ABSTRACT:The attachment of thiolated DNA to gold nanoparticles (AuNPs) has enabled many landmark works in nanobiotechnology. This conjugate chemistry is typically performed using a salt-aging protocol, where in the presence of an excess amount of DNA, NaCl is gradually added to increase DNA loading over 1-2 days. To functionalize large AuNPs, surfactants need to be used, which may generate difficulties for downstream biological applications. We report herein a novel method using a pH 3.0 citrate buffer to complete the attachment process in a few minutes. More importantly, it allows for quantitative DNA adsorption, eliminating the need to quantify the number of adsorbed DNA and allowing the adsorption of multiple DNAs with different sequences at predetermined ratios. The method has been tested for various DNAs over a wide range of AuNP sizes. Our work suggests a synergistic effect between pH and salt in DNA attachment and reveals the fundamental kinetics of AuNP aggregation versus DNA adsorption, providing a novel means to modulate the interactions between DNA and AuNPs.

Single-stranded DNA can be adsorbed by citrate capped gold nanoparticles (AuNPs), resulting in increased AuNP stability, which forms the basis of a number of biochemical and analytical applications, but the fundamental interaction of this adsorption reaction remains unclear. In this study, we measured DNA adsorption kinetics, capacity, and isotherms, demonstrating that the adsorption process is governed by electrostatic forces. The charge repulsion among DNA strands and between DNA and AuNPs can be reduced by adding salt, reducing pH or by using non-charged peptide nucleic acid (PNA).Langmuir adsorption isotherms are obtained, indicating the presence of both adsorption and desorption of DNA from AuNPs. While increasing salt concentration facilitates DNA adsorption, the desorption rate is also enhanced in higher salt due to DNA compaction. DNA adsorption capacity is determined by both DNA oligomer length, DNA concentration and salt. Previous studies indicated faster adsorption of short DNA oligomers by AuNPs, we find that once adsorbed, longer DNAs are much more effective in protecting AuNPs from aggregation. DNA adsorption is also facilitated by using low pH buffers and high alcohol concentrations. A model based on electrostatic repulsion on

For many applications, it is desirable to stabilize colloids over a wide range of buffer conditions while still retaining surface accessibility for adsorption and reaction. Commonly used charge or steric stabilization cannot achieve this goal since the former is sensitive to salt and the latter blocks the particle surface. We use depletion stabilization in the presence of high molecular weight polyethylene glycol (PEG) to stabilize a diverse range of nanomaterials including gold nanoparticles (from 10 to 100 nm), graphene oxide, quantum dots, silica nanoparticles, and liposomes in the presence of Mg 2+ (>1.6 M), heavy metal ions, extreme pH (pH 1-13), organic solvents and adsorbed nucleosides and drugs. At the same time, the particle surface remains accessible for adsorption of both small molecules and macromolecules. Based on this study, high loading of thiolated DNA was achieved in one step with just 2% PEG 20000 in 2 h.Stabilization of colloidal systems is one of the most important and fundamental aspects of nanoscience, enabling a diverse range of applications in physical and biological desciplines. 1 Charge stabilization is easy to achieve by means of electrostatic repulsion. For example, citrate-capped gold nanoparticles (AuNPs) are negatively charged and stable in <10 mM Na + for many years. 2 With a slight increase in salt concentration (e.g. >30 mM Na + ), aggregation starts to occur because of charge screening and AuNPs can approach each other to experience London attractive force. 3,4 Therefore, charge stabilization is limited by several factors including susceptibility to salt and the need for polar solvents. If a particle surface is coated with polymers such as thiolated polyethylene glycol (PEG), and the size of the polymer is greater than the London interaction range, steric stabilization might be achieved. 5 However, the coated polymers also block the particle surface from adsorbing other molecules. A combination of charge and steric stabilization is also possible, where AuNP capped by thiolated DNA is a good example. 6 For many applications involving nanoparticles, such as surface enhanced spectroscopy, nanoparticle bioconjugation, catalysis, heavy metal detection, and drug delivery, eliminating the colloidal stability problem while still retaining surface accessibility is highly desirable. Charge or steric stabilization, however, cannot achieve this goal.When dispersed in a non-adsorbing polymer solution, nanoparticles may experience a depletion force originated from the excluded volume effect, for which no specific binding between the nanoparticle and polymer is required. 7 Theoretic calculations suggest both short-ranged depletion attraction and long-ranged depletion repulsion. 8 Depletion repulsion occurs when the nanoparticle separation is greater than the correlation length ξ of polymer concentration fluctuation in the bulk solution. In a semi-dilute polymer solution, ξ is much smaller than the size of the polymer. 9 Therefore, dispersed nanoparticles are repelled by each other ...

The last 16 years have witnessed the landmark development of polyvalent thiolated DNA functionalized gold nanoparticles (AuNPs) possessing striking properties within the emerging field of nanobiotechnology. Many novel properties of this hybrid nanomaterial are attributed to the dense DNA shell. However, the question of whether non-thiolated polyvalent DNA-AuNP could be fabricated with high DNA density and similar properties as its thiolated counterpart has not been explored in detail.Herein, we report that by simply tuning the pH of the DNA/AuNP mixture, an ultrahigh capacity of nonthiolated DNA can be conjugated to AuNPs in a few minutes, resulting in polyvalent DNA-AuNP conjugates with cooperative melting behavior, a typical property for polyvalent thiolated DNA functionalized AuNPs. With this method, large AuNPs (e.g., 50 nm) can be functionalized to achieve colorimetric detection of sub-nM DNA. Further, this fast and stable DNA loading was employed to separate AuNPs of different size. We propose that a large fraction of the attached DNAs are adsorbed via one or a few terminal bases to afford the high loading capacity and the ability to hybridize with the complementary DNA. This discovery not only offers a time-and cost-effective way to functionalize AuNPs with a high density of non-thiolated DNA, but also provides new insights into the fundamental understanding of how DNA strands with different sequences interact with AuNPs.

Adsorption of a fluorophore-labeled DNA probe by graphene oxide (GO) produces a sensor that gives fluorescence enhancement in the presence of its complementary DNA (cDNA). While many important analytical applications have been demonstrated, it remains unclear how DNA hybridization takes place in the presence of GO, hindering further rational improvement of sensor design. For the first time, we report a set of experimental evidence to reveal a new mechanism involving non-specific probe displacement followed by hybridization in the solution phase. In addition, we show quantitatively that only a small portion of the added cDNA molecules undergo hybridization while most are adsorbed by GO to play the displacement role. Therefore, it is possible to improve signaling by raising hybridization efficiency. A key innovation herein is using probes and cDNA with a significant difference in their adsorption energy by GO. This study offers important mechanistic insights into the GO/DNA system. At the same time, it provides simple experimental methods to study biomolecular reaction dynamics and mechanism on surface, which may be applied for many other biosensor systems.3