Ultrasensitive DNA detection using oligonucleotide-silver nanoparticle conjugates

Oligonucleotide-gold nanoparticle (OGN) conjugates are powerful tools for the detection of target DNA sequences due to the unique properties conferred upon the oligonucleotide by the nanoparticle. Practically all the research and applications of these conjugates have used gold nanoparticles to the exclusion of other noble metal nanoparticles. Here we report the synthesis of oligonucleotide-silver nanoparticle (OSN) conjugates and demonstrate their use in a sandwich assay format. The OSN conjugates have practically identical properties to their gold analogues and due to their vastly greater extinction coefficient both visual and absorption analyses can occur at much lower concentrations. This is the first report of OSN conjugates being successfully used for target DNA detection and offers improved sensitivity which is of interest to a range of scientists.     

Multiplexed nanoflares: mRNA detection in live cells

We report the development of the multiplexed nanoflare, a nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are spherical nucleic acid (SNA) gold nanoparticle (Au NP) conjugates consisting of densely packed and highly oriented oligonucleotide sequences, many of which are hybridized to a reporter with a distinct fluorophore label and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nanoflares, these structures allow one to determine more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.     

Formation of 1D and 2D gold nanoparticle arrays by divalent DNA-gold nanoparticle conjugates

Divalent DNA-AuNP (gold nanoparticle) conjugates comprising two DNA strands at diametrically opposed positions are prepared. Highly linear 1D and tetragonal lattice-like 2D AuNP arrays are constructed using the conjugates and DNA assemblies based on T- and double-crossover motifs and the Holliday junction.     

PLGA Spherical Nucleic Acids

A new class of polymer spherical nucleic acid (SNA) conjugates comprised of poly(lactic‐co‐glycolic acid) (PLGA) nanoparticle (NP) cores is reported. The nucleic acid shell that defines the PLGA‐SNA exhibits a half‐life of more than 2 h in fetal bovine serum. Importantly, the PLGA‐SNAs can be utilized to encapsulate a hydrophobic model drug, coumarin 6, which can then be released in a polymer composition‐dependent tunable manner, while the dissociation rate of the nucleic acid shell remains relatively constant, regardless of core composition. Like prototypical gold NP conjugate SNAs, PLGA‐SNAs freely enter Raw‐Blue cells and can be used to activate toll‐like receptor 9 in a sequence‐ and dose‐dependent manner. Taken together, the data show that this novel nanoconstruct provides a means for controlling the release kinetics of encapsulated cargos in the context of the SNA platform, which may be useful for developing combination therapeutics.     

Isolation of discrete nanoparticle-DNA conjugates for plasmonic applications

Discrete DNA-gold nanoparticle conjugates with DNA lengths as short as 15 bases for both 5 and 20 nm gold particles have been purified by anion-exchange HPLC. Conjugates comprising short DNA (<40 bases) and large gold particles (> or =20 nm) are difficult to purify by other means and are potential substrates for plasmon coupling experiments. Conjugate purity is demonstrated by hybridizing complementary conjugates to form discrete structures, which are visualized by TEM.     

Quantitative surface acoustic wave detection based on colloidal gold nanoparticles and their bioconjugates

The immobilization scheme of monodispersed gold nanoparticles (10-nm diameter) on piezoelectric substrate surfaces using organosilane molecules as cross-linkers has been developed for lithium niobate (LiNbO3) and silicon oxide (SiO2)/gold-covered lithium tantalate (LiTaO3) of Rayleigh and guided shear horizontal- (guided SH) surface acoustic wave (SAW) sensors. In this study, comparative measurements of gold nanoparticle adsorption kinetics using high-resolution field-emission scanning electron microscopy and SAW sensors allow the frequency responses of SAW sensors to be quantitatively correlated with surface densities of adsorbed nanoparticles. Using this approach, gold nanoparticles are used as the "nanosized mass standards" to scale the mass loading in a wide dynamical range. Rayleigh-SAW and guided SH-SAW sensors are employed here to monitor the surface mass changes on the device surfaces in gas and liquid phases, respectively. The mass sensitivity ( approximately 20 Hz.cm2/ng) of Rayleigh-SAW device (fundamental oscillation frequency of 113.3 MHz in air) is more than 2 orders of magnitude higher than that of conventional 9-MHz quartz crystal microbalance sensors. Furthermore, in situ (aqueous solutions), real-time measurements of adsorption kinetics for both citrate-stabilized gold nanoparticles and DNA-gold nanoparticle conjugates are also demonstrated by guided SH-SAW (fundamental oscillation frequency of 121.3 MHz). By comparing frequency shifts between the adsorption cases of gold nanoparticles and DNA-gold nanoparticle conjugates, the average number of bound oligonucleotides per gold nanoparticle can also be determined. The high mass sensitivity ( approximately 6 Hz.cm2/ng) of guided SH-SAW sensors and successful detection of DNA-gold nanoparticle conjugates paves the way for real-time biosensing in liquids using nanoparticle-enhanced SAW devices.     

Silver nanoparticle-oligonucleotide conjugates based on DNA with triple cyclic disulfide moieties

We report a new strategy for preparing silver nanoparticle-oligonucleotide conjugates that are based upon DNA with cyclic disulfide-anchoring groups. These particles are extremely stable and can withstand NaCl concentrations up to 1.0 M. When silver nanoparticles functionalized with complementary sequences are combined, they assemble to form DNA-linked nanoparticle networks. This assembly process is reversible with heating and is associated with a red shifting of the particle surface plasmon resonance and a concomitant color change from yellow to pale red. Analogous to the oligonucleotide-functionalized gold nanoparticles, these particles also exhibit highly cooperative binding properties with extremely sharp melting transitions. This work is an important step toward using silver nanoparticle-oligonucleotide conjugates for a variety of purposes, including molecular diagnostic labels, synthons in programmable materials synthesis approaches, and functional components for nanoelectronic and plasmonic devices.     

The Impact of Protein Corona Formation on the Macrophage Cellular Uptake and Biodistribution of Spherical Nucleic Acids

The effect of serum protein adsorption on the biological fate of Spherical Nucleic Acids (SNAs) is investigated. Through a proteomic analysis, it is shown that G‐quadruplexes templated on the surface of a gold nanoparticle in the form of SNAs mediate the formation of a protein corona that is rich in complement proteins relative to SNAs composed of poly‐thymine (poly‐T) DNA. Cellular uptake studies show that complement receptors on macrophage cells recognize the SNA protein corona, facilitating their internalization, and causing G‐rich SNAs to accumulate in the liver and spleen more than poly‐T SNAs in vivo. These results support the conclusion that nucleic acid sequence and architecture can mediate nanoparticle–biomolecule interactions and alter their cellular uptake and biodistribution properties and illustrate that nucleic acid sequence is an important parameter in the design of SNA therapeutics.     

Amplified voltammetric detection of DNA hybridization via oxidation of ferrocene caps on gold nanoparticle/streptavidin conjugates

Gold nanoparticle/streptavidin conjugates covered with 6-ferrocenylhexanethiol were attached onto a biotinylated DNA detection probe of a sandwich DNA complex. Due to the elasticity of the DNA strands, the ferrocene caps on gold nanoparticle/streptavidin conjugates are positioned in close proximity to the underlying electrode modified with a mixed DNA capture probe/hexanethiol self-assembled monolayer and can undergo reversible electron-transfer reactions. A detection level, down to 2.0 pM (10 amol for the 5 microL of sample needed) for oligodeoxynucleotide samples was obtained. The amplification of the voltammetric signals was attributed to the attachment of a large number of redox (ferrocene) markers per DNA duplex formed. The ferrocene oxidation current increased with the target concentration and began to level off at a target concentration of 10 nM. An Excellent linearity was found within the range between 6.9 and 150.0 pM and reasonable relative standard deviations (between 3.0 and 13.0%) were obtained. The amenability of this method to the analyses of polynucleotides (i.e., PCR products of the pre-S gene of hepatitis B virus in serum samples) was also demonstrated. The method is shown to be simple, selective, reproducible, and cost-effective and does not require labeling of the DNA targets.     

Homogeneous detection of nucleic acids based upon the light scattering properties of silver-coated nanoparticle probes

Herein we report the development of a simple, rapid, homogeneous, and sensitive detection system for DNA based on the scattering properties of silver-amplified gold nanoparticle probes. The assay uses DNA-functionalized magnetic particle probes that act as scavengers for target DNA, which can be collected via a magnetic field. Once the DNA targets are isolated from the initial sample, they are sandwiched via hybridization by a second set of probes. The latter probes are 13-nm gold nanoparticles modified with a different target complementary DNA. Excess probes are removed through repetitive washing steps. The gold particles are dispersed in solution by dehybridization, corresponding to an assumed 1:1 ratio with the target DNA. Electroless deposition of silver on the surface of the gold probes results in particle growth, which increases their scattering efficiency with time. The scattering efficiency and the extinction signatures of the particle sizes are monitored as a function of time and correlated with target concentration. The limit of detection for this novel assay was determined to be 10 fM.     

Modulation of Interparticle Distance in Discrete Gold Nanoparticle Dimers and Trimers by DNA Single‐Base Pairing

Self‐assembled structures of metallic nanoparticles with dynamically changeable interparticle distance hold promise for the regulation of collective physical properties. This paper describes gold nanoparticle dimers and trimers that exhibit spontaneous and reversible changes in interparticle distance. To exploit this property, a gold nanoparticle is modified with precisely one long DNA strand and approximately five short DNA strands. The long DNA serves to align the nanoparticles on a template DNA via hybridization, while the short DNAs function to induce the interparticle distance changes. The obtained dimer and trimer are characterized with gel electrophoresis, dynamic light scattering measurements, and transmission electron microscopy (TEM). When the complementary short DNA is added to form the fully matched duplexes on the particle surface in the presence of MgCl₂, spontaneous reduction of the interparticle distance is observed with TEM and cryo‐electron microscopy. By contrast, when the terminal‐mismatched DNA is added, no structural change occurs under the same conditions. Therefore, the single base pairing/unpairing at the outermost surface of the nanoparticle impacts the interparticle distance. This unique feature could be applied to the regulation of structures and properties of various DNA‐functionalized nanoparticle assemblies.     

Ultra-Stable oligonucleotide-gold and -silver nanoparticle conjugates prepared by a facile silica reinforcement method

We report a simple method of enhancing the chemical stability of monothiol-modified oligonucleotide-gold and -silver nanoparticle conjugates by a thin silica reinforcement coating. Conventional conjugates prepared by chemisorption of monothiol-modified oligonucleotides onto nanoparticle surfaces undergo rapid aggregation in the presence of thiol-containing small molecules (e.g., dithiothreitol) due to ligand exchange reactions. When the conjugates are treated with (3-mercaptopropyl)trimethoxysilane, a thin silica layer is formed on the nanoparticle surface, thereby entrapping and reinforcing the thiol-gold/-silver linkage. These silica-modified oligonucleotide-gold and -silver nanoparticle conjugates become much more stable toward dithiothreitol as compared to the unmodified conjugates. Moreover, the silica layer significantly hinders the gold/silver core from oxidative dissolution by sodium cyanide. Importantly, the unique hybridization-induced color change property of the oligonucleotide-gold and -silver nanoparticle conjugates is preserved even under harsh condition (i.e., high concentrations of dithiothreitol). Taken together, these ultra-stable oligonucleotide-nanoparticle conjugates hold promise for new diagnostics and therapeutics.      

Two-dimensional nanoparticle arrays show the organizational power of robust DNA motifs

The bottom-up spatial organization of potential nanoelectronic components is a key intermediate step in the development of molecular electronics. We describe robust three-space-spanning DNA motifs that are used to organize nanoparticles in two dimensions. One strand of the motif ends in a gold nanoparticle; only one DNA strand is attached to the particle. By using two of the directions of the motif to produce a two-dimensional crystalline array, one direction is free to bind gold nanoparticles. Identical motifs, tailed in different sticky ends, enable the two-dimensional periodic ordering of 5 and 10 nm diameter gold nanoparticles.     

Critical flocculation concentrations, binding isotherms, and ligand exchange properties of peptide-modified gold nanoparticles studied by UV-visible, fluorescence, and time-correlated single photon counting spectroscopies

Protocols for modifying gold nanoparticles with peptide-bovine serum albumin (BSA) conjugates are described within. The resulting constructs were characterized using a number of techniques including static fluorescence spectroscopy and time-correlated single photon counting spectroscopy (TCSPC) in order to quantify peptide-BSA binding isotherms, exchange rates, critical flocculation concentrations, and the composition of mixed peptide-BSA monolayers on gold nanoparticles. TCSPC has proven to be a powerful technique for observing the microenvironment of protein-gold nanoparticle conjugates because it can distinguish between surface-bound and solution-phase species without the need for separation steps. Full characterization of the composition and stability of peptide-modified metal nanoparticles is an important step in their use as intracellular delivery vectors and imaging agents.     

The Sequence‐Specific Cellular Uptake of Spherical Nucleic Acid Nanoparticle Conjugates

The sequence‐dependent cellular uptake of spherical nucleic acid nanoparticle conjugates (SNAs) is investigated. This process occurs by interaction with class A scavenger receptors (SR‐A) and caveolae‐mediated endocytosis. It is known that linear poly(guanine) (poly G) is a natural ligand for SR‐A, and it has been proposed that interaction of poly G with SR‐A is dependent on the formation of G‐quadruplexes. Since G‐rich oligonucleotides are known to interact strongly with SR‐A, it is hypothesized that SNAs with higher G contents would be able to enter cells in larger amounts than SNAs composed of other nucleotides, and as such, cellular internalization of SNAs is measured as a function of constituent oligonucleotide sequence. Indeed, SNAs with enriched G content show the highest cellular uptake. Using this hypothesis, a small molecule (camptothecin) is chemically conjugated with SNAs to create drug‐SNA conjugates and it is observed that poly G SNAs deliver the most camptothecin to cells and have the highest cytotoxicity in cancer cells. Our data elucidate important design considerations for enhancing the intracellular delivery of spherical nucleic acids.     

Electrochemically programmed release of biomolecules and nanoparticles

The controlled release of molecules or nanoparticle conjugates is an important tool for a wide range of applications in science and engineering. Here we demonstrate electrochemically programmed release of biomolecules and nanoparticles immobilized on patterned gold electrodes using the thiol-gold linkage. This technique exploits the reductive desorption of self-assembled monolayers and allows both spatially controlled release and regeneration of small molecules (e.g., drugs), biopolymers (e.g., peptides, proteins, DNA), protein assemblies (e.g., viruses), and nanoparticles (e.g., particle-DNA conjugates). Fluorescence microscopy is used to image the release of avidin and nanoparticles in phosphate-buffered saline and to determine the kinetics of desorption. We also demonstrate that the electrodes can be regenerated using the same conjugation scheme.     

Designed hybridization properties of DNA-gold nanoparticle conjugates for the ultraselective detection of a single-base mutation in the breast cancer gene BRCA1

We have investigated the hybridization properties of DNA-gold nanoparticle conjugates and have discovered that the hybridization properties are dramatically affected by controlling various synthetic and environmental conditions. We have further demonstrated that moderate DNA loading instead of high loading per nanoparticle significantly enhances the hybridization rates of DNA-gold nanoparticle conjugates, which allows one to precisely design their hybridization properties to distinguish a single-nucleotide polymorphism (SNP). A diagnostic application for the colorimetric detection of an SNP associated with a mutation in the breast cancer gene BRCA1 has been carefully designed and demonstrated.     

DNA-controlled assembly of a NaTl lattice structure from gold nanoparticles and protein nanoparticles

The formation of diamond structures from tailorable building blocks is an important goal in colloidal crystallization because the non-compact diamond lattice is an essential component of photonic crystals for the visible-light range. However, designing nanoparticle systems that self-assemble into non-compact structures has proved difficult. Although several methods have been proposed, single-component nanoparticle assembly of a diamond structure has not been reported. Binary systems, in which at least one component is arranged in a diamond lattice, provide alternatives, but control of interparticle interactions is critical to this approach. DNA has been used for this purpose in a number of systems. Here we show the creation of a non-compact lattice by DNA-programmed crystallization using surface-modified Qβ phage capsid particles and gold nanoparticles, engineered to have similar effective radii. When combined with the proper connecting oligonucleotides, these components form NaTl-type colloidal crystalline structures containing interpenetrating organic and inorganic diamond lattices, as determined by small-angle X-ray scattering. DNA control of assembly is therefore shown to be compatible with particles possessing very different properties, as long as they are amenable to surface modification.     

Sharpening the Thermal Release of DNA from Nanoparticles: Towards a Sequential Release Strategy

Unlike the sharp melting behavior of DNA‐linked nanoparticle aggregates, the melting of DNA strands from individual gold nanoparticles is broad despite the high surface density of bound DNA. Here, it is demonstrated how sharpened melting can be achieved in colloidal nanoparticle systems using branched DNA–doubler structures hybridized with complementary DNA‐doublers bound to the gold nanoparticle. Moreover, sharpened transitions are observed when DNA‐doublers are hybridized with linear DNA‐modified gold nanoparticles. This result suggests that the DNA density on nanoparticles is intrinsically great enough to form cooperative structures with the DNA‐doublers. Finally, by introducing abasic destabilizing groups, the melting temperature of these DNA‐doublers decreases without decreasing the sharpness. Consequently, by varying the temperature, two DNA‐doublers with different stabilities dissociate sequentially from the gold nanoparticle surface, without overlapping and within a narrow temperature window. Owing to the excellent thermal selectivities exhibited by this system, the implementation of DNA‐doublers in sequential photothermal therapies and with other nanomedicine delivery agents that rely on DNA dissociation as the mechanism of selective release is anticipated.     

Nanoparticle-enhanced surface plasmon resonance detection of proteins at attomolar concentrations: comparing different nanoparticle shapes and sizes

The application of biofunctionalized nanoparticles possessing various shapes and sizes for the enhanced surface plasmon resonance (SPR) detection of a protein biomarker at attomolar concentrations is described. Three different gold nanoparticle shapes (cubic cages, rods and quasi-spherical) with each possessing at least one dimension in the 40-50 nm range were systematically compared. Each nanoparticle (NP) was covalently functionalized with an antibody (anti-thrombin) and used as part of a sandwich assay in conjunction with a Au SPR chip modified with a DNA-aptamer probe specific to thrombin. The concentration of each NP-antibody conjugate solution was first optimized prior to establishing that the quasi-spherical nanoparticles resulted in the greatest enhancement in sensitivity with the detection of thrombin at concentrations as low as 1 aM. When nanorod and nanocage antibody conjugates were instead used, the minimum target concentrations detected were 10 aM (rods) and 1 fM (cages). This is a significant improvement (>10(3)) on previous NP-enhanced SPR studies utilizing smaller (~15 nm) gold NP conjugates and is attributed to the functionalization of both the NP and chip surfaces resulting in low nonspecific adsorption as well as a combination of density increases and plasmonic coupling inducing large shifts in the local refractive index at the chip surface upon nanoparticle adsorption.