Rapid Immobilization of Oligonucleotides at High Density on Semiconductor Quantum Dots and Gold Nanoparticles

Abstract

Oligonucleotide-coated nanoparticles (NPs) have been used in numerous applications such as bioassays, as intracellular probes, and for drug delivery. One challenge that is confronted in the preparation of oligonucleotide-NP conjugates is derived from surface charge because nanoparticles are often stabilized and made water-soluble with a coating of negatively charged capping ligands. Therefore, an electrostatic repulsion is present when attempting to conjugate oligonucleotides. The result is that the conjugation can be a slow process, sometimes requiring 1 to 2 days to equilibrate at the highest surface density. The effect is compounded by electrostatic repulsion between neighboring oligonucleotide strands on the NP surfaces, which tends to lower the surface density. Herein, we report a novel method that enables conjugation in less than 1 min with a surface density of oligonucleotides up to the theoretical physical limit of occupancy. Negatively charged NPs are first adsorbed onto the surface of positively charged magnetic beads (MBs) to create MB-NP conjugates. Oligonucleotides are subsequently electrostatically adsorbed onto the MB surfaces when added to a suspension of MB-NP conjugates. This creates an oligonucleotide concentration 10 to 10 greater than in bulk solution in the vicinity of the nanoparticles, resulting in the promotion of the kinetics by over 1000-fold and achieving the maximum density possible for the conjugation reaction.

Enhanced Immunoassay Using a Rotating Paper Platform for Quantitative Determination of Low Abundance Protein Biomarkers

“…The R-oligo is attached by hybridization with its complementary immobilized strand (C-oligo), and D-Ab is immobilized by interaction with Protein G. Given the critical role of BNPs in the sensitivity and specificity of the enzyme amplification method, surface immobilization strategies have been used that allow for adequate control of the packing density and orientation of these reagents. An interfacial NP decoration method using magnetic beads, called the MBL method, that we have recently reported was used to immobilize C-oligos (BNP-1) and to subsequently hybridize R-oligo to the surface of AuNPs (BNP-2). In addition to the rapid immobilization kinetics, the MBL method allowed for maximization of the packing density of C-oligos, and hence maximized the R-oligo loading capacity of BNPs.…”

“…In addition to the rapid immobilization kinetics, the MBL method allowed for maximization of the packing density of C-oligos, and hence maximized the R-oligo loading capacity of BNPs. The average loading of R-oligos on the AuNP surfaces as well as the fraction of those R-oligos released upon dispersion of NPs in DI water were determined using previously reported methods . We have determined that the average loading was 94 ± 12 and 326 ± 29 R-oligos on the surfaces of AuNPs with a 15 and 40 nm diameter, respectively.…”

“…Conjugation of oligonucleotides to the surface of AuNPs with 15 and 40 nm diameters were done using the magnetic bead-loading (MBL) method that was reported previously. , In this method, the negatively charged nanoparticles and DNA oligonucleotides were electrostatically loaded on the surfaces of the positively charged magnetic beads. The accumulation of oligonucleotides in the vicinity of nanoparticles at the magnetic bead surface led to high density nanoparticle-DNA conjugates within seconds. , Briefly, 0.3 mg of magnetic beads was dispersed in 200 μL of TBS buffer (tris-borate 100 mM, 1 M NaCl, pH 7.4) in a 2-mL Eppendorf tube, vortexed, and isolated using a magnet. The procedure was repeated once again in TBS buffer and then twice in PB (10 mM, pH 7.4).…”

“…Water-soluble glutathione-coated QDs (GSH-QD) were prepared using a previously reported method . The immobilization MB probes on QD surfaces was done using the magnetic loading method . Briefly, 5 pmol GSH-QD was added to 0.1 mg of MB in 100 μL of TBS buffer (Tris-borate 100 mM, pH 7.4), and the tube was agitated for 30 s. Then, 50 pmol of H6-MB was added to the solution, and the tube was agitated for another 30 s. The MBs were isolated using a magnet and redispersed in BBS.…”

Interface-Driven Hybrid Materials Based on DNA-Functionalized Gold Nanoparticles

“…65 Sedighi and Krull reported a facile method using positively charged magnetic particles. 66 The positive surface generated a high local concentration of DNA and AuNPs, achieving fast adsorption (1,000-fold enhancement). Inserting an oligoethylene glycol spacer between the thiol group and DNA also allowed rapid and quantitative attachment of the DNA.…”