Green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance DNA hybridization biosensing

Sensitive electrochemical impedance assay of DNA hybridization by using a novel graphene sheets platform was achieved. The graphene sheets were firstly functionalized with 3,4,9,10-perylene tetracarboxylic acid (PTCA). PTCA molecules separated graphene sheets efficiently and introduced more negatively-charged -COOH sites, both of which were beneficial to the decoration of graphene with gold nanoparticles. Then amine-terminated ionic liquid (NH?-IL) was applied to the reduction of HAuCl? to gold nanoparticles. The green-synthesized gold nanoparticles, with the mean diameter of 3 nm, dispersed uniformly on graphene sheets and its outer layer was positively charged imidazole termini. Due to the presence of large graphene sheets and NH?-IL protected gold nanoparticles, DNA probes could be immobilized via electrostatic interaction and adsorption effect. Electrochemical impedance value increased after DNA probes immobilization and hybridization, which was adopted as the signal for label-free DNA hybridization detection. Unlike previously anchoring DNA to gold nanoparticles, this label-free method was simple and noninvasive. The conserved sequence of the pol gene of human immunodeficiency virus 1 was satisfactorily detected via this strategy.     

A Visualized Assay for Quercetin Based on the Formation of Silver–Gold Alloy Nanoparticles

A visualized assay for quercetin (QU) was first developed based on the formation of silver–gold alloy nanoparticles in this contribution. With the ability to reduce metal ions to metal substances, QU could reduce Ag⁺ absorbed on the surface of gold nanoparticles to metallic silver. The thickness of the formed Ag shell and the color change of the solution were proportional to the concentration of QU. Therefore, visualized detection of QU could be realized by studying the surface resonance plasmon absorption spectra of the analytical systems after addition of different concentration of QU. Under optimum conditions, trace amount of QU could be detected in the linear range 9.0?×?10^?7–1.0?×?10^?4 mol L^?1 with a detection limit of 6.5?×?10^?7 mol L^?1. The present assay was applied in the determination of QU in human serum and satisfactory results were obtained. This assay is simple, rapid, and cost-effective, and it is a powerful complement for the spectroscopy assays for QU. Also, it is the first visualized spectroscopic assay of QU until now.     

High sensitivity DNA detection using gold nanoparticle functionalised polyaniline nanofibres

Polyaniline (PANI) nanofibres (PANI-NF) have been modified with chemically grown gold nanoparticles to give a nanocomposite material (PANI-NF-AuNP) and deposited on gold electrodes. Single stranded capture DNA was then bound to the gold nanoparticles and the underlying gold electrode and allowed to hybridise with a complementary target strand that is uniquely associated with the pathogen, Staphylococcus aureus (S. aureus), that causes mastitis. Significantly, cyclic voltammetry demonstrates that deposition of the gold nanoparticles increases the area available for DNA immobilisation by a factor of approximately 4. EPR reveals that the addition of the Au nanoparticles efficiently decreases the interactions between adjacent PANI chains and/or motional broadening. Finally, a second horseradish peroxidase (HRP) labelled DNA strand hybridises with the target allowing the concentration of the target DNA to be detected by monitoring the reduction of a hydroquinone mediator in solution. The sensors have a wide dynamic range, excellent ability to discriminate DNA mismatches and a high sensitivity. Semi-log plots of the pathogen DNA concentration vs. faradaic current were linear from 150×10(-12) to 1×10(-6) mol L(-1) and pM concentrations could be detected without the need for molecular, e.g., PCR or NASBA, amplification.     

Radiosensitization of DNA by gold nanoparticles irradiated with high-energy electrons

Zheng, Y., Hunting, D. J., Ayotte, P. and Sanche, L. Radiosensitization of DNA by Gold Nanoparticles Irradiated with High-Energy Electrons. Radiat. Res. 168, 19-27 (2008). Thin films of pGEM-3Zf(-) plasmid DNA were bombarded by 60 keV electrons with and without gold nanoparticles. DNA single- and double-strand breaks (SSBs and DSBs) were measured by agarose gel electrophoresis. From transmission electron micrographs, the gold nanoparticles were found to be closely linked to DNA scaffolds, probably as a result of electrostatic binding. The probabilities for formation of SSBs and DSBs from exposure of 1:1 and 2:1 gold nanoparticle:plasmid mixtures to fast electrons increase by a factor of about 2.5 compared to neat DNA samples. For monolayer DNA adsorbed on a thick gold substrate, the damage increases by an order of magnitude. The results suggest that the enhancement of radiosensitivity is due to the production of additional low-energy secondary electrons caused by the increased absorption of ionizing radiation energy by the metal, in the form of gold nanoparticles or of a thick gold substrate. Since short-range low-energy secondary electrons are produced in large amounts by any type of ionizing radiation, and since on average only one gold nanoparticle per DNA molecule is needed to increase damage considerably, targeting the DNA of cancer cells with gold nanoparticles may offer a novel approach that is generally applicable to radiotherapy treatments.     

DNA gold nanoparticle conjugates incorporating thiooxonucleosides: 7-deaza-6-thio-2'-deoxyguanosine as gold surface anchor

A new protocol for the covalent attachment of oligonucleotides to gold nanoparticles was developed. Base-modified nucleosides with thiooxo groups were acting as molecular surface anchor. Compared to already existing conjugation protocols, the new linker strategy simplifies the synthesis of DNA gold nanoparticle conjugates. The phosphoramidite of 7-deaza-6-thio-2'-deoxyguanosine (6) was used in solid-phase synthesis. Incorporation of the sulfur-containing nucleosides can be performed at any position of an oligonucleotide; even multiple incorporations are feasible, which will increase the binding stability of the corresponding oligonucleotides to the gold nanoparticles. Oligonucleotide strands immobilized at the end of a chain were easily accessible during hybridization leading to DNA gold nanoparticle network formation. On the contrary, oligonucleotides immobilized via a central position could not form a DNA-AuNP network. Melting studies of the DNA gold nanoparticle assemblies revealed sharp melting profiles with a very narrow melting transition.     

Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0×10(-14) M without the need for a spectrometer or a scanning stage.     

Enhancing the efficiency of a PCR using gold nanoparticles

We found that the PCR could be dramatically enhanced by Au nanoparticles. With the addition of 0.7 nM of 13 nm Au nanoparticles into the PCR reagent, the PCR efficiency was increased. Especially when maintaining the same or higher amplification yields, the reaction time could be shortened, and the heating/cooling rates could be increased. The excellent heat transfer property of the nanoparticles should be the major factor in improving the PCR efficiency. Different PCR systems, DNA polymerases, DNA sizes and complex samples were compared in this study. Our results demonstrated that Au nanoparticles increase the sensitivity of PCR detection 5- to 10-fold in a slower PCR system (i.e. conventional PCR) and at least 10⁴-fold in a quicker PCR system (i.e. real-time PCR). After the PCR time was shortened by half, the 100 copies/µl DNA were detectable in real-time PCR with gold colloid added, however, at least 10⁶ copies/µl of DNA were needed to reach a detectable signal level using the PCR reagent without gold colloid. This innovation could improve the PCR efficiency using non-expensive polymerases, and general PCR reagent. It is a new viewpoint in PCR, that nanoparticles can be used to enhance PCR efficiency and shorten reaction times.     

Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo

Biotinylated nucleotides (bio-11-dCTP, bio-11-dUTP, and bio-7-dATP) were microinjected into unfertilized and fertilized Xenopus laevis eggs. The amounts introduced were comparable to in vivo deoxy-nucleoside triphosphate pools. At various times after microinjection, DNA was extracted from eggs or embryos and subjected to electrophoresis on agarose gels. Newly synthesized biotinylated DNA was analyzed by Southern transfer and visualized using either the BluGENE or Detek-hrp streptavidin-based nucleic acid detection systems. Quantitation of the amount of biotinylated DNA observed at various times showed that the microinjected biotinylated nucleotides were efficiently incorporated in vivo, both into replicating endogenous chromosomal DNA and into replicating microinjected exogenous plasmid DNA. At least one biotinylated nucleotide could be incorporated in vivo for every eight nucleotides of DNA synthesized. Control experiments also showed that heavily biotinylated DNA was not subjected to detectable DNA repair during early embryogenesis (for at least 5 h after activation of the eggs). The incorporated biotinylated nucleotides were visualized by electron microscopy by using streptavidin-colloidal gold or streptavidin-ferritin conjugates to bind specifically to the biotin groups projecting from the newly replicated DNA. The incorporated biotinylated nucleotides were thus made visible as electron-dense spots on the underlying DNA molecules. Biotinylated nucleotides separated by 20-50 bases could be resolved. We conclude that nascent DNA synthesized in vivo in Xenopus laevis eggs can be visualized efficiently and specifically using the techniques described.     

A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin

A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1–18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.     

Optical detection of DNA hybridization based on fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles

A novel system for the detection of DNA hybridization in a homogeneous format is developed. This method is based on fluorescence quenching by gold nanoparticles used as both nanoscaffolds for the immobilization of capture sequences and nanoquenchers of fluorophores attached to detection sequences. The oligonucleotide-functionalized gold nanoparticles are synthesized by derivatizing the colloidal gold solution with 5'-thiolated 12-base oligonucleotides. Introduction of sequence-specific target DNAs (24 bases) into the mixture containing dye-tagged detection sequences and oligonucleotide-functionalized gold nanoparticles results in the quenching of carboxytetramethylrhodamine-labeled DNA fluorescence because DNA hybridization occurs and brings fluorophores into close proximity with oligonucleotide-functionalized gold nanoparticles. The quenching efficiency of fluorescence increases with the target DNA concentration and provides a quantitative measurement of sequence-specific DNA in sample. A linearity is obtained within the range from 1.4 to 92 nM. The target sequence is detected down to 2 nM. This new system not only overcomes many of the drawbacks inherent in radioisotopic measurement or enzyme-linked assay but also avoids the requirement for the stem-loop structure compared with conventional molecular beacons. Furthermore, the background signal that is defined as fluorescence quenching arising from electrostatic attraction between positively charged fluorophores and negatively charged gold nanoparticles is comparatively low due to electrostatic repulsion between negatively charged oligonucleotides. In addition, this is a homogeneous assay that can offer the potential to be monitored in real time, be amenable to automation, eliminate washing steps, and reduce the risk of contamination.     

Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases

The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly.     

Simple colorimetric DNA detection based on hairpin assembly reaction and target-catalytic circuits for signal amplification

A simple strategy of colorimetric DNA detection is presented based on a hairpin assembly reaction and target-catalytic DNA circuits to achieve enzyme-free signal amplification. The method employed two hairpin species (H1 and H2), which were stable and unable to hybridize in the absence of target. In the presence of target, the target hybridized with hairpin H1 and the opened hairpin H1 hybridized with hairpin H2, allowing the target to be displaced. H1 and H2 were respectively attached to gold nanoparticles, allowing the duplex formed from H1 and H2 to be visualized with the naked eye. The displaced target again triggered the next round of strand exchange reaction to achieve signal amplification. The method may have a wide range of sensor applications because it is enzyme-free and simple to perform.     

Linear clusters of gold nanoparticles in quasinematic layers of DNA liquid-crystalline dispersion particles

The effects of small size (～2 nm) gold nanoparticles on the properties of particles of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules were analyzed. It has been shown that gold nanoparticles induce two different processes. First, they facilitate reorganization of the spatial cholesteric structure of dispersion particles to nematic one. This process is accompanied by the fast decrease in the amplitude of abnormal band in the CD spectrum. Second, they can form ensembles consisting of gold nanoparticles. This process is accompanied by the development and displacement of surface plasmon resonance band in the visible region of the absorption spectrum. The appearance of this band is analyzed by considering two different models of the formation of ensembles consisting of gold nanoparticles. By small-angle X-ray scattering we performed structural analysis of phases formed by DNA cholesteric liquid-crystalline dispersion particles treated with gold nanoparticles. As a result of this study it was possible to prove the formation of linear clusters of gold nanoparticles in the “free space” between the adjacent DNA molecules fixed in the quasinematic layers of liquid-crystalline particles. It has been hypothesized that the formation of linear clusters of gold nanoparticles is most likely related to DNA molecules, ordered in the spatial structure of quasinematic layers, and the toxicity of these nanoparticles in biological systems hypothesized.     

Naked eye detection of mutagenic DNA photodimers using gold nanoparticles

We developed a method to detect mutagenic DNA photodimers by the naked eye using gold nanoparticles. The stability of gold nanoparticles in a high ionic strength solution is maintained by straight ssDNA adsorbed physically on the AuNPs. However, we found that UV-irradiated DNA was less adsorptive onto gold nanoparticles because of a conformational change of UV-irradiated DNA. The accumulated deformation of the DNA structure by multiple-dimer formation triggered aggregation of the gold nanoparticles mixed with the UV-irradiated DNA and thus red to purple color changes of the mixture, which allowed colorimetric detection of the DNA photodimers by the naked eye. No fragmented mass and reactive oxygen species production under the UVB irradiation confirmed that the aggregation of gold nanoparticles was solely attributed to the accumulated deformation of the UV irradiated DNA. The degree of gold nanoparticles-aggregation was dependent on the UVB irradiated time and base compositions of the UV-irradiated oligonucleotides. In addition, we successfully demonstrated how to visually qualify the photosensitizing effect of chemical compounds in parallel within only 10 min by applying this new method. Since our method does not require any chemical or biochemical treatments or special instruments for purifying and qualifying the DNA photolesions, it should provide a feasible tool for the studies of the UV-induced mutagenic or carcinogenic DNA dimers and accelerate screening of a large number of drug candidates.     

Electrochemical biosensor for the detection of cauliflower mosaic virus 35 S gene sequences using lead sulfide nanoparticles as oligonucleotide labels

Lead sulfide (PbS) nanoparticles were synthesized in aqueous solution and used as oligonucleotide labels for electrochemical detection of the 35 S promoter from cauliflower mosaic virus (CaMV) sequence. The PbS nanoparticles were modified with mercaptoacetic acid and could easily be linked with CaMV 35 S oligonucleotide probe. Target DNA sequences were covalently linked on a mercaptoacetic acid self-assembled gold electrode, and DNA hybridization of target DNA with probe DNA was completed on the electrode surface. PbS nanoparticles anchored on the hybrids were dissolved in the solution by oxidation of HNO₃ and detected using a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used to monitor the hybridization reaction. The CaMV 35 S target sequence was satisfactorily detected with the detection limit as 4.38 × 10⁻¹² mol/L (3σ). The established method extends nanoparticle-labeled electrochemical DNA analysis to specific sequences from genetically modified organisms with higher sensitivity and selectivity.     

STUDIES ON THE ATTACHMENT OF DNA TO SILICA-COATED NANOPARTICLES THROUGH A DIELS-ALDER REACTION

A new method has been investigated for the functionalization of gold nanoparticles with DNA. Silica-coated nanoparticles functionalized with a maleimide have been prepared. These particles are designed to react with modified DNA containing a diene functionality at one end of the molecule. The result would be the formation of a more stable attachment of the DNA to the particle through a Diels-Alder reaction. This covalent attachment would not be susceptible to ligand exchanges, which are known to occur in the conventional DNA functionalization of gold nanoparticles.     

Association temperature governs structure and apparent thermodynamics of DNA-gold nanoparticles

Apparent thermodynamics of association of DNA-modified gold nanoparticles has been characterized by UV spectroscopy and dynamic light scattering (DLS). Extinction coefficients of unlabelled and DNA-labelled gold nanoparticles have been determined to permit quantitative analysis of the absorption measurements. In contrast to previous studies the associating gold nanoparticles were furnished with complementary oligonucleotide DNA single strands. This resulted in direct complex formation between the nanoparticles on mixing without the requirement of a DNA linker sequence for initiation of cluster formation. Melting curves of the nanoparticle assemblies formed at different temperatures were subjected to two-state analysis. A comparison of the apparent thermodynamic parameters obtained for the dissociation of these aggregates suggests that both thermodynamically and structurally different nanoparticle clusters are obtained depending on the temperature at which assembly proceeds. The van't Hoff enthalpies permit an estimate of the DNA duplexes: gold nanoparticle ratio involved in network formation.     

Biomedical potential of silver nanoparticles synthesized from calli cells of Citrullus colocynthis (L.) Schrad

Background

Transmission electron microscopy (TEM) remains an important technique to investigate the size, shape and surface characteristics of particles at the nanometer scale. Resulting micrographs are two dimensional projections of objects and their interpretation can be difficult. Recently, electron tomography (ET) is increasingly used to reveal the morphology of nanomaterials (NM) in 3D. In this study, we examined the feasibility to visualize and measure silica and gold NM in suspension using conventional bright field electron tomography.

Results

The general morphology of gold and silica NM was visualized in 3D by conventional TEM in bright field mode. In orthoslices of the examined NM the surface features of a NM could be seen and measured without interference of higher or lower lying structures inherent to conventional TEM. Segmentation by isosurface rendering allowed visualizing the 3D information of an electron tomographic reconstruction in greater detail than digital slicing. From the 3D reconstructions, the surface area and the volume of the examined NM could be estimated directly and the volume-specific surface area (VSSA) was calculated. The mean VSSA of all examined NM was significantly larger than the threshold of 60 m²/cm³. The high correlation between the measured values of area and volume gold nanoparticles with a known spherical morphology and the areas and volumes calculated from the equivalent circle diameter (ECD) of projected nanoparticles (NP) indicates that the values measured from electron tomographic reconstructions are valid for these gold particles.

Conclusion

The characterization and definition of the examined gold and silica NM can benefit from application of conventional bright field electron tomography: the NM can be visualized in 3D, while surface features and the VSSA can be measured.     

A colorimetric method for point mutation detection using high-fidelity DNA ligase

The present study reported proof-of-principle for a genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) through the gold nanoparticle assembly and the ligase reaction. By incorporating the high-fidelity DNA ligase (Tth DNA ligase) into the allele-specific ligation-based gold nanoparticle assembly, this assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single-base discrimination without the need of precise temperature control. Additionally, the ligase reaction can be performed at a relatively high temperature, which offers the benefit for mitigating the non-specific assembly of gold nanoparticles induced by interfering DNA strands. The assay could be implemented via three steps: a hybridization reaction that allowed two gold nanoparticle-tagged probes to hybrid with the target DNA strand, a ligase reaction that generates the ligation between perfectly matched probes while no ligation occurred between mismatched ones and a thermal treatment at a relatively high temperature that discriminate the ligation of probes. When the reaction mixture was heated to denature the formed duplex, the purple color of the perfect-match solution would not revert to red, while the mismatch gave a red color as the assembled gold nanoparticles disparted. The present approach has been demonstrated with the identification of a single-base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild-type and mutant type were successfully scored. To our knowledge, this was the first report concerning SNP detection based on the ligase reaction and the gold nanoparticle assembly. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in practical clinical diagnosis of gene-mutant diseases.     

Studies on the attachment of DNA to silica-coated nanoparticles through a Diels-Alder reaction

A new method has been investigated for the functionalization of gold nanoparticles with DNA. Silica-coated nanoparticles functionalized with a maleimide have been prepared. These particles are designed to react with modified DNA containing a diene functionality at one end of the molecule. The result would be the formation of a more stable attachment of the DNA to the particle through a Diels-Alder reaction. This covalent attachment would not be susceptible to ligand exchanges, which are known to occur in the conventional DNA functionalization of gold nanoparticles.