Attaching DNA to Gold Nanoparticles With a Protein Corona

Abstract

DNA-functionalized gold nanoparticles (AuNPs) have been widely used in directed assembly of materials, biosensors, and drug delivery. This conjugate may encounter proteins in these applications and proteins may affect not only DNA adsorption but also the function of the attached DNA. Bovine serum albumin (BSA) with many cysteine residues can strongly adsorb on AuNPs and this conjugate showed high colloidal stability against salt, acid and base. Similar protection effects were also observed with a few other common proteins including catalase, hemoglobin, glucose oxidase, and horseradish peroxidase. DNA oligonucleotides without a thiol label can hardly displace adsorbed BSA, and BSA cannot displace pre-adsorbed DNA either, indicating a strongly kinetically controlled system. Thiolated DNA can be attached at a low density on the AuNPs with a BSA corona. The BSA corona did not facilitate the hybridization of the conjugated DNA, while a smaller peptide, glutathione allowed faster hybridization. Overall, proteins increase the colloidal stability of AuNPs, and they do not perturb the gold-thiol bond in the DNA conjugate, although a large protein corona may inhibit the hybridization function of DNA.

Keywords: aptamers; bioconjugation; biointerface; biosensors; nanomaterials.

Figure 1

(A) The structures of BSA and AuNP/BSA conjugates. (B) The UV–vis spectra of citrate-capped AuNP and AuNP/BSA conjugates. (C) Adsorption kinetics of FITC-labeled BSA (50 nM) by 13 nm AuNPs (0.4 nM added at 1 min). (D) The UV–vis spectra of AuNP/BSA conjugates prepared by mixing 13 nm AuNPs with 10 μM BSA in up to 300 mM NaCl. Inset: photographs citrate-AuNPs with 50 mM NaCl (left), and AuNP/BSA with 300 mM NaCl. (E) Photographs of AuNP/BSA in different solutions. (F) The UV–vis spectra of AuNPs capped with various proteins conjugates in 300 mM NaCl. All samples were in HEPES buffer (10 mM, pH 7.4).

Figure 2

Desorbed (A) A₁₅ and (B) T₁₅ from their AuNP conjugates after overnight incubation with 100 mM NaCl, 1 μM BSA, or their mixture. (C) Desorbed BSA to each AuNP conjugate after overnight incubation with 100 mM NaCl, 1 μM A₁₅, T₁₅, or their combinations. All samples were in HEPES buffer (10 mM, pH 7.4).

Figure 3

Adsorption kinetics of FAM-labeled thiolated DNA (FAM-9A5-SH) on (A) AuNP/BSA, and (B) citrate-capped AuNPs in 10 mM HEPES buffer (pH 7.4) containing various concentrations of NaCl. The AuNP/BSA conjugates were added at ~1 min. Schematic representation for thiolated DNA adsorption onto AuNP/BSA (C) without and (D) with NaCl.

Figure 4

(A) The number of DNA strands on each AuNP as a function of the initial concentration of BSA with 100 mM NaCl. The first four samples showed purple color and the rest showed red color. (B) Effect of the ratio (DNA to AuNP) on DNA loading with the addition of 300 mM NaCl, and all of these samples were stable. (C) The UV–vis spectra of AuNP/BSA/DNA conjugates without or with the addition of NaCl. The conjugates were prepared with different ratios of AuNP/BSA to DNA. (D) No thiolated FAM-DNA desorbed from AuNP/BSA/DNA conjugates induced by buffer or 300 mM NaCl.

Figure 5

(A) The number of cDNA or rDNA hybridized to AuNP/DNA containing various protein corona. (B) The cDNA and rDNA hybridization kinetics on AuNP/DNA and AuNP/BSA/DNA. Control experiments were performed by adding citrate-AuNP for confirming the decrease of fluorescence from DNA hybridization instead from the inner-filter effect of AuNPs. (C) The number of cDNA or rDNA hybridized to AuNP/DNA dispersed in BSA and GSH solutions. (D) The cDNA and rDNA hybridization kinetics to AuNP/DNA in BSA and GSH solutions. (E) AuNPs conjugates analyzed by 0.5% agarose gel electrophoresis in 5 mM HEPES buffer, and the DNA was 9A5-SH. (F) Scheme of hybridization of AuNP/protein/DNA and AuNP/DNA/GSH conjugates.