Determining Density of Oligonucleotide Functionalized Gold Nanoparticles
Background
Gold nanoparticles loaded with DNA oligonucleotides have many practical uses in analytical chemistry and biotechnology including: biosensing, drug delivery, cell imaging and for fundamental understanding of nanoscience.
Classic methods to conjugate DNA to the surface of gold nanoparticles such as “salt-aging” are laborious and time-consuming processes. In contrast, Cytodiagnostics Maleimide-Activated Gold Nanoparticle Conjugation Kits and OligoREADY™ Conjugation Kits have been optimized for high efficiency one-step conjugation of thiol-containing ligands such as oligonucleotides to gold nanoparticles. Conjugation of the oligonucleotide is achieved by the formation of a strong and stable gold-thiol bond without any additional linkers for OligoREADY™ and through a PEG-linker for maleimide particles. The method described below is suitable for determining the oligonucleotide density of conjugates prepared with all conjugation strategies described above.
For several downstream applications it might be necessary to estimate the number of conjugated oligonucleotides that are present per gold nanoparticle in you conjugate. One method of doing this is by functionalizing fluorophore labeled version of your oligonucleotide onto the surface of the gold and incubating with DTT to displace the DNA from the surface of the gold and measuring fluorescence signal in comparison to a standard curve. The result obtained is the absolute number of the oligonucleotides per gold nanoparticle in solution.
Practically, this is done by incubating the fluorophore-labeled DNA functionalized gold nanoparticles with DTT. When mixed, the -SH groups on the DTT displace the -SH group on the DNA molecule (or PEG-SH-linker) from the surface of gold, forming a dense monolayer and freeing the DNA into solution. Initially, the fluorescent signal produced by the fluorophore is quenched by the gold when functionalized, but once the oligonucleotide is cleaved by the DTT, the fluorescent signal is freed from the quenching power of the gold nanoparticles and can be measured by a fluorometer. The method to achieving density of DNA on any size of gold nanoparticles is outlined below.
Materials and Equipment
- 1M Dithiothreitol (DTT)
- Oligonucleotide Conjugated Gold Nanoparticles
- Oligonucleotide stock solution for creating standard curve
- Fluorometer
Procedure
- Dilute stock DTT solution (usually 1M) to 75mM with H2O.
- Create a standard curve (Ex. 0nM – 1000nM for a 20nm DNA-functionalized gold nanoparticles) using the same DNA as conjugated to the gold nanoparticles, diluting the DNA using 75mM DTT (9 DTT : 1 DNA, 100L total volume).
- Mix the oligo labeled gold nanoparticles with the 75mM DTT (9 DTT : 1 DNA, 100L total volume).
- Incubate the standard curve* solutions and the gold nanoparticle solution(s) until the next day, covering all solutions in foil to prevent light exposure to fluorophores.
- Measure the fluorescence signals of all solutions using a fluorometer.
* The standard curve (see figure 1) is used to determine concentration of oligonucleotides on the surface of the gold. Dividing by the concentration of the gold nanoparticle solution will give you the total # of DNA/gold nanoparticle.
Figure 1. Example of a standard curve generated from multiple DNA samples of various concentrations in step 2 above. The total DNA concentration in your oligonucleotide gold conjugate sample can be derived from the standard curve by extrapolating the fluorescent signal measured to the corresponding DNA concentration on the standard curve (grey lines in graph).