A novel automated assay with dual-color hybridization for single-nucleotide polymorphisms genotyping on gold magnetic nanoparticle array

A high-throughput and cost-effective single-nucleotide polymorphism (SNP) genotyping method based on a gold magnetic nanoparticle (GMNP) array with dual-color hybridization has been designed. Biotinylated single-strand polymerase chain reaction (PCR) products containing the SNP locus were captured by the GMNPs that were coated with streptavidin. The GMNP array was fabricated by immobilizing single-stranded DNA (ssDNA)-GMNP complexes onto a glass slide using a magnetic field, and SNPs were identified with dual-color fluorescence hybridization. Three different SNP loci from 24 samples were genotyped successfully using this platform. This procedure allows the user to directly analyze the bead fluorescence to determine the SNP genotype, and it eliminates the need for background subtraction for signal determination. This method also bypasses tedious PCR purification and concentration procedures, and it facilitates large-scale SNP studies by using a method that is highly sensitive, simple, labor-saving, and potentially automatable.     

Detection of protein-DNA interaction and regulation using gold nanoparticles

The interaction between protein and DNA is usually regulated by a third species, an effector, which can be either a protein or a small molecule. Convenient methods capable of detecting protein-DNA interaction and its regulation are highly desirable research tools. In the current study, we developed a method to directly “visualize” the interaction between a protein-DNA pair and its effector through the coupling with gold nanoparticles (AuNPs). As a proof-of-concept experiment, we constructed a model system based on the interaction between the lac repressor (protein) and operator (DNA) and its interplay with the lac operon inducer isopropyl β-d-1-thiogalactopyranoside (IPTG, which inhibits the interaction between the lac repressor and operator). We coated AuNPs with the lac operator sequences and mixed them with the lac repressor. Because the lac repressor homotetramer contains two DNA binding modules, it bridged the particles and caused them to aggregate. We demonstrated that the assembly of DNA-modified AuNPs correlated with the presence of the corresponding protein and effector in a concentration-dependent manner. This AuNP-based platform has the potential to be generalized in the creation of reporter and detection systems for other interacting protein-DNA pairs and their effectors.     

Typing single-nucleotide polymorphisms using a gel-based sequencer

Single-nucleotide polymorphisms (SNPs) are increasingly used as genetic markers. Although a high number of SNP-genotyping techniques have been described, most techniques still have low throughput or require major investments. For laboratories that have access to an automated sequencer, a single-base extension (SBE) assay can be implemented using the ABI SNaPshot™ kit. Here we present a modified protocol comprising multiplex template generation, multiplex SBE reaction, and multiplex sample analysis on a gel-based sequencer such as the ABI 377. These sequencers run on a Macintosh platform, but on this platform the software available for analysis of data from the ABI 377 has limitations. First, analysis of the size standard included with the kit is not facilitated. Therefore a new size standard was designed. Second, using Genotype (ABI), the analysis of the data is very tedious and time consuming. To enable automated batch analysis of 96 samples, with 10 SNPs each, we developed SNPtyper. This is a spreadsheet-based tool that uses the data from Genotyper and offers the user a convenient interface to set parameters required for correct allele calling. In conclusion, the method described will enable any lab having access to an ABI sequencer to genotype up to 1000 SNPs per day for a single experimenter, without investing in new equipment.     

Homogeneous and label-free fluorescence detection of single-nucleotide polymorphism using target-primed branched rolling circle amplification

We present a simple, sensitive, and cost-effective fluorescent assay of single-nucleotide polymorphism (SNP) with target-primed branched rolling circle amplification (TPBRCA). Designed padlock probe is circularized after perfect hybridization to mutant DNA. Then rolling circle amplification (RCA) reaction can be initiated from the mutant DNA that acts as primer and generates a long tandem single-stranded DNA (ssDNA) product. At the same time, the introduction of a reverse primer complementary to the target-primed RCA products leads to the branched RCA and eventually generates the various lengths of ssDNA and double-stranded DNA products, which are sensitively detected using SYBR Green I (SG) fluorescence dye. In contrast, the wild DNA contains a single mismatched base with the padlock probe and primes only a limited extension with the unligated padlock probe, generating weak background fluorescence with the addition of SG. Due to the excellent specificity and powerful amplification of TPBRCA reaction, the mutant DNA was distinctively differentiated from the wild DNA in a homogeneous and label-free manner. The assay is sensitive and specific enough to detect 5-amol (8.6-fM) mutant DNA strands. It was possible to accurately determine the mutant allele frequency as low as 1.0%.     

A sensitive assay of mercury using fluorescence correlation spectroscopy of gold nanoparticles

We described a new and sensitive method for the determination of mercury ions (Hg²⁺) on the basis of fluorescence correlation spectroscopy (FCS) and recognition of oligonucleotides. In this assay, 30‐nm gold nanoparticles (GNPs) were modified with oligonucleotides containing thymine bases (T) as fluorescent probes, and the principle of this assay was based on the specific binding of Hg²⁺ by two DNA thymine bases. When two GNPs labelled with different oligonucleotides were mixed with a sample containing Hg²⁺, the T‐Hg²⁺‐T binding reaction should cause GNPs to form dimers (or oligomers), which would lead to a significant increase in the characteristic diffusion time of GNPs in the detection volume. The FCS method is a single molecule detection method and can sensitively detect the change in the characteristic diffusion time of GNPs before and after binding reactions. The quantitative analysis was performed according to the relation between the change in the characteristic diffusion time of GNPs and the concentration of Hg²⁺. Under optimal conditions, the linear range of this method was from 0.3 nM to 100 nM, and the detection limit was 0.14 nM for Hg²⁺. This new method was successfully applied for direct determination of Hg²⁺ levels in water and cosmetics samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Aptamer biosensor for protein detection using gold nanoparticles

Combining gold nanoparticles (GNPs) as fluorescence quencher and aptamer as probe, we have developed protein biosensors by using DNA-modified GNPs. We examined how the experimental design, such as the type of interaction between DNA strands and GNPs, temperature, and microenvironment of aptamer, influences the recognition ability of the biosensor. Under our experimental conditions, the recognition of protein by the complex of dye-labeled DNA hybridized with aptamer that is immobilized on GNPs (Ap-Im-GNPs) shows the best character in protein detection.     

Site-specific labeling of T7 DNA polymerase with a conformationally sensitive fluorophore and its use in detecting single-nucleotide polymorphisms

Like most enzymes, DNA polymerases undergo a large conformational change on the binding of a correct nucleotide. To determine how the conformational change contributes to substrate specificity, we labeled the T7 DNA polymerase with a conformationally sensitive fluorophore at a position that provides a signal coincident with structural changes following nucleotide binding and distinguishes correct base pairs from incorrect ones by the sign of the fluorescence change. Here we describe methods to document that only one site on the polymerase was labeled with the fluorophore based on mass spectral analysis of tryptic peptides. In addition, we show by equilibrium titrations of opposing signals that mismatches and correct bases compete for the same site. This analysis forms an essential basis for characterization of a fluorescently labeled enzyme intended for mechanistic studies. Finally, we show that the labeled enzyme can be used to identify single-nucleotide mutations in a procedure that could be automated.     

Production of gold nanoparticles by electrode-respiring Geobacter sulfurreducens biofilms

The goal of this work was to synthesize gold nanoparticles (AuNPs) using electrode-respiring Geobacter sulfurreducens biofilms. We found that AuNPs are generated in the extracellular matrix of Geobacter biofilms and have an average particle size of 20 nm. The formation of AuNPs was verified using TEM, FTIR and EDX. We also found that the extracellular substances extracted from electrode-respiring G. sulfurreducