DNA AuNPs Research Articles

Designed Diblock Oligonucleotide for the Synthesis of Spatially Isolated and Highly Hybridizable Functionalization of DNA–Gold Nanoparticle Nanoconjugates

Conjugates of DNA and gold nanoparticles (AuNPs) typically exploit the strong Au-S chemistry to self-assemble thiolated oligonucleotides at AuNPs. However, it remains challenging to precisely control the orientation and conformation of surface-tethered oligonucleotides and finely tune the hybridization ability. We herein report a novel strategy for spatially controlled functionalization of AuNPs with designed diblock oligonucleotides that are free of modifications. We have demonstrated that poly adenine (polyA) can serve as an effective anchoring block for preferential binding with the AuNP surface, and the appended recognition block adopts an upright conformation that favors DNA hybridization. The lateral spacing and surface density of DNA on AuNPs can also be systematically modulated by adjusting the length of the polyA block. Significantly, this diblock oligonucleotide strategy results in DNA-AuNPs nanoconjugates with high and tunable hybridization ability, which form the basis of a rapid plasmonic DNA sensor.

Cascade signal amplification for ultra-sensitive impedimetric detection of DNA hybridization using a hairpin DNA as probe

An ultra-sensitive impedimetric DNA sensor based on cascade signal amplification in sandwich manner was developed. A hairpin DNA was immobilized on a gold electrode and was acted as a probe DNA to improve the selectivity of the device. Without the target DNA, the hairpin DNA probe was in a “closed” state. However, in the presence of a target DNA, the hybridization between the loop moiety and the target broke the stem duplex. Consequently, the stem moiety at the 3′-end of the probes was moved away from the electrode surface and available for hybridization with the reporter DNA–Au nanoparticles conjugates (reporter DNA–AuNPs). The thiolated reporter DNA was complementary to the stem moiety at the 3′-end of the probe. After the probe DNA/target DNA/reporter DNA–AuNPs sandwich complex was formed on the electrode surface, the AuNPs were enlarged by immersing the electrode in a growth solution containing a negatively charged surfactant. Cascaded signal amplification was achieved because the electrode surface became more negatively charged after each assembly step. The electron-transfer resistance (Ret) of the DNA sensor increased with the increase in target DNA concentration through a wide linear range from 0.1fM to 300fM. Under optimized conditions, the DNA sensor could detect as low as 30aM of the fully matched target DNA. The sensor also showed excellent differentiation ability for single-base mismatch.

Direct electrochemistry and electrocatalysis of myoglobin immobilized on DNA-gold nanoparticle clusters composite film

Novel gold nanoparticle clusters conjugated with DNA (DNA–AuNP Clusters), which were synthesized by controlling the cross-linking capability of dithiothreitol (DTT) and particle-stabilizing effect of monothiol DNA, were applied for the immobilization and direct electrochemistry studies of Mb. The DNA–AuNP Clusters-based biocomposite film modified electrode was fabricated through layer-by-layer coating. The morphology, structure and electrochemical properties of the DNA–AuNP Clusters and their biocomposite film were characterized by dynamic light scattering, field emission transmission electron microscopy, UV–vis spectroscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The Mb immobilized in the composite film kept its bioactivity and exhibited greatly enhanced direct electrochemical behavior, which is mainly attributed to the good conductivity and excellent biocompatibility of DNA–AuNP Clusters. The modified electrode displayed good electrocatalytic activity to the reduction of H2O2. Therefore, with combining the advantages of the AuNPs and DNA, this DNA–AuNP Clusters-based composite film could be used as a new promising platform for protein immobilization and novel biosensor fabrication.

Instantaneous and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA Using a pH-Assisted and Surfactant-Free Route

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.

An electrochemical signal ‘off–on’ sensing platform for microRNA detection

The abnormal expression of microRNAs (miRNAs) in many solid tumors makes miRNAs potential biomarkers for disease diagnosis and highlights the need for the sensitive and selective detection of miRNAs. In the present work, an 'off-on' signaling genosensor platform for miRNA-21 detection was well developed. This tactic was based on a locked nucleic acid-integrated nucleic acid hairpin probe, a biotin-labeled bridge DNA-AuNPs-bio-barcode signal amplification unit and enzymatic signal amplification. The test is simple, fast and ultrasensitive with a linear range of 0.01-700 pM. The detection limit was estimated to be 6 fM. The overexpression of miRNA-21 was confirmed in total RNA extracted from human hepatocarcinoma cells BEL-7402 and human HeLa cells compared with the control sample extracted from normal human hepatic L02 cells. This method does not need miRNA-21 labeling, isolation, enrichment or PCR amplification. The performance of the assay developed here could satisfy the need for rapid, easy, sensitive and specific early cancer diagnosis in clinical diagnostics.

Photothermal imaging and measurement of protein shell stoichiometry of single HIV-1 Gag virus-like nanoparticles.

Virus life stages often constitute a complex chain of events, difficult to track in vivo and in real-time. Challenges are associated with spatial and time limitations of current probes: most viruses are smaller than the diffraction limit of optical microscopes while the entire time scale of virus dynamics spans over 8 orders of magnitude. Thus, virus processes such as entry, disassembly, and egress have generally remained poorly understood. Here we discuss photothermal heterodyne imaging (PHI) as a possible alternative to fluorescence microscopy in the study of single virus-like nanoparticle (VNP) dynamics, with relevance in particular to virus uncoating. Being based on optical absorption rather than emission, PHI could potentially surpass some of the current limitations associated with fluorescent labels. As proof-of-principle, single VNPs self-assembled from 60 nm DNA-functionalized gold nanoparticles (DNA-Au NPs) encapsulated in a Gag protein shell of the human immunodeficiency virus (HIV-1) were imaged, and their photothermal response was compared with DNA-Au NPs. For the first time, the protein stoichiometry of a single virus-like particle was estimated by a method other than electron microscopy.

Selective Enhancement of Nucleases by Polyvalent DNA-Functionalized Gold Nanoparticles

We demonstrate that polyvalent DNA-functionalized gold nanoparticles (DNA-Au NPs) selectively enhance ribonuclease H (RNase H) activity while inhibiting most biologically relevant nucleases. This combination of properties is particularly interesting in the context of gene regulation, since high RNase H activity results in rapid mRNA degradation and general nuclease inhibition results in high biological stability. We have investigated the mechanism of selective RNase H activation and found that the high DNA density of DNA-Au NPs is responsible for this unusual behavior. This work adds to our understanding of polyvalent DNA-Au NPs as gene regulation agents and suggests a new model for selectively controlling protein-nanoparticle interactions.

Highly stable, amphiphilic DNA-encoded nanoparticle conjugates for DNA encoding/decoding applications

We synthesized highly stable, amphiphilic polymer-protected DNA–Au nanoparticle conjugates (DNA–AuNPs) and developed a DNA encoding/decoding strategy using these particles. Here, DNA–AuNPs were encapsulated by amphiphilic poly(N-vinyl-2-pyrrolidone) (PVP) to make them more stable and soluble in both organic and aqueous solvents. These particles are stable in a wide range of salt concentrations, pH and temperatures and sustain their stability even after the addition of D,L-dithiothreitol. Finally, this polymer layer was readily removed and particle-modified DNA was decoded via the microarrayed chip and dark-field-based AuNP imaging technique.

A highly selective and sensitive on–off sensor for silver ions and cysteine by light scattering technique of DNA-functionalized gold nanoparticles

DNA-functionalized gold nanoparticles are shown to act as a light-scattering switch. Ag(+) ion turns on the switch through the DNA-Au NPs conjugates based on the formation of cytosine-Ag(+)-cytosine base pairs, whereas cysteine turns off the light-scattering signal because it competitively binds to Ag(+).

