Source: UNIV OF WISCONSIN submitted to NRP
COLLOIDAL METAL PARTICLES FOR HIGH RESOLUTION BIOLOGICAL LABELING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0187000
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2000
Project End Date
Sep 30, 2004
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
ANIMAL SCIENCES
Non Technical Summary
An understanding of cell function requires knowledge of the molecular organization within the cell. Cells are little machines and in order to understand how the machine works we need to know how the parts fit together. This project seeks to develop effective labeling strategies that will allow simultaneous visualization of multiple molecular species so the way in which they fit together in the cell can be determined.
Animal Health Component
15%
Research Effort Categories
Basic
85%
Applied
15%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20670101030100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
7010 - Biological Cell Systems;

Field Of Science
1030 - Cellular biology;
Goals / Objectives
Objectives: The proposed studies are designed to develop novel methodologies for labeling of biological specimens at subcellular, molecular, and submolecular levels of resolution. The new technology will have the potential to substantially increase the number of labels that can be used simultaneously and will offer greater flexibility in cell labeling compared to existing techniques. The organization of molecules and macromolecular assemblies within the cell is a key factor in cell function. In order to help determine intracellular organization considerable effort has been directed toward the development of fluorescent probes having differing emission wavelengths. In this way specific cellular elements can be selectively stained with a dye of a unique color. Thus the relationships of different molecules and structures, stained by different colored fluorescent dyes, can be viewed simultaneously and spatial relationships determined. This approach is effective in fluorescent light or confocal fluorescent light (photon) based imaging systems and is limited to the resolution attainable with such systems. Determining relationships at molecular or submolecular levels of spatial resolution requires instrumentation capable of considerably higher levels of spatial resolution. Principal among these are electron based imaging systems. Until recently there has been no ability to easily recognize different wavelength (in essence different "color") electrons. Hence simultaneous labeling of multiple molecular species in electron microscopes has relied primarily on spherical colloidal gold nanoparticles of differing sizes. However the number of particles providing resolution in the molecular size range is generally limited to one or two; then particles become too large. The ability to use labels that are all of the same very small size but can be distinguished from on another based on properties other than size would be of considerable benefit to studies investigating molecular level organization within the cell. The specific aims of this study are as follows: Aim 1. To synthesize colloidal nanoparticles of uniform size from Au and other metals including Ag, Pd, Pt, Rh, Ru, Mo, Cd, Ni, Cu, and various of the lanthanides. Aim 2. To use electron energy loss spectroscopy to distinguish between particles of different metal composition. Aim 3. To synthesize colloidal metal particles of different, unique shapes which can be identified in conventional transmission electron microscopy, scanning electron microscopy, and scanned force microscopy. Aim 4. To conjugate antibodies, and other ligands or small active fragments of antibodies and ligands to colloidal particles in preparation for subsequent cell labeling.
Project Methods
Approach Aim 1. To synthesize colloidal nanoparticles of uniform size from Au and other metals. Colloidal phase preparations of Au, and other metals, will be synthesized via the reduction of a metal salt. The metal initially forms crystalline nuclei around which condenses additional reduced metal. Pursuant to this aim are the following goals: (1) we will attempt to form colloids of Ag, Cd, Cu, Mo, Ni, Pd, Pt, Rh, Ru and the lanthanides by using the citrate, tannic acid, white phosphorus, sodium borohydride, and thiocyanate methods used in the synthesis of cAu; (2) several other reduction methods that are specific to certain of the metals of interest will be used; and (3) we will attempt to control particle size and size distribution by altering the reducing agent and metal salt concentrations, along with reaction temperature, pH, and the presence of protecting polymers such as sodium polyacrylate and polyvinyl alcohol. Aim 2. To use electron energy loss spectroscopy (EELS) to distinguish between metal particles. EELS enables the detection of small numbers of atoms of a single element within a specimen. The metals under consideration as potential colloidal labels have been chosen based in part upon the unique energy loss spectrum of each. To date we have used EELS to detect cNi, cCu, cPt, cRh, cAu, cAg, and cPd particles. Current studies will be directed as follows: (1) techniques to identify particles synthesized from the various metals will be refined to permit particles in the 2nm to 30nm size range to be dentified; (2) studies will be pursued to enable the discrimination between individual metal particles in a mixture of two or more colloids; and (3) strategies will be devised to allow particle discrimination in cases where energy loss overlap occurs. Aim 3. To synthesize colloidal metal particles of different unique shapes. In this study we will pursue the synthesis of nanoparticles having unique shapes. The metallic particles will retain the properties which facilitate detection in the TEM, SEM, SFM and AFM but additionally the different shapes will permit differentiation of similarly sized particles attached to different identifier molecules. Studies will be directed toward the following: (1) production of nanoparticles of cAu in uniform small sizes but with different morphologies including spherical, polygonal, and irregular or lobate, and (2) production of nanoparticles of Pt, Pd, and Rh with unique polygonal and rough or lobate morphologies. Aim 4. To conjugate antibodies and other ligands to colloidal particles for cell labeling. The utility of any of the colloidal metal nanoparticles for labeling of biological systems requires that identifier molecules attach tightly to the surface and remain active. The binding of molecular species to metallic surfaces via hydrophobic interactions provides a virtually irreversible conjugation that, in theory, and generally in practice, retains the activity of the conjugated species. Our specific goals toward this aim are (1) to develop methodologies for conjugating proteins to the other colloidal metals, and (2) to use these conjugates to label model cellular systems.

Progress 10/01/00 to 09/30/04

Outputs
The work here dealt specifically with the development of synthetic pathways and detection systems for a variety of nanoparticles principally for cell labeling purposes. The project was successful in meeting the goals originally put forward. A wide range of reducing agents and conditions were investigated. Nano-particles were generated in the molecular and sub-molecular size range (3nm to 30nm) having specific, narrowly defined, sizes, unique shapes, and different elemental compostions. For the first time this allows for simultaneous, in situ, labeling and co-localization of multiple (currently in the range of 5 to 7)different molecular species with both the scanning electron and transmission electron microscope. Particle shapes (spheres, popcorn, pyramid, square/trapezoid) can be clearly recognized in both high resolution field emission SEM and in TEM. Particles can be differentitated on the basis of composition( Au, Ag, Pd, Pt, Rh, Fe) via high resolution EDX-STEM and by energy filtering (EELS)TEM. Successful methodology for conjugation of identifier species such as the Fab fragment of antibody molecules or the active fragment of ligands was also developed. Most recently core-shell particles have been synthesized. Cores of spectrally distinct elements are surrounded by a thin but continuous layer of gold. Cores, of different elements, provide unique labels while the gold shell provides a stable, non-toxic surface which can be readily conjugated to identifier species such as antibody or ligand. The combined use of the different, unique particle labels, permits high resolution qualitative and quantitative analysis of molecule numbers and organization at the level of cell ultrastructure. The individual particles in the 10nm and greater range are detectable and trackable using interference base light microscopy so that labeling and tracking of specific molecular species is possible in living cells which can then be fixed and prepared for ultrastructural level studies. Tracking in whole animals or plants is also possible. Neutron activation can be used to measure bulk concentration of the particles at parts per trillion levels. This facilitates studies on distribution of particle-conjugated or labeled species in whole animals and in environmental studies. Samples with particles identified by neutron activation can subsequently be examined by electron microscopy to identify the exact tissue, cellular, or subcellular location of molecular species of interest.

Impacts
The principal impact of the nanoparticles discovered in this study will be the ability of investigators to specifically and simultanously label different individual molecules and even specific parts of molecules for high resolution visualization in scanning and transmisson electron microscopes. The particles also have a variety of other potential uses from more effective drug delivery agents in animals and humans as anti-cancer agents and as tracking markers for molecules and organisms in living animals and in environmental studies.

Publications

  • Meyer, D.A. and R.M. Albrecht. Size Selective Synthesis of Colloidal Platinum Nanoparticles for use as High Resolution EM Labels. Micros.Microanal.8 (Suppl.2) pp124-125, 2002.
  • Albrecht, R.M. and D.A. Meyer. All that Glitters is not Gold: Approaches to Labeling for EM. Micros.Microanal. 8 (Suppl.2) pp194-195, 2002.
  • Meyer, D.A. and R.M. Albrecht. Multiple Labeling for EM Colloidal Nanoparticles of Different Shapes and Elemental Compositions. Proceedings 15th Int. Congress on Electron Microscopy, Vol 2, pp53-54, 2002.
  • R.M. Albrecht and D.A. Meyer. All that glitters is not gold: Approaches to labeling for EM. Microscopy Today, Issue #02-3, pp 24-26, 2002.
  • Meyer, D.A. and R.M. Albrecht. Sodium Ascorbate Method for the Synthesis of Colloidal Palladium Particles of Different Sizes. Micros. Microanal. 9 (Suppl 3) pp 1190-1191, 2003.
  • Albrecht, R.M. Correlative Microscopy: One Specimen, Many Microscopies, Lots of Info. Micros. and Microanal. 9 (Suppl 3) pp 1552-1553, 2003.
  • Kandela, I.K., D.A. Meyer, P.E. Oshel, E. Rosa-Molinar, and R.M. Albrecht. Fluorescence Quenching by Colloidal Heavy Metals: Implications for Correlative Fluorescence and Electron Microscopic Studies. Micros. Microanal. 9 (Suppl. 3) pp 1194-1195, 2003.
  • Malecki, M., R.J. Casper, R.K. Noll, D.A. Meyer, and R.M. Albrecht. Molecular Markers of Different Elemental Compositions Bioengineered for Simultaneous Detection and Localization of Multiple Biomolecules with Energy and Wavelength Dispersive Spectroscopic Imaging. Micros. Microanal. 9 (Suppl 3) pp 1224-1225, 2003.
  • Bleher, R., D.A. Meyer, and R.M. Albrecht. Multiple Labeling for EM using Colloidal Particles of Gold, Palladium, and Platinum as Markers. Micros. & Microanaly, 10 (suppl 2):158-159. 2004.
  • Albrecht, R.M. and D. A. Meyer. The Potential for Correlative and Multiple Labeling With Colloidal Particles of differing Shapes. Microscopy and Microanalysis 6, Supplement 2: 318-319, 2000
  • Meyer, D.A. and R.M. Albrecht. Identification of Multiple Colloidal Labels of Various Metallic Composition by Means of Electron Energy Loss Spectroscopy.Microscopy and Microanalysis, 6:322-323 (2000)
  • Meyer DA and Albrecht RM. Multiple Labeling for EM Using Colloidal Particles of Different Shapes and Elemental Compostions. Proceedings of the Annual Symposium of the Iowa Microscopy Society, 2001. 9/20/2001 University of Iowa, Iowa City IA.
  • Meyer, D.A. and R.M. Albrecht. The Feasibility of High Resolution, Multiple Labeling Using Colloidal Particles of Similar Size but Different Shapes and Elemental Compositions. Microscopy and Microanalysis 7:1032-1033.(2001)


Progress 01/01/03 to 12/31/03

Outputs
We have continued to refine a core-shell method for the synthesis of colloidal metal particles of various elements, particularly Au, Pd, and Pt, having discreet diameters and narrow size distributions. Small core particles are initially formed by the reduction of the metal salt with any of a variety of agents which promote rapid particle nucleation. These core particles are subsequently used as nuclei or seeds around which additional metal is condensed using a different reducing agent which promotes particle growth as opposed to initiating additional nucleation. Although these preparations are already monodisperse, they may be further subdivided by means of sucrose or glycerol density gradient ultracentrifugation into three or four samples each of a different particle diameter with even narrower size distributions. Additionally, the effect of the aging of ascorbic acid solutions on colloidal Pd morphology has been investigated. Studies involving neutron activation in conjunction with visible light spectroscopy have also been initiated with the goal of measuring the actual particle concentrations of various colloidal suspensions. Such data as these are essential in order to conduct future multiple labeling experiments. Labeling efficiency is dependent in part upon the concentrations of the labels that are used, and equal concentrations of different labels must be applied to the specimen in order to draw valid conclusions from comparative analysis of labeling patterns.

Impacts
Impact: The precise control with respect to the size of colloidal metal particles greatly enhances the versatility of our high resolution labeling approach. It now becomes feasible to employ tailor-made labels of equal valence in order to perform valid and reliable comparative and quantitative analyses. The availability of colloidal particles of various elemental and morphologic compositions over a broad range of different, non-overlapping sizes greatly increases the total number of labels available in the repertoire.

Publications

  • Meyer, D.A. and R.M. Albrecht. 2003. Sodium ascorbate method for the synthesis of colloidal palladium particles of different sizes. Microscopy and Microanalysis 9 (Suppl. 2):1190.
  • Malecki, M., R.J. Casper, R.K. Noll, D.A. Meyer, and R.M. Albrecht. 2003. Molecular markers of different elemental compositions bioengineered for simultaneous detection and localization of multiple biomolecules with energy and wavelength dispersive spectroscopic imaging. Microscopy and Microanalysis 9 (Suppl. 2):1188.
  • Kandela, I.K., D.A. Meyer, P.E. Oshel, E. Rosa-Molinar, and R.M. Albrecht. 2003. Fluorescence quenching by colloidal heavy metals: implications for correlative fluorescence and electron microscopy studies. Microscopy and Microanalysis 9 (Suppl. 2):1194.


Progress 01/01/02 to 12/31/02

Outputs
This past year we have concentrated on the synthesis and characterization of highly size-specific colloidal palladium, platinum, and gold particles. Particles in the range of 3nm. to 150nm. can be produced with size variabilities in the range of +/-1% by a two step process in which metal is condensed around a prefabricated core particle. This is opposed to the current methodology which seeks to produce particles of a single size via a one step process (with corresponding size varibility in the +/- 8% to 15% range). The two step process also can be used to prepare hybrid particles with one or more metals in the shell in addition to the original core metal. These particles can be easily (as well as reliably) differentiated based on size. The opportunity for the hybrid multi-element particles substantially increases the number of different particles which can be used in the labeling process. Each particle has a specific size and then within a given size there can be several particle subtypes based on the particular metal composition. We have demonstrated that the particles of 3nm. and larger having different compositions can be identified by EELS using an energy filtering TEM or via a high resolution EDX in SEM. This substantially increases the number of molecular and submolecular species that can be simultaneously identified and localized for cell structure/function analyses.

Impacts
Development of the highly uniform controlled size/composition colloidal particles will provide additional opportunities for multiple co-localization studies at the molecular level of resolution. They also have potential use in transepithelial absorption studies which deal with mechanisms of absorption and subsequent localization of absorbed molecular species, colloids, and aggregates based on size.

Publications

  • Meyer, D.A. and R.M. Albrecht. Size Selective Synthesis of Colloidal Platinum Nanoparticles for use as High Resolution EM Labels. Micros.Microanal.8 (Suppl.2) pp124-125, 2002.
  • Albrecht, R.M. and D.A. Meyer. All that Glitters is not Gold: Approaches to Labeling for EM. Micros.Microanal. 8 (Suppl.2) pp194-195, 2002.
  • Meyer, D.A. and R.M. Albrecht. Multiple Labeling for EM: Colloidal Nanoparticles of Different Shapes and Elemental Compositions. Proceedings 15th Int. Congress on Electron Microscopy, Vol 2, pp53-54, 2002.


Progress 01/01/01 to 12/31/01

Outputs
The work involves the generation of monodisperse colloidal metal particles of relatively biologically inert metals and metal combinations. The particle sizes are in the 2nm to 100nm size range. When conjujgated to biologically active molecular species they have a variety of potential uses. These include: as labels to identify, localize, and track molecules in biological systems; as markers for correlative microscoopic studies on living and fixed tissue; and as carriers for pharmacologically active compounds, antigens, or antibodies. Using single label types in conjunction with electon microscopy and instrumental neutron activation analysis we have worked with investigators on the fate of follicular fluid in horses, development of bone microcracks in dogs and horses, the distribution and binding of LPS/e.coli endotoxin in pigs and in vitro cell culture, and a number of other studies. The utility of the technology will be greatly expanded if multiple labels can be used simultaneously. To this end we are proceeding with the synthesis of particles of different shapes (other than spheres) and labels of different metal composition. (Bulk analysis via neutron activation and microscopic analysis via energy filtering TEM permit a clear differentiation of the different metal particles.) Particle utility depends on 1. having the particles and 2. being able to conjugate the particles to active molecular species. This past year we have concentrated on the synthesis of particles. We have developed methodology to produce spherical and star shaped particles in the 3 to 50nm size range from several metals including gold, palladium,platinum, silver, and rhodium. We have also been successful in producing colloidal palladium/molybdenum and silver/molybdenum particles. The colloidal alloys greatly increase the number of distinct particles that can be used. Also alloys can utilize metals that don't normally form stable aqueous colloids and which have energy loss spectra not seen with the other single element particles. In additon we have demonstrated the simultaneous labeling of 2 distinct surface integrins on platelet surfaces with ligand and antibody conjugates of colloidal gold and colloidal palladium. We also reported the use of single particles of different sizes to demonstrate mechanisms of absorption. This utilized high resolution electron microscopy and neutron activiation analysis to determine absorption and whole body distribution of absorbed colloidal particles. A specific new absorptive pathway was described.

Impacts
The new colloids represent a large step in our goal to provide technology for simultaneous multiple labeling of different cell molecules. Preliminary studies have shown the new labels will prove useful in specific labeling and in bulk labeling applications.

Publications

  • Meyer,D.A., and Albrecht, R.M. 2001. The feasibility of high resolution, multiple labeling using colloidal particles of similar size but different shapes and elemental compositions. Microscopy and Microanalysis 7 (supplement):1032-1033.
  • Hillyer, J.F., and Albrecht, R.M. 2001. Gastrointestinal persorption and tissue distribution of differently sized colloidal gold nanoparticles. J. Pharm. Sci. 90:1927-1936.
  • Olorundare, O.E., Peyruchaud, O., Albrecht, R.M., and Mosher, D.F. 2001. Assembly of a fibronectin matrix by adherent platelets stimulated by lysophosphatidic acid and other agonists. Blood 98:117-124.
  • Kutuzova,G.D., Albrecht, R.M.,Erickson,C.M., and Qureshi, N. 2001. Diphosphoryl Lipid A from Rhodobacter sphaeroides Blocks the Binding and Internalization of Lipopolysaccharide in RAW 264.7 Cells. J Immunol 167: 482-489.