DNA-functionalized Gold Nanoparticles Research Articles

Visual detection of kanamycin with DNA-functionalized gold nanoparticles probe in aptamer-based strip biosensor

Kanamycin has been widely used to treat human and animal diseases. The excessive use of kanamycin causes its accumulation in animal-derived foods, and eventually threats human health. In the present study, we develop a lateral flow strip biosensor for fast and sensitive detection of kanamycin. The strip biosensor combines the easy separation of magnetic microspheres (MMS) with target-mediated chain displacement of single-stranded DNA and the capture of the visible DNA-functionalized gold nanoparticles (AuNPs) probe. The presence of kanamycin can competitively bind to the aptamer and release cDNA to the supernatant. The concentration of free cDNA, which is the direct target of the strip, is proportional to the concentration of kanamycin. The capture of DNA-functionalized AuNPs on the test zone of the strip through cDNA-induced hybridization provides a visual detection signal. The assay can be completed within 20 min. The visual detection limit by naked eyes of the strip is 50 nM. A linear detection range of 5–500 nM is derived for quantitative determination, with the detection limit of 4.96 nM (S/N = 3). This lateral flow strip biosensor can quickly and sensitively detect kanamycin in different food samples, which holds great application potential in medicine and daily life.

Naked-Eye Detection of Grapevine Red-Blotch Viral Infection Using a Plasmonic CRISPR Cas12a Assay.

Herein, we described a novel plasmonic CRISPR Cas12a assay for the visual, colorimetric detection of grapevine viral infections. Our assay generates rapid and specific colorimetric signals for nucleic acid amplicons by combining the unique target-induced incriminate single-stranded DNase activity of Cas12a with plasmon coupling of DNA functionalized gold nanoparticles. The practical applicability of our plasmonic assay was successfully demonstrated through the detection of emerging red-blotch viral infections in grapevine samples collected from commercial vineyards.

DNA-Conjugated Gold Nanoparticles as High-Mass Probes in Imaging Mass Cytometry.

We employ imaging mass cytometry (IMC) to investigate in vitro uptake and cellular distribution of DNA-functionalized gold nanoparticles (AuNPs). IMC enables the multiparametric imaging of cell components and allows for the detection of AuNPs in cells with >100 times higher sensitivity than conventional confocal fluorescence imaging, as each nanoparticle contains thousands of atoms for signal amplification. Changes in the accumulation of nanoparticles in cells due to oligonucleotide sequence-dependent interactions are exploited to examine a model biomarker for hypoxia-microRNA-210. We find that AuNPs functionalized with microRNA-210-targeting sequence accumulate in hypoxic cells 3 to 4-fold compared to normoxic cells. The work examines the potential use of DNA-AuNP as high-mass probes for the analysis of nonabundant nucleic acids.

An aptamer based aggregation assay for the neonicotinoid insecticide acetamiprid using fluorescent upconversion nanoparticles and DNA functionalized gold nanoparticles.

An acetamiprid-binding aptamer (ABA), gold nanoparticles (AuNPs) and upconversion nanoparticles (UCNPs) are used in a colorimetric and fluorometric method for the ultrasensitive and selective detection of the pesticide acetamiprid. The ABA is first configured into a duplex with a complementary DNA covalently attached to AuNPs. The resulting dsDNA-functionalized AuNP probe is not stable in 0.15M NaCl solution and aggregates. This causing the color to change from red to purple. In the presence of acetamiprid, the ABA undergoes a structural switch from a DNA duplex to an aptamer-acetamiprid complex and consequently dissociates from the AuNPs. The partially unhybridized AuNPs are stable against salt-induced aggregation and show red color. The ratio of absorbances at 524nm (red) and 650nm (purple blue) varies with the concentration of acetamiprid in the 0.025-10μM concentration range. The colorimetric signal can be further amplified by introducing DNA-modified carboxylated UCNPs (silica-coated NaYF4:Yb,Er) which display red and green fluorescence under 980nm excitation. An inner filter effect occurs between DNA-modified UCNPs and dsDNA-modified AuNPs. The fluorometric assay is based on the measurement of the ratio of red (654nm) and green (540nm) fluorescence and works in the 0.025 to 1μM acetamiprid concentration range and has a 0.36nM detection limit (at a signal-to-noise ratio of 3). Because of the specificity of the aptamer, the assay is high selective. It was successfully used to quantify acetamiprid in contaminated real samples. Graphical abstract Schematic presentation of an upconversion fluorescent assay for acetamiprid. It involves the principle of analyte-triggered structural switch of aptamers, salt-induced AuNP aggregation, and signal amplification from UCNP.

Three-Dimensional Molecular Transfer from DNA Nanocages to Inner Gold Nanoparticle Surfaces.

It is of great interest to construct DNA-functionalized gold nanoparticles (DNA-AuNPs) with a controllable number of DNA strands and relative orientations. Herein, we describe a three-dimensional (3D) molecular transfer strategy, in which a pattern of DNA strands can be transferred from a DNA icosahedron cage (I-Cage) to the wrapped AuNP surface. The results show that DNA-AuNPs produced by this method inherit DNA pattern information encoded in the transient I-Cage template with high fidelity. Controllable numbers and positions of DNA on the surface of AuNPs can be simultaneously realized by direct "printing" of a DNA pattern from the nanoshell (I-Cage) to the nanocore (AuNP), further expanding the applications of DNA nanotechnology to nanolithography. Prospectively, the customized DNA-printed nanoparticles possess great potential for constructing programmable architectures for optoelectronic devices as well as smart biosensors for biomedical applications.

Label-free and amplified electrogenerated chemiluminescence biosensing for the detection of thymine DNA glycosylase activity using DNA-functionalized gold nanoparticles triggered hybridization chain reaction

Effective detection of thymine DNA glycosylase (TDG) activity is extremely crucial and urgent for epigenetic research. Herein, a novel label-free electrogenerated chemiluminescence (ECL) biosensing method was developed for the detection of TDG activity using DNA-functionalized gold nanoparticles (DNA-AuNPs) triggered hybridization chain reaction (HCR). In this assay, the thiol modified hairpin probe DNA (hp-DNA) with 5′ overhangs and one mismatched base pair of guanines: thymine (G: T) in the stem part was boned onto gold electrode. TDG specifically removed T base of the G: T mismatch to produce apyrimidinic (AP) sites through the N-glycosidic bond hydrolysis. The AP site was then cleaved by the catalysis of Endonuclease IV (EnIV) to generate dsDNA containing a free 3’ end in the long sequence, which serves as a complementary sequence to hybridize with the specific sequence (ssDNA1) of DNA-AuNPs. Then, the functionalized DNA-AuNPs with initiator strands (ssDNA2) could trigger HCR to form nicked double helices DNA polymer which can embed numerous ECL indicator, Ru(phen)32+, resulting in significantly increased ECL signal. The proposed strategy combined the amplification function of DNA-AuNPs triggered HCR and the inherent high sensitivity of the ECL technique, a detection limit of 1.1 × 10−5 U/μL (0.0028 ng/mL) for TDG determination was obtained. In addition, this method was successfully applied to evaluate TDG activity in cancer cell, which provides great possibility for TDG activity assay in related clinical diagnostics.

Enhanced Secretion of Functional Insulin with DNA-Functionalized Gold Nanoparticles in Cells.

We have previously shown the use of gold nanoparticles (AuNPs) functionalized with DNA (AuNP-DNA) to increase insulin mRNA translation in a cell-free system. In this study, we translate the concept into a whole cell system to demonstrate functionality despite the additional complexity of intracellular delivery and mRNA translation inside living cells. We selected an insulin-secreting pancreatic islet cell line, RIN-5F, as our model and designed a DNA oligomer (insDNA) that is complementary to the 3'-untranslated region of insulin mRNA for conjugation to AuNPs (AuNP-insDNA). AuNP-insDNA was stable in the extracellular environment of RIN-5F cells for up to 24 h, without eliciting any cell toxicity. Upon cellular entry, AuNP-insDNA was able to sustain enhanced insulin secretion from 6 to 12 h post-incubation, peaking at 10 h with an enhancement factor of 1.69-fold. This enhancement was not observed when insDNA was removed or replaced with poly thymine or poly adenine DNAs. The enhanced insulin secreted was 100% functional and capable of binding to its insulin receptor. The outcome of this study demonstrated the feasibility of AuNP-DNA to enhance the synthesis of proteins in whole cells and could serve as a new direction of invoking a patient's own beta cells to increase insulin secretion for treatment of diabetes.

Characterizing the non-crosslinked aggregation of DNA-modified gold nanoparticles: effects of DNA length and terminal base pair.

We previously reported that fully complementary DNA duplexes formed on gold nanoparticle (GNP) surfaces aggregate at high salt concentrations. We previously reported that DNA-functionalized gold nanoparticles (GNPs) aggregate by hybridization with fully complementary DNA at high salt concentrations. Although this behavior has been applied to some precise naked-eye colorimetric analyses of DNA-related molecules, the aggregation mechanism is still unclear and comprehensive studies are needed. In this paper, we reveal the key factors that influence GNP aggregation. The effects of temperature, electrolyte concentration, probe length, and particle size, which control the stabilities of double-stranded DNAs and GNPs, were investigated. Larger GNPs aggregated more easily, and GNP aggregates were easily formed with ∼15-mer-long probes, while longer probes prevented aggregation, perhaps by preventing the formation of rigid double-stranded DNA layers, compared to shorter probes. Furthermore, GNPs with purine bases at their 5' ends aggregated more easily than those with these bases at their 3' ends. This phenomenon is different from that based on the melting-temperature trend calculated using the nearest-neighbor method.

Colorimetric sensor assay for discrimination of proteins based on exonuclease I-triggered aggregation of DNA-functionalized gold nanoparticles.

Proteins play a key role in disease diagnosis, and protein discrimination is an important but difficult issue. Here, we report a novel strategy for improving protein discrimination through a facile colorimetric sensor array, which is based on DNA-gold nanoparticle (AuNP) conjugates manipulated by exonuclease I (Exo I). Different proteins exhibit diverse affinities toward the three DNAs, and the DNA-protein binding is resistant to the digestion of Exo I and protects the AuNPs from aggregation in high concentrations of NaCl media, forming distinct response patterns of the array. These response patterns as "fingerprints" can be acquired on the sensor array and then identified by linear discriminant analysis (LDA). The sensor array achieved the correct discrimination of 15 proteins at a 10 nM level in buffer solution and real serum samples. Also, the sensor array had the capability to discriminate individual proteins and the mixtures of them. Remarkably, the practicability of the sensor array was further confirmed by the identification of 35 unknown protein samples with 100% accuracy.

Poly(adenine)-mediated DNA-functionalized gold nanoparticles for sensitive detection of mercury ions in aqueous media.

In this work, a facile and sensitive colorimetric sensor for Hg2+ ions based on poly (adenine)-mediated DNA-functionalized gold nanoparticles (Au NPs) is reported. One DNA sequence consisting of poly-A and T-rich DNA was designed rationally. Poly-A was used as an anchoring block to bind tightly to Au NPs, and T-rich DNA was utilized for specific recognition of Hg2+ ions. With the assistance of poly-A, T-rich DNA was easily introduced onto the surface of Au NPs and kept an upright orientation. In the presence of Hg2+ ions, T base binding with Hg2+ ions results in the formation of “T–Hg2+–T” among the Au NPs, which caused aggregation of the Au NPs and a subsequent change in the color of the solution, from wine red to grayish blue. On this occasion, the limit of detection (LOD) was 3.75 nM Hg2+ ions with a linear range from 5 nM to 200 nM, as measured by UV-Vis spectroscopy. Moreover, successful application of this method for the detection of Hg2+ ions in real samples was demonstrated.

Liquid-cell scanning transmission electron microscopy and fluorescence correlation spectroscopy of DNA-directed gold nanoparticle assemblies

In the use of solution-based 3D nanoarchitectures for optics, drug delivery, and cancer treatment, the precise nanoparticle architecture morphologies, architecture sizes, interparticle distances, and the assembly stability are all critical to their functionality. 3D nanoparticle architectures in solution are difficult to characterize, as few techniques can provide individualized information on interparticle spacing (defined by linkage molecule), nanoparticle assembly size, morphology, and identification of false aggregation. Bulk characterization techniques, including small angle x-ray scattering, can provide architecture sizes, though they are unable to precisely measure differences within interparticle spacings for individual architectures and can falsely measure assemblies caused by non-linkage grouped nanoparticles. Two solution-based characterization techniques were used to determine which assembly type and linkage length would produce the fastest assembly rate for large DNA-directed gold nanoparticle assemblies. In-situ liquid-cell scanning transmission electron microscopy (LC-STEM), measured interparticle spacings between DNA-functionalized nanoparticles, and fluorescence correlation spectroscopy provided the bulk volume fraction of large and small assemblies for nanoparticle architectures that were assembled using two different types: (1) the hybrid assemblies join two complementary single-stranded DNA linkages, and (2) the bridged assemblies are comprised of single-stranded DNA (bridging component) that is double the length of two different complementary single-stranded DNA-functionalized gold nanoparticles. Assembly times were tested at 24-hrs intervals over 3 days. Statistics derived from the in-situ LC-STEM images provided data for interparticle distance measurements, which identified the fraction of nanoparticles within the images acquired that were at the expected double-stranded DNA-binding distance of the linkages (varied in three distances for each of the two different architectures). In general, longer linkage lengths assembled in the shortest amount of time. The bridged assemblies formed fewer large architectures at 24-hrs but ultimately assembled a greater fraction of nanoparticles, which was due to the longer functionalized DNA lengths for individual nanoparticles. Fluorescence correlation spectroscopy provided a bulk average of the gold nanoparticle assembly sizes over time, which supported the conclusions drawn from the in-situ LC-STEM data. The microscopy provided sub-2 nm precision in the interparticle distances between gold nanoparticles in a solution environment. This coupled microscopy and spectroscopy characterization approach can provide more detailed information than bulk characterization methods.

Highly Sensitive and Selective Colorimetric Detection of Methylmercury Based on DNA Functionalized Gold Nanoparticles.

A new colorimetric detection of methylmercury (CH3Hg+) was developed, which was based on the surface deposition of Hg enhancing the catalytic activity of gold nanoparticles (AuNPs). The AuNPs were functionalized with a specific DNA strand (HT7) recognizing CH3Hg+, which was used to capture and separate CH3Hg+ by centrifugation. It was found that the CH3Hg+ reduction resulted in the deposition of Hg onto the surface of AuNPs. As a result, the catalytic activity of the AuNPs toward the chromogenic reaction of 3,3,5,5-tetramethylbenzidine (TMB)-H2O2 was remarkably enhanced. Under optimal conditions, a limit of detection of 5.0 nM was obtained for CH3Hg+ with a linear range of 10–200 nM. We demonstrated that the colorimetric method was fairly simple with a low cost and can be conveniently applied to CH3Hg+ detection in environmental samples.

Polyvalent Spherical Nucleic Acids for Universal Display of Functional DNA with Ultrahigh Stability.

For nanomaterials that are difficult to functionalize by covalent attachment of DNA, we herein communicate a general method taking advantage of the high avidity of polyvalent binding and the 3D structure of densely functionalized spherical nucleic acids (SNAs). Using DNA-functionalized gold nanoparticles, simple mixing leads to the formation of highly stable conjugates on 11 different materials including metals, metal oxides, metal-organic frameworks, transition-metal dichalcogenides, nanocarbons, and polymers. The adsorption affinity of SNAs can be over thousand-fold higher than that of free DNA of the same sequence, and practically irreversible conjugates are formed withstanding various denaturing agents. The surface attachment and molecular recognition functions of DNA are spatially separated, showing a key advantage of SNAs. The functionalized materials possess the properties of both the substrate and the SNA, allowing specific DNA hybridization in buffer and in serum.

In Situ Visualization of hERG Potassium Channel via Dual Signal Amplification.

Dysfunction of the human ether-a-go-go related gene (hERG)-encoded potassium channel is identified as a major cause of the long QT syndrome, a marker for lethal cardiac arrhythmia. Furthermore, recent studies revealed that hERG K+ channel is a regulator of tumor cell apoptosis and proliferation. Herein, an ultrasensitive fluorescence assay combining DNA-functionalized gold nanoparticles and rolling circle amplification (RCA) was attempted for the first time to visualize hERG channels in living cells. The spherical nucleic acid gold nanoparticles, which can anchor on hERG channels in the cell membrane, not only act as the primary amplification elements but also trigger the subsequent RCA reaction to achieve the secondary amplification. Within 30 min, the ratio of reporter to target can reach up to 104, realizing the detection of hERG channels in cells with low-level expression. Therefore, the strategy provides a valuable tool for hERG-related studies. More importantly, it opens a new horizon for imaging various membrane proteins which possess specific aptamers or antibodies.

Utilizing Design Principles of Electrochemical DNA Aptamer Biosensors in Multiple Optical Assay Methodologies

DNA aptamers, short oligonucleotides with high specificity and affinity for a variety of molecular targets, have been widely adopted as capture probes and detection elements for a variety of assay and biosensor applications. In particular, electrochemical DNA (E‐DNA) biosensors have been demonstrated for many applications, ranging from the detection of metal ions (e.g., uranium and mercury), to small molecules (e.g., doxorubicin and other antibiotics), and even to large proteins (e.g., botulinum toxin and transcription factors). E‐DNA biosensors essentially consist of an aptamer oligonucleotide region that is incorporated into a larger oligonucleotide scaffold, with an anchoring modification (typically a thiol) and a reporter modification (typically the dye and redox reporter methylene blue) covalently appended. Recent efforts in our lab have focused on taking insights from E‐DNA biosensor design and incorporation of aptamers into functional probes, and applying them to a variety of alternative, optical assays repurposing existing E‐DNA probes. One of these approaches is the demonstration of Raman spectroscopic detection through dye‐labeled DNA functionalized gold nanoparticle assemblies that dissociate in a dose‐dependent response to the presence of the target molecule. A second approach has been the creation of portable, do‐it‐yourself 3D‐printed total internal reflectance fluorescence based assays using dye‐labeled DNA arrays on acrylate microscope slides where fluorescence intensity, which can be captured on a smart phone, reflects target presence. A third approach has focused on the characterization of rapid lateral flow assay (“dipstick”) assays based on DNA functionalized gold nanoparticles and nitrocellulose arrays, where target presence is reflected in the intensity of a nanoparticle‐based color line. Ultimately, these projects illustrate the high transferability of DNA aptamer biosensor design parameters between different sensing modalities, offering insight into engineering successful biosensor designs.Support or Funding InformationThis work is supported by internal grant funding from the Metropolitan State University of Denver.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nonlinear optical behavior of DNA-functionalized gold nanoparticles

The third-order nonlinear optical properties of gold nanoparticles embedded in the DNA-based composites were investigated by means of the third harmonic generation. With this purpose, the thin films comprising DNA-based complexes and Au nanoparticles were spin-deposited on glass substrate and their optical and nonlinear optical features were studied using the Maker-fringe technique at a laser fundamental wavelength of 1064 nm. The values of the third-order susceptibility χ(3)(− 3ω; ω, ω, ω) of the composite films based on DNA complex doped with 5 wt% of N-ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo)aniline were found to be significantly higher than those for pure composite films. Meanwhile, the presence of Au nanoparticles noticeable decreases the third-order nonlinear response of DNA-based composite mainly due to the enhanced absorption and scattering of laser and generated beam, respectively.

A technique for highly sensitive detection of mercury ions using DNA-functionalized gold nanoparticles and resonators based on a resonance frequency shift

Mercury is widely used in various research and industrial areas. However, due to the ionization of mercury to the mercury ion, the influence of this ion on human health and the environment is cause for concern. Gold nanoparticles (AuNPs) are the most widely studied nanomaterial because of their unique optical, chemical, electrical, and catalytic properties and have attracted much interest regarding the applications to detection of toxic metal ions. In this paper, we propose a technique for detection of divalent mercury using AuNPs and Mercury-specific-oligonucleotide–conjugated resonators (MSOIRs). The detection technique is based on the measurement of a resonance frequency shift in the resonators that results from a specific interaction between the thymine–thymine base pair and mercury ion as well as the adsorption of AuNPs that act as a mass amplifier. Our proposed method can quantify mercury ions with the limit of detection of 100 pM, which is 10-fold better than that of the existing assays. Furthermore, MSOIR can detect mercury ions in real tap water. These results imply that the mercury-detecting sensor may be used to enhance drinking-water quality.

Aptamer based voltammetric determination of ampicillin using a single-stranded DNA binding protein and DNA functionalized gold nanoparticles.

An aptamer based method is described for the electrochemical determination of ampicillin. It is based on the use of DNA aptamer, DNA functionalized gold nanoparticles (DNA-AuNPs), and single-stranded DNA binding protein (ssDNA-BP). When the aptamer hybridizes with the target DNA on the AuNPs, the ssDNA-BP is captured on the electrode surface via its specific interaction with ss-DNA. This results in a decreased electrochemical signal of the redox probe Fe(CN)63- which is measured best at a voltage of 0.188mV (vs. reference electrode). In the presence of ampicillin, the formation of aptamer-ampicillin conjugate blocks the further immobilization of DNA-AuNPs and ssDNA-BP, and this leads to an increased response. The method has a linear reposne that convers the 1 pM to 5nM ampicillin concentration range, with a 0.38 pM detection limit (at an S/N ratio of 3). The assay is selective, stable and reproducible. It was applied to the determination of ampicillin in spiked milk samples where it gave recoveries ranging from 95.5 to 105.5%. Graphical abstract Schematic of a simple and sensitive electrochemical apta-biosensor for ampicillin detection. It is based on the use of gold nanoparticles (AuNPs), DNA aptamer, DNA functionalized AuNPs (DNA-AuNPs), and single-strand DNA binding protein (SSBP).

Electrical Modulation of Graphene by the Self-Assembly of DNA-Functionalized Gold Nanoparticles

Electrical Modulation of Graphene by the Self-Assembly of DNA-Functionalized Gold Nanoparticles

Real-time imaging of VCAM-1 mRNA in TNF-α activated retinal microvascular endothelial cells using antisense hairpin-DNA functionalized gold nanoparticles

Vascular cell adhesion molecule 1 (VCAM-1) is an important inflammatory biomarker correlating with retinal disease progression. Thus, detection of VCAM-1 mRNA expression levels at an early disease stage could be an important predictive biomarker to assess the risk of disease progression and monitoring treatment response. We have developed VCAM-1 targeted antisense hairpin DNA-functionalized gold nanoparticles (AS-VCAM-1 hAuNP) for the real time detection of VCAM-1 mRNA expression levels in retinal endothelial cells. The AS-VCAM-1 hAuNP fluorescence enhancement clearly visualized the TNF-α induced cellular VCAM-1 mRNA levels with high signal to noise ratios compared to normal serum treated cells. The scrambled hAuNP probes were minimally detectable under same image acquisition conditions. Intracellular hAuNPs were detected using transmission electron microscopy (TEM) analysis of the intact cells. In addition, the AS-VCAM-1 hAuNP probes exhibited no acute toxicity to the retinal microvascular endothelial cells as measured by live-dead assay.