Preparation of Gold Nanoparticle–DNA Conjugates

T. Andrew Taton1

1 University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 12.2
DOI:  10.1002/0471142700.nc1202s09
Online Posting Date:  August, 2002
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Abstract

This unit describes the preparation of conjugates between nanometer‐scale gold particles and synthetic oligonucleotides. Oligonucleotide‐functionalized gold nanoparticles are finding increased use in both the construction of complex, tailored nanostructures and the optimization of DNA sequence analysis. The protocols in this unit outline the synthesis, purification, and characterization of nanoparticle‐DNA conjugates for applications in nanotechnology and biotechnology. Separate procedures are presented for nanoparticles functionalized with just one or a few oligonucleotide strands and for nanoparticles functionalized with a dense layer of oligonucleotide strands. The different physical and chemical properties of these two types of conjugates are discussed, as are their stability and utility in different environments.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Preparation of Gold Nanoparticle–DNA Conjugates Containing One to Several DNA Strands Per Particle
  • Basic Protocol 2: Preparation of Gold Nanoparticle–DNA Conjugates Containing Many DNA Strands Per Particle
  • Support Protocol 1: Synthesis of Aqueous Citrate‐Protected Gold Colloid
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Gold Nanoparticle–DNA Conjugates Containing One to Several DNA Strands Per Particle

  Materials
  • Oligonucleotide: ∼l mM synthetic 5′‐ or 3′‐disulfide‐containing or thiol‐containing oligonucleotide (see ), dissolved in water
  • Aqueous gold nanoparticle solution (British Biocell, Ted Pella; or see protocol 3)
  • Phosphine: 4,4′‐(phenylphosphinidene)bis(benzenesulfonic acid), dipotassium salt hydrate (Aldrich), solid and 0.5 M aqueous solution
  • NaCl, solid and 1 M aqueous solution
  • Methanol
  • 5× TBE electrophoresis buffer ( appendix 2A)
  • 30% (v/v) glycerol
  • UV/vis spectrophotometer
  • Quartz cuvette
  • Razor blade, sterilized
  • Glass‐fiber filter paper, 1.2‐µm retention (e.g., GF/C; Whatman)
  • Dialysis membrane, MWCO 10,000 (e.g., SpectraPor Biotech RC; Spectrum Laboratories)
  • Centrifugal filter device, 0.45‐µm pore size (e.g., Ultrafree‐MC; Amicon)
  • Additional reagents and equipment for agarose gel electrophoresis (e.g., CPMB UNIT )

Basic Protocol 2: Preparation of Gold Nanoparticle–DNA Conjugates Containing Many DNA Strands Per Particle

  Materials
  • Oligonucleotide: ∼1 mM synthetic 5′‐ or 3′‐disulfide‐containing or thiol‐containing oligonucleotide (see ), dissolved in water
  • Aqueous gold nanoparticle solution (British Biocell or see protocol 3)
  • 1 M NaCl
  • 0.1 M sodium phosphate buffer, pH 7 ( appendix 2A)
  • 0.1 M NaCl/10 mM sodium phosphate buffer, pH 7
  • 0.3 M NaCl/0.01% (w/v) sodium azide/10 mM sodium phosphate buffer, pH 7
  • Additional reagents and equipment for quantitating oligonucleotide, nanoparticle, and conjugate solutions (see protocol 1)
CAUTION: Sodium azide is poisonous and explosive in solid form; wear gloves and handle with care.

Support Protocol 1: Synthesis of Aqueous Citrate‐Protected Gold Colloid

  Materials
  • Aqua regia: 3:1 (v/v) concentrated HCl/concentrated HNO 3
  • 1 mM HAuCl 4 (Aldrich)
  • 38.8 mM sodium citrate (Aldrich)
  • 1‐L round‐bottom flask
  • Reflux condenser
  • Heating mantle
  • 0.45‐µm nylon filter
CAUTION: Aqua regia is noxious and extremely caustic. Handle with extreme care in a well‐ventilated fume hood.
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Figures

Videos

Literature Cited

Literature Cited
   Alivisatos, A.P., Johnsson, K.P., Peng, X., Wilson, T.E., Loweth, C.J., Bruchez, M.P. Jr., , and Schultz, P.G. 1996. Organization of ‘nanocrystal molecules’ using DNA. Nature 382:609‐611.
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   Taton, T.A., Mirkin, C.A., and Letsinger, R.L. 2000. Scanometric DNA detection with nanoparticle probes. Science 289:1757‐1760.
   Taton, T.A., Lu, G., and Mirkin, C.A. 2001. Two‐color labeling of oligonucleotide arrays via size‐selective scattering of nanoparticle probes. J. Am. Chem. Soc. 123:5164‐5165.
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Internet Resources
   www.basic.nwu.edu/biotools/oligocalc.html
  Provides an extinction coefficient calculator for oligonucleotides.
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