DNA hybridization of pathogenicity island of vancomycin-resistant Enterococcus faecalis with discretely functionalized gold nanoparticles in organic solvent mixtures

Short single-strand (ss) DNA sensors that involve mono-functionalized gold nanoparticles are reported for the first time. By way of an example, the hybridization and detection of a 10 base-pair oligonucleotide (TGACTCTTAA), which relates to the pathogenicity island of vancomycin-resistant Enterococcus faecalis, has been demonstrated in the organic solvent mixture (DMF/CHCl3/H2O/NaCl) at sub-nanomole concentrations. The formation of the resulting double-strand (ds) DNA–AuNP complexes which involve either tail-to-tail or head-to-head alignment, are sensitive to salt (NaCl) and solvent (CHCl3) concentrations. By UV/visible absorption measurements, it has been found that the melting temperature of the dsDNA–AuNP complexes increases with increasing salt concentrations as well as the chloroform ratio, for both tail-to-tail and head-to-head alignments. Transmission electron microscopic images of the dsDNA–AuNP complexes with mono-functionalized AuNPs reveal significant amounts of dimeric AuNP species while that of randomly functionalized AuNPs reveal an agglomerated AuNP nanostructures.

In vitro transcription and translation inhibition via DNA functionalized goldnanoparticles

The use of gold nanoparticles (AuNPs) has been gaining momentum as vectors for genesilencing strategies, combining the AuNPs’ ease of functionalization with DNA and/orsiRNA, high loading capacity and fast uptake by target cells. Here, we used AuNPfunctionalized with thiolated oligonucleotides to specifically inhibit transcriptionin vitro, demonstrating the synergetic effect between AuNPs and a specific antisensesequence that blocks the T7 promoter region. Also, AuNPs efficiently protect theantisense oligonucleotide against nuclease degradation, which can thus retain itsinhibitory potential. In addition, we demonstrate that AuNPs functionalizedwith a thiolated oligonucleotide complementary to the ribosome binding site andthe start codon, effectively shut down in vitro translation. Together, these twoapproaches can provide for a simple yet robust experimental set up to test for efficientgene silencing of AuNP–DNA conjugates. What is more, these results show thatappropriate functionalization of AuNPs can be used as a dual targeting approach to anenhanced control of gene expression—inhibition of both transcription and translation.

Scavenger Receptors Mediate Cellular Uptake of Polyvalent Oligonucleotide-Functionalized Gold Nanoparticles

Mammalian cells have been shown to internalize oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs or siRNA-Au NPs) without the aid of auxiliary transfection agents and use them to initiate an antisense or RNAi response. Previous studies have shown that the dense monolayer of oligonucleotides on the nanoparticle leads to the adsorption of serum proteins and facilitates cellular uptake. Here, we show that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. We identify the pathway for DNA-Au NP entry in HeLa cells. Biochemical analyses indicate that DNA-Au NPs are taken up by a process involving receptor-mediated endocytosis. Evidence shows that DNA-Au NP entry is primarily mediated by scavenger receptors, a class of pattern-recognition receptors. This uptake mechanism appears to be conserved across species, as blocking the same receptors in mouse cells also disrupted DNA-Au NP entry. Polyvalent nanoparticles functionalized with siRNA are shown to enter through the same pathway. Thus, scavenger receptors are required for cellular uptake of polyvalent oligonucleotide functionalized nanoparticles.

Ultrasensitive nucleic acid biosensor based on enzyme–gold nanoparticle dual label and lateral flow strip biosensor

In this article, we describe an ultrasensitive nucleic acid biosensor (NAB) based on horseradish peroxidase (HRP)–gold nanoparticle (Au-NP) dual labels and lateral flow strip biosensor (LFSB). The results presented here expand on prior work ( Mao et al., 2009a) by optimizing the preparation of HRP–Au-NP–DNA conjugates. It was found that sodium dodecyl sulfate (SDS) and the immobilization sequence of thiolated DNA and HRP on the Au-NP surface played very important roles to improve the sensitivity of the assay. After systematic optimization, the detection limit of current approach is 1000 times lower than that in prior work. Deposition of insoluble enzymatic catalytic product (red colored chromogen) on the captured Au-NPs at the test zone of LFSB offers a dramatic visual enhancement. Combining enzyme catalytic amplification with unique optical properties of Au-NPs, the NAB was capable of detecting of 0.01-pM target DNA without instrumentation. The NAB thus provides a rapid, sensitive, low-cost tool for the detection of nucleic acid samples. It shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents.

Detection of Hepatitis B Virus Deoxyribonucleic Acid Based on Gold Nanoparticle Probe Chip

Abstract Nanoparticle-based bio-barcode amplification (BCA) approach was developed for the sensitive detection of hepatitis B virus (HBV) DNA. The BCA approach employed two sets of particles: (1) two-component oligonucleotide-modified gold nanoparticles (AuNPs): one oligonucleotide probe called signal probe (barcode DNA) is partially complementary with the target sequence of HBV DNA, and another one called detection probe is partially complementary with the signal probe; (2) single component oligonucleotide-modified magnetic microparticles (MMPs). In the presence of the target molecule, the gold nanoparticles and magnetic particles formed sandwich hybrids. Along with the isolation of sandwiched hybrids from the sample using a magnet, nonspecific-bound gold nanoparticles were removed, ensuring that only target-bound gold nanoparticles were collected. Subsequently, the bar-code DNAs were dehybridized from the gold nanoparticle by using the dithiothreitol (DTT). The detection probe with AuNP mixed with bar-code DNA solution was then added to a bar-code capture DNA-modified chip, and the spots on chip were labeled with bar-code DNA strands and AuNP probes. Finally, the scanometric detection of silver stain was introduced to further amplify the signal. The results showed that it was possible in a format that offers low fM (10−15 M) sensitivity in the detection of HBV DNA sample, and there is a good linear relationship between target concentration and spot intensity on chip. The detection of the assay could be fulfilled within 1.5 h. The method could provide a new generation of diagnostic assays for HBV or the other infectious disease.

Delivery of shRNA using gold nanoparticle–DNA oligonucleotide conjugates as a universal carrier

The efficient delivery of nucleic acids into mammalian cells is a central aspect of research involving cell biology and medical applications, including the clinical treatment of genetic disorders. We report an efficient small hairpin RNA (shRNA) delivery system that utilizes a single species of gold nanoparticle–DNA oligonucleotide conjugate (AuNP–DNA oligo) as a universal carrier. In vitro synthesized shRNA that is specific to the p53 gene was efficiently delivered into HEK293 and HeLa human cell lines using an AuNP–DNA oligo. The delivery resulted in an 80–90% knockdown of p53 expression. The same AuNP–DNA oligo was also efficient for the delivery of another shRNA, which is specific to the Mcl-1 gene, as well as the repression of MCL-1 expression. The knockdown efficiency of shRNA that was delivered using an AuNP–DNA oligo was comparable with that of a liposome-based shRNA delivery method. Our results offer an alternate delivery system for shRNA that can be used on any gene of interest.

Theophylline detection using an aptamer and DNA–gold nanoparticle conjugates

A detection system for theophylline that combined the recognition properties of an aptamer and the plasmonic response of gold nanoparticles (AuNPs) is presented. The aptamer was used as a linker for AuNPs functionalized with complementary sequences to the aptamer (DNA–AuNPs), producing supramolecular complexes that disassemble when exposed to theophylline due to aptamer binding. The detection event was reported as a change in the AuNPs plasmonic peak and intensity. Addition of a spacer on the DNA immobilized on the AuNPs facing the aptamer binding site improved the aggregates’ response, doubling the detection range of system response to theophylline. Modification of the oligonucleotides immobilized on the AuNPs that reduced the interparticle distance in the aggregated state suppressed their response to theophylline and addition of the spacer recovered it. This work demonstrated that the design of oligonucleotides immobilized on the AuNPs could be used to improve their plasmonic response without affecting aptamer performance.

Colloidal Au-enhanced surface plasmon resonance imaging: application in a DNAhybridization process

The detection of the DNA hybridization mechanism using monodispersed goldnanoparticles as labels is an interesting alternative to increase the sensitivityof the SPR imaging technique. DNA-modified Au nanoparticles (DNA-Au NPs)containing single-stranded (ss) portions of DNA were prepared by monitoringtheir monolayer formation by UV–vis spectroscopy. The hybridization processbetween specific thio-oligonucleotides immobilized on the DNA–Au NPs and thecorresponding complementary strands is reported and compared with the traditionalhybridization process on properly self-assembled thin gold films deposited on glasssubstrates. A remarkable signal amplification is observed, following the incorporation ofcolloidal Au into a SPR biosensing experiment, resulting in an increased SPRresponse to DNA–DNA interactions. In particular Fusarium thiolated DNA (5′HS poly(T)15ATC CCT CAA AAA CTG CCG CT-3) and trichothecenes complementary DNA (5′-AGC GGC AGTTTT TGA GGG AT-3′) sequences have been explored due to their possible application to agro-industry for thecontrol of food quality.

Noncovalent DNA decorations of graphene oxide and reduced graphene oxide toward water-soluble metal–carbon hybrid nanostructuresviaself-assembly

Non-covalent DNA decorations on the basal planes of graphene oxide and reduced graphene oxide nanosheets are realized. The resulting DNA–carbon bioconjugates (DNA–GO or DNA–RGO) bearing multiple thiol groups tagged on DNA strands are then employed to scaffold the two-dimensional self-assembly of gold nanoparticles (AuNPs) into metal–carbon hybrid nanostructures (namely AuNP–DNA–GO or AuNP–DNA–RGO) that may find important applications in various aspects. The resulting heteronanostructures incorporating metal nanoparticles obtained by self-assembly are highly stable and water-soluble, and can be easily isolated by gel electrophoresis to guarantee high purity. Thanks to the noncovalent features of this method, either GO or RGO do not suffer from any permanent alterations of their structures and properties. In addition, the nanoparticles still maintain their optical absorbance after being assembled, and the assembly process is highly specific. This self-assembly based method for constructing heterostructured materials is excellent at overcoming any incompatibilities between nanoparticle syntheses and the formation of hybrid structures. As a result, this strategy is easily adaptable to various other materials other than gold nanoparticles and also favors the combinatorial assembly of multiple nanophases on a single nanosheet.

Salt concentration-induced dehybridisation of DNA–gold nanoparticle conjugate assemblies for diagnostic applications

We present the cooperative dehybridisation of DNA-gold nanoparticle conjugate (DNA-AuNP) assemblies induced by reduced salt concentration ([salt]), which can be precisely controlled by various conditions. The detection of Ag(+) based upon the [salt]-induced dehybridisation of DNA-AuNP assemblies is five times more sensitive than that achieved under conventional thermal melting conditions.

Polyvalent Oligonucleotide Gold Nanoparticle Conjugates as Delivery Vehicles for Platinum(IV) Warheads

Amine-functionalized polyvalent oligonucleotide gold nanoparticles (DNA-Au NPs) were derivatized with a cisplatin prodrug, and the resulting DNA-Au NP conjugates were used to internalize multiple platinum centers. A platinum(IV) complex, c,c,t-[Pt(NH(3))(2)Cl(2)(OH)(O(2)CCH(2)CH(2)CO(2)H)], was tethered to the surface of DNA-Au NPs through amide linkages. The platinum-tethered gold nanoparticles were taken into several cancer cells. The drop in intracellular pH facilitated reductive release of cisplatin from the prodrug, which then formed 1,2-d(GpG) intrastrand cross-links in the cell nuclei, as confirmed by an antibody specific for this adduct. The cytotoxicity of the platinum(IV) complex increases significantly in several cancer cell lines when the complex is attached to the surface of the DNA-Au NPs and in some instances exceeds that of cisplatin.