side light scatter (resonant Rayleigh scattering) with excitation at 633 nm but no enhancement (same light scatter intensity as uncoated polystyrene beads) at excitation wavelengths of 544, 488, and 458 nm. On the other hand, nanoparticles of silver greater than about 50 nm in diameter on polystyrene beads showed enhanced 90° or side light scatter with excitation wavelengths of 458, 488, 544, and 633 nm but no enhancement with 351-365 nm excitation. Amplification of elastic scatter from both silver and gold colloids was maximally achieved with structures of 50-200 nm diameter, as shown in forward versus side scatter histograms obtained by flow cytometry. Side scatter enhancements of 2- to 10-fold were observed for gold- or silver-coated polystyrene beads over uncoated polystyrene beads of the same diameter. The origin of this wavelengthdependent, light scattering amplification that was observed by flow cytometry has been identified with excitation into surface plasmon resonance bands of gold and silver nanoparticles on polystyrene latex beads.
Introduction The present CD4- or CD8-antibody-polystyrene latex beadbased analyses of targeted cell populations in whole blood rely on changes in size and shape of the cell-bead complex versus the original cell and the original bead to enumerate cell populations.1-3 A thin metallic coating on polystyrene beads of about two microns in diameter has the ability to enormously change the light scattering properties of the parent beads without changing their size or shape to any large degree. The metalcoated and uncoated polystyrene beads can ultimately be used as light scatter probes for differentiating various subpopulations of white blood cells. Together with fluorescent markers for cells, they will be able to increase the number of simultaneous probes that can be used with a single blood sample and flow cytometer to enumerate cell populations. Although our work has been disclosed as issued patents4,5 in the past five years, and was recently orally presented,6 no previous publication has appeared. Conjugation of antibody to the silver and gold nanoparticleaminodextran-polystyrene bead complexes and their applications to flow cytometric analyses of white blood cell subsets have been described in a separate article.7 * Author to whom correspondence should be addressed. E-mail:
[email protected]. † Advanced Technology, Beckman Coulter, Inc. ‡ Reagents Applications & Development, Beckman Coulter, Inc.
The area of silver/gold nanoparticle research has experienced a burst of new activity or “renaissance” in the past few years. Many new applications have sprung forth in analysis of oligonucleotides,8-9 in arrays of metal nanoparticles and surfaceenhanced Raman scattering sensing,10-15 in surface plasmon spectroscopy,16-19 in colloidal metal sensors,20-21 and in colloidal metal deposition on polystyrene microspheres.22-23 Since light scattering in the visible wavelength region is a surface phenomenon, a surface coating alone on the original beads is sufficient to change its light scattering properties. Most materials are insulators and have a real refractive index; however, conductors and semiconductors may have a large imaginary part to their refractive index, which is also dependent on the wavelength of incident light.24 Silver is the most nearly perfect conductor and silver mirrors are the best reflectors of light. Gold, however, is more chemically inert as it does not tarnish in air, and still exhibits excellent electrical conduction and light scatter properties. Both silver and gold have an extra benefit in biological cell studies in that they are also excellent anti-microbial agents.25 Thus, suspensions of silver- and goldcoated beads do not require any extraneous anti-microbial agent for preservation. Small particles of conducting metals show “plasmon resonance”,26 which is also shown by other small structures of metals such as thin films, island films, roughened electrode surfaces,
10.1021/jp000255z CCC: $19.00 © 2000 American Chemical Society Published on Web 10/03/2000
9796 J. Phys. Chem. B, Vol. 104, No. 42, 2000 and microfabricated arrays. Excitation with light into a “plasmon resonance band” can give enhanced light scatter intensities or resonant Rayleigh scattering. These “plasmon resonances” arise from oscillations of the “free” conduction electron(s) of the metal. Silver and gold each have a single outer s subshell conducting electron, and their “plasmon resonance bands” happen to be located in the visible spectral region, accessible with common laser excitation wavelengths. The bands can be located by measuring the extinction () absorption + scattering) spectra of silver- and gold-coated beads. Selective enhancements of light scatter can, then, be obtained by choosing the excitation wavelength properly to excite the “plasmon resonance band”. In the small particle limit at 0.95 mm or (H - h)2/H2 > 0.8. This showed full development of a parabolic, electroosmotic flow profile of the standard particles in the DELSA sample chamber. Other DELSA instrument parameters were chosen as follows: frequency range,
Cytometry on Metal Nanoparticle-Amdex-PS Beads
Figure 2. Electrophoretic mobility versus pH data for suspensions of aldehyde/sulfate polystyrene latex beads, uncoated or coated with 1Xaminodextran, gold nanoparticle-1X-aminodextran, and silver nanoparticle-1X-aminodextran.
500 Hz; frequency shift, 250 Hz; current, 0.7 mA unless otherwise noted; on time, 2.0 s; off time, 0.5 s. The pH dependence of the mobilities of gold-1X-AmdexPS beads and their parent beads, raw PS aldehyde/sulfate and 1X-Amdex-PS beads, is presented in Figure 2. Prior to electrophoretic measurements, the raw PS and 1X-Amdex-PS beads were suspended in distilled water and the gold-1XAmdex-PS beads, in 0.010 M aqueous sodium citrate solution. For measurements, all beads were diluted from either 4 or 1% w/v solids to 4 × 10-4 w/v solids with 0.010 M aqueous potassium nitrate solution. The pH of the diluted bead suspensions was adjusted with 0.10 M aqueous potassium hydroxide or 0.10 M aqueous nitric acid solutions. Conductivities of the bead suspensions ranged from 1.3 to 1.6 mS/cm. As shown in Figure 2, 1X-Amdex-coated polystyrene aldehyde/sulfate latex beads showed nearly constant mobility of about -1 cm2 V-1 s-1 in the pH 7-10 range of measurements, reflecting the presence of positively charged protonated amino groups that have replaced some neutral aldehyde groups to counter the preexisting negatively charged sulfate groups on the surface of the latex beads. Note that the raw polystyrene aldehyde/sulfate latex beads were originally negatively charged and showed in Figure 2a very high negative mobility of -6 to -7 cm2 V-1 s-1 in the pH 7-10 range. After gold colloid was introduced onto the 1X-Amdex-PS beads, the steep drop in mobility of the resulting particles from about +2 to -4 cm2 V-1 s-1 in the pH 4-6 range showed the presence of adsorbed citrate on gold1X-Amdex-PS beads even after dilution of the beads with aqueous sodium nitrate solution. The acid dissociation constants of citric acid as reflected in the stepwise pKa values of 3.14, 4.77, and 6.39 at 20° C, albeit modified for adsorbed citrate, occur in the same pH region as the large drop in mobilities. In addition, an artifact at about +0.3 cm2 V-1 s-1 was present throughout the pH 4-10 range in mobility profiles of 1XAmdex-PS and gold-1X-Amdex-PS beads, especially at low currents (applied electric field)
