Preparation of Gold Nanoparticle–DNA Conjugates

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 HNO₃
1 mM HAuCl₄ (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

Figure 12.2.1 Schematic results for horizontal gel electrophoresis of oligonucleotide‐nanoparticle conjugates. The first, third, and fifth wells were loaded with unmodified gold nanoparticle standards, and the second and fourth wells with the nanoparticle/conjugate mixture (see , step ). The numbers to the right of the gel indicate the number of attached oligonucleotides. The unmodified particles migrate at the zero mark. Each band of successively decreasing mobility corresponds to a nanoparticle with an additional oligonucleotide covalently attached.

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Figure 12.2.2 Assembly of gold nanoparticle conjugates, functionalized with oligonucleotide sequences a and b, onto complementary oligonucleotide templates a′b′. (A ) Nanoparticle conjugates bearing only one oligonucleotide strand assemble selectively into dimers in the presence of the template. More complex structures can be generated from templates with additional recognition segments. (B ) Nanoparticle conjugates bearing many oligonucleotide strands assemble into polymeric macrostructures in the presence of the complementary template. The optical changes associated with this polymeric assembly make this system particularly effective as a colorimetric DNA hybridization sensor.

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Figure 12.2.3 Scheme for conjugation of gold nanoparticles with disulfide‐modified oligonucleotides. An organic mercaptoalcohol is also incorporated into the conjugate.

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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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	Mirkin, C.A., Letsinger, R.L., Mucic, R.C., and Storhoff, J.J. 1996. A DNA‐based method for rationally assembling nanoparticles into macroscopic materials. Nature 382:607‐609.
	Storhoff, J.J., Elghanian, R., Mucic, R.C., Mirkin, C.A., and Letsinger, R.L. 1998. One‐pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticle probes. J. Am. Chem. Soc. 120:1959‐1964.
	Sugimoto, N., Nakano, S., Yoneyama, M., and Honda, K. 1996. Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucl. Acids Res. 24:4501‐4505.
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	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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Preparation of Gold Nanoparticle–DNA Conjugates

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited