DNA-functionalized Gold Nanoparticles Research Articles

Freeze-Driven Adsorption of Poly-A DNA on Gold Nanoparticles: From a Stable Biointerface to Plasmonic Dimers.

Increasing attention is paid to poly-adenine (poly-A) DNA-functionalized gold nanoparticles due to the high cost of thiols. Freezing is an effective approach for immobilizing poly-A DNA on gold nanoparticles, but its mechanism remains elusive. To cope with this issue, in this paper, some experimental insights are provided. It is shown that (1) the DNA loading density is independent of the length of poly-A. (2) DNA is densely packed on gold nanoparticles, and the biointerface is peculiarly stable, which is not in line with the existing “wrapping” model. (3) Using a DNA-staining dye, thiazole orange, it is shown that poly-A duplex structures are formed on the surface of gold nanoparticles, with evidence given by fluorescence and Raman measurements. An alternative model involving stable poly-A duplexes anchored by finite terminal adenines is proposed. Based on it, a strategy for constructing plasmonic dimers is developed, using freeze-driven adsorption of a DNA sequence with poly-adenine at both ends. This work provides insights into the reaction between poly-A DNA and AuNPs upon freezing and is expected to facilitate related research in biosensor development and nanotechnology.

Building Polyvalent DNA-Functionalized Anisotropic AuNPs using Poly-Guanine-Mediated In-Situ Synthesis for LSPR-Based Assays: Case Study on OncomiR-155.

DNA-functionalized gold nanoparticles (DNA-AuNPs) hold great promise for numerous biomedical applications, especially the building of well-defined nanosystems. Previously reported methods for the preparation of DNA-AuNPs all rely on the use of DNA-bearing free thiol or disulfide groups at their 3'/5' ends. But here we report a novel polyvalent DNA-AuNPs conjugation approach by in-situ fast synthesis of AuNPs at the polyguanine (G12 ) strands. As confirmed by both TEM images and gel electrophoresis analysis, many poly G strand can form an individual anisotropic AuNP and so each AuNP functionalized with a dense layer of DNA, resulting in the formation of polyvalent (p)DNA-AuNPs. The general applicability of this novel approach was further verified in hybridization test and UV-Vis spectroscopy results show that pDNA-AuNPs conjugation is more attractive in biomedical diagnosis and specific sequence detection like microRNA-155 by using an extra-strand poly G with "sticky end" that are complementary to the target sequence.

RGB color analysis of formaldehyde in vegetables based on DNA functionalized gold nanoparticles and triplex DNA.

A highly sensitive and selective RGB color analysis for the detection of formaldehyde (FA) was developed by using a DNA functionalized gold nanoparticle (AuNPs-DNA) probe. When complementary oligonucleotides (oligo 2 and oligo 3) and a silver ion (Ag+) were added to the AuNPs-DNA solution, triplex DNA was formed, resulting in the aggregation of AuNPs, and accompanied by a solution color change from red to purple. With the addition of formaldehyde, it reacted with Ag+, decreased the stability of triplex DNA between AuNPs-DNA, induced the dispersion of AuNPs, and the color of AuNPs recovered to red. Therefore, the formaldehyde concentration could be estimated with the RGB (red, green, blue) values of the AuNP solution by using a smartphone application (APP). The R value of the system was proportional to the concentration of formaldehyde within the range of 0.23-4.50 mg L-1, with a detection limit of 0.14 mg L-1. The method has been successfully applied to detect the residues of formaldehyde in vegetable samples and has the potential of the on-site determination of formaldehyde.

Exploring the size of DNA functionalized gold nanoparticles for high efficiency exosome uptake and sensitive biosensing

Exosomes are nanoscale lipid vesicles that play important roles in oncogenesis and tumor heterogeneity. Recently, more attentions have been attracted for the analysis of exosomes by using AuNPs-based nanoprobes due to their unique physicochemical properties. In this regard, the geometrical parameters of AuNPs have high impact on the performance for exosome sensing, whereas the related mechanistic studies have not been reported so far. Herein in this work, we symmetrically explored the size influence of DNA functionalized AuNPs on their uptake efficiency by exosomes, and found that the uptake efficiency was highly size-dependent. Based on these interesting discoveries, we developed AuNPs-based nanoprobes for the in-situ detection of miRNA in exosomes. The nanoprobe prepared using 5 nm AuNPs exhibits the highest sensitivity with a detection limit of 6.53 × 103 particles/μL, which is about 2 orders of magnitude lower than that of traditional 13 nm AuNPs. Furthermore, the nanoprobe based on 5 nm AuNPs was successfully used in clinical serum samples to differentiate cancer patients from healthy individuals. All these results advise that DNA-AuNPs can be easily tailored through size modulation to design functional nanoprobe with excellent exosome uptake properties and improved performance in exosome sensing applications.

A fluorescence and localized surface plasmon resonance dual-readout sensing strategy for detection of acetamiprid and organophosphorus pesticides

In this work, a fluorescence/localized surface plasmon resonance (Fluorescence/LSPR) dual-readout sensing system that using DNA functionalized gold nanoparticles (AuNPs) as nanoprobe was designed for acetamiprid (ACE) and organophosphorus pesticides (OPs) detection. The ACE detection was based on the fluorescence resonance energy transfer (FRET) between fluorochrome Cy3-labeled aptamer and AuNPs of nanoprobe. The high specificity of ACE allowed Cy3-labeled aptamer to release from nanoprobe, resulting in a significantly fluorescence recovery related to the concentration of ACE. On the other hand, the strategy for OPs detection relied on the acetylcholinesterase (AChE)-mediated oxidative polymerization blocking of dopamine on AuNPs surface and the inhibition of OPs toward AChE. The degree of polymerization of dopamine depended on the concentration of OPs, which led to a tremendous LSPR spectral shift and scattering color change of nanoprobe. The dual-readout sensing system showed great sensitivity and selectivity for ACE and OPs (used chlorpyrifos as model) with a detection limit of 166.7 pg mL−1 and 0.17fgmL−1, respectively. Moreover, two distinct color changes could be visual recognized to indicate the content of ACE and OPs, which was considered to be a conceivable way for pesticides residues assay. Therefore, this work paved a new avenue for rapid detection of multi-pesticides via a Fluorescence/LSPR dual signal response and visual screening, and hold great potential for noxious substance monitoring.

Nanoparticle Superlattices through Template-Encoded DNA Dendrimers.

The chemical interactions that lead to the emergence of hierarchical structures are often highly complex and difficult to program. Herein, the synthesis of a series of superlattices based upon 30 different structurally reconfigurable DNA dendrimers is reported, each of which presents a well-defined number of single-stranded oligonucleotides (i.e., sticky ends) on its surface. Such building blocks assemble with complementary DNA-functionalized gold nanoparticles (AuNPs) to yield five distinct crystal structures, depending upon choice of dendrimer and defined by phase symmetry. These DNA dendrimers can associate to form micelle-dendrimers, whereby the extent of association can be modulated based upon surfactant concentration and dendrimer length to produce a low-symmetry Ti5Ga4-type phase that has yet to be reported in the field of colloidal crystal engineering. Taken together, colloidal crystals that feature three different types of particle bonding interactions-template-dendron, dendrimer-dendrimer, and DNA-modified AuNP-dendrimer-are reported, illustrating how sequence-defined recognition and dynamic association can be combined to yield complex hierarchical materials.

Introducing DNA Nanosensor to Undergraduate Students: Rapid Non-Cross-Linking Aggregation of DNA-Functionalized Gold Nanoparticles for Colorimetric DNA Assay

Demonstration of a colorimetric approach for nucleic acid detection represents an attractive educational experiment for chemistry undergraduate students in the time of coronavirus pandemic. Herein, a rapid and vivid detection method that visualizes the presence of a specific DNA sequence is described. The plasmon resonance of gold nanoparticles (AuNPs) (∼20 nm in diameter) endows a red color to DNA-functionalized gold nanoparticles (DNA-AuNPs) in a colloidal solution. Simply upon addition of a sample containing the complementary DNA sequence, the DNA-AuNPs dispersed in a strong ionic solution can spontaneously aggregate in minutes and turn to be purple in color, which can be detected using UV–visible spectroscopy as well as the naked eye. In contrast, the DNA-AuNPs remain highly dispersed as a reddish colloid even at high ionic strength when the DNA sequence is terminally mismatched or overhung. According to the teaching experience, this DNA testing experiment can be accomplished by students in pairs or groups under instruction in a three-day lab session. Given the rapid and fail-proof DNA testing, the testing method is also well-suited for hands-on introduction of analytical chemistry and nanotechnology to chemistry freshman and high school students.

Stimuli-Responsive Three-Dimensional DNA Nanomachines Engineered by Controlling Dynamic Interactions at Biomolecule-Nanoparticle Interfaces.

Stimuli-responsive nanomachines are attractive tools for biosensing, imaging, and drug delivery. Herein, we demonstrate that the orientation of macromolecules and subsequent dynamic interactions at the biomolecule-nanoparticle (bio-nano) interfaces can be rationally controlled to engineer stimuli-responsive DNA nanomachines. The success of this design principle was demonstrated by engineering a series of antibody-responsive DNA walkers capable of moving persistently on a three-dimensional track made of DNA functionalized gold nanoparticles. We show that drastically different responses to antibodies could be achieved using DNA walkers of identical sequences but with varying number or sites of modifications. We also show that multiple interfacial factors could be combined to engineer stimuli-responsive DNA nanomachines with high sensitivity and modularity. The potential of our strategy for practical uses was finally demonstrated for the amplified detection of antibodies and small molecules in both buffer and human serum samples. Unlike many DNA-based nanomachines, the performance of which could be significantly hindered by the matrix of serum, our system shows a matrix-enhanced sensitivity as a result of the engineering approach at the bio-nano interface.

Probing and modulating the interactions of the DNAzyme with DNA-functionalized nanoparticles

DNA-functionalized gold nanoparticles are one of the most versatile bionanomaterials for biomedical and clinical diagnosis. Herein, we discovered that the performance of DNAzyme cleaving the substrate is highly related to its length. This intriguing phenomenon only appears at the interfaces of DNA-functionalized gold nanoparticles. We systematically investigated the causes of this phenomenon. We conjectured that the DNAzyme with extended nucleotides that do not match its substrate strand is vulnerable to non-specific adsorption, electrostatic repulsion, and steric hindrance. Based on our improved understanding of this phenomenon, we have successfully developed a highly sensitive and specific amplifiable biosensor to detect human apurinic/apyrimidinic endonuclease 1.

Tuning optical properties of self-assembled nanoparticle network with external optical excitation

DNA-driven self-assembly enables precise positioning of the colloidal nanoparticles owing to specific Watson–Crick interactions. Another important feature of this self-assembly method is its reversibility by controlling the temperature of the medium. In this work, we study the potential of another mechanism to control the binding/unbinding process of DNA-functionalized gold nanoparticles. We employ laser radiation that can be absorbed by the gold nanoparticles to heat their network and disassociate it. Here, we show that we can actively control the optical properties of the nanoparticle network by external optical excitation. We find out that by irradiating the structure with a green hand-held laser, the total transmittance can increase by ∼30% compared to the transmittance of the sample not irradiated by the laser. Similarly, the optical microscopy images indicate the transformation of the nanoparticle network from opaque to transparent, while the nanoparticles formed a network again after the laser irradiation stopped. Our results prove that the optical excitation can be used to tailor the structure and thus the optical properties of the DNA-self-assembled nanoparticle networks.

Rapid and sensitive colorimetric detection of microsporidian Enterocytozoon hepatopenaei (EHP) based on spore wall protein (SWP) gene using loop-mediated isothermal amplification combined with DNA functionalized gold nanoparticles as probes

Enterocytozoon hepatopenaei (EHP) is a newly emerging microsporidian parasite that causes growth retardation in cultured shrimp. Currently, EHP infection can be detected by standard polymerase chain reaction (PCR)-based methods, as well as loop-mediated isothermal amplification (LAMP) methods that target EHP's small subunit (SSU) ribosomal RNA (rRNA) gene (LAMP-SSU). However, this gene target has a propensity for generating false positive test results due to cross-reactivity of LAMP primers with SSU-rRNA genes from closely related microsporidia that infect other aquatic organisms. Herein, we describe the development of a simple, specific and sensitive method for detection of EHP by the naked eye. The method is a LAMP technique targeting the spore wall protein (SWP) gene combined with DNA-functionalized gold nanoparticle probes (LAMP-SWP). It has analytical sensitivity comparable to that of nested-PCR (10 copies of plasmid DNA; 1 pg of total DNA) and other reported LAMP methods. Furthermore, no cross-reactivity was observed with other pathogens known to infect shrimp. LAMP-SWP had a sensitivity of 97% (95% CI: 88.2–99.9%) and specificity of 100% (95% CI: 88.4–100%)for EHP detection, while the corresponding sensitivity and specificity of the LAMP-SSU we used for comparison were 100% (95% CI: 91.9–100%) and 56% (95% CI: 37.4–74.5%), respectively. Hence, our novel colorimetric LAMP-SWP combines high specificity for EHP (due to the SWP target), high sensitivity for 10 target copies (similar to conventional nested-PCR), confirmation of PCR amplicons by DNA hybridization and colorimetric visualization of PCR amplicons by the naked-eye (i.e., no electrophoresis or fluorometric equipment needed). It is a rapid method that could allow for real-time management decisions at shrimp hatcheries and farms without the need for sophisticated and expensive laboratory equipment.

DNA-Functionalized Gold Nanoparticles with Toehold-Mediated Strand Displacement for Nucleic Acid Sensors

We present a study of gold nanoparticles linked by DNA that undergo toehold-mediated strand displacement for nucleic acid sensing. The factors influencing colloidal aggregation, thermal melting, an...

Dual MicroRNA-Triggered Drug Release System for Combined Chemotherapy and Gene Therapy with Logic Operation.

Combination therapy via stimulus-responsive drug release is known to improve treatment efficacy and minimize side effects. However, the use of low-abundance cancer biomarkers as molecular triggers to induce efficient drug release for combination therapy still remains a challenge. Herein, we developed a dual microRNA-responsive drug nanocarrier for catalytic release of doxorubicin (Dox) and small interfering RNA (siRNA) in cancerous cells for combined chemotherapy and gene therapy with logic operation. The nanocarrier is constructed by assembling two duplexes of DNA/RNA and Dox molecules onto DNA-functionalized gold nanoparticles. Two microRNA molecules (miRNA-21 and miRNA-10b overexpressed in MDA-MB-231) could alternatively catalyze the disassembly of the nanocarrier through a thermodynamically driven entropy gain process, during which Dox molecules are released, and the two pairs of released DNA/RNA duplex hybridize to generate siRNA (siBcl-2) in situ by strand displacement reactions. Quantum dots are used to track the process in living cells. The AND logic gate-based drug release system allows effective treatment of specific cancer cell types according to miRNA expression patterns. This strategy represents an effective means to overcome multidrug resistance and improve therapeutic effects.

Attaching DNA to Gold Nanoparticles With a Protein Corona.

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.

A high local DNA concentration for nucleating a DNA/Fe coordination shell on gold nanoparticles.

Preparing DNA/Fe coordination nanoparticles in solution requires a high concentration of DNA. Herein we grew a DNA/Fe shell on DNA-functionalized gold nanoparticles. Taking advantage of the high local DNA density, the required DNA concentration decreased 60-fold, and the size can be controlled. This hybrid material allowed drug loading and colorimetric sensing.

Accelerated non-crosslinking assembly of DNA-functionalized nanoparticles in alcoholic solvents: for application in the identification of clear liquors.

Colorimetric detection of various target molecules in aqueous solutions based on the non-crosslinking assembly of DNA-functionalized Au nanoparticles (DNA-AuNPs) has been well established in recent years. The extension of DNA-AuNPs to other solvents remains much less explored, despite the practical importance of detection in non-aqueous solutions, such as those containing an organic ingredient that is required or not removable in many contexts. However, the general consideration that DNA is easily denatured and precipitated in organic solvents has been hampering the use of DNA-AuNPs in low polar solvents. Herein, we report a more rapid non-crosslinking assembly of double-stranded (ds) DNA-AuNPs in alcoholic solvents than in aqueous solvents. When the concentration of ethanol in the disperse medium is increased from 0% to 20% (v/v), the rate of non-crosslinking assembly is distinctly increased by a factor of 5-6, whereas the rate is sharply decreased when the ethanol concentration is further increased to 40%. This biphasic kinetics trend could be attributed to the competitive balance between the enhanced intermolecular attraction between dsDNAs and the increased propensity for melting of dsDNA. Rapid naked-eye identification of clear liquors that are encoded by oligonucleotide additives has also been demonstrated by using the alcoholic non-crosslinking assembly of dsDNA-AuNPs as a proof-of-concept.

DNA-functionalized gold nanoparticle assemblies for Surface Enhanced Raman Scattering

The programmable assembly of DNA strands is a promising tool for building tailored bottom-up nanostructures. Here, we present a plasmonic nanosystem obtained by the base-pairing mediated aggregation of gold nanoparticles (NPs) which are separately functionalized with two different single-stranded DNA chains. Their controlled assembly is mediated by a complementary DNA “bridge” sequence. We monitor the formation of DNA assembled NP aggregates in solution, and we study their Surface Enhanced Raman Scattering (SERS) response by comparison with the single NP constituents. We interpret the revealed SERS signatures in terms of the molecular and NP organization at the nanoscale, demonstrating that the action of the DNA bridge molecule yields regular NP aggregates with controlled interparticle distance and reproducible SERS response. In perspective, this demonstrates the potential of the present system as a stable, biocompatible, and recyclable SERS sensor.

An analytical method to control the surface density and stability of DNA-gold nanoparticles for an optimized biosensor

DNA functionalized gold nanoparticles (DNA-AuNPs) have shown great potential for biosensing as they combine the excellent optical properties of gold nanoparticles and the molecular recognition function of DNA. Since the DNA density determines the assay performance and the stability of the conjugate, a precise control of the surface density of DNA-AuNP is crucial for an optimized biosensor. Here we report a simple assay for quantifying multiple unlabeled DNAs on AuNPs. The assay relies on potassium cyanide (KCN) to first dissolve the AuNPs, which then releases surface bound DNA for quantification through a double-stranded DNA dye. Using this analytical quantification method, we investigated several strategies to control the surface density of DNA-AuNPs. Besides the precise control of DNA density, the stability of DNA-AuNPs after conjugation is also important in developing a biosensor with optimal performance. Without proper storing conditions, DNA-AuNPs are unstable and aggregate over time. To overcome this problem, we developed a long-term storage solution to ensure the stability and quality of DNA-AuNPs after conjugation which would benefit any DNA-AuNP-based biosensor.

Construction of Bio/Nanointerfaces: Stable Gold Nanoparticle Bioconjugates in Complex Systems.

The real application of DNA-functionalized gold nanoparticles (DNA-Au NPs) was limited by decreased stability and irreversible aggregation in high-ionic strength solutions and complex systems. Therefore, exploring a kind of DNA-Au NPs with excellent stability in high-ionic strength solutions and complex systems is challenging and significant. Herein, a novel universal bioconjugate strategy for constructing ultrastable DNA-Au NPs was designed based on the combination of polydopamine (PDA) shell and DNA linker. The obtained DNA-linked Au@polydopamine nanoparticles (DNA-Au@PDA NPs) showed colloidal stability in high-ionic strength solution and complex systems (such as human serum and cell culture supernatant). Moreover, the nanoparticles still maintained good dispersion after multiple freeze-thaw cycles. The high stability of DNA-Au@PDA NPs may be attributed to increasing the electrostatic and steric repulsions among nanoparticles through the effect of both PDA shell and DNA linker on Au@PDA NPs. For investigating the application of such nanoparticles, a highly sensitive assay for miRNA 141 detection was developed using DNA-Au@PDA NPs coupled with dynamic light scattering (DLS). Comparing with the regular DNA-Au NPs, DNA-Au@PDA NPs could detect as low as 50 pM miRNA 141 even in human whole serum. Taken together, the features of Bio/Nanointerface make the nanoparticle suitable for various applications in harsh biological and environmental conditions due to the stability. This work may provide a universal modification method for obtaining stable nanoparticles.

Effects of Small Molecules on DNA Adsorption by Gold Nanoparticles and a Case Study of Tris(2-carboxyethyl)phosphine (TCEP).

DNA-functionalized gold nanoparticles (AuNPs) often encounter various small molecules and ions such as backfilling agents, bifunctional cross-linkers, stabilizers, and molecules from biological fluids both during and after the DNA conjugation process. Small molecules and ions can influence the stability and property of the conjugate, but such interactions are yet to be fully explored. In this work, eight important molecules were studied and compared, including tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS), 6-hydroxy-1-hexanethiol (MCH), l-glutathione (GSH), bromide (Br-), bis(p-sulfonatophenyl)phenylphosphine (BSPP), and thiocyanate (SCN-). Depending on the size, charge, and adsorption affinity on the AuNPs, they can either stabilize or destabilize the AuNPs. Their ability to displace thiolated DNA from AuNPs follows the order of MCH > SPDP > GSH > SCN- > TCEP > Br- > BSPP > GMBS. BSPP has the best stabilization effect for the colloidal stability of AuNPs, while it does not displace the adsorbed DNA. TCEP can be adsorbed on AuNPs and enhance the adsorption of A/C rich DNA in low-salt conditions. This work indicates that the effects of small molecules and ions cannot be ignored when studying the DNA-functionalized AuNPs, which ensures optimal applications and correct interpretation of the data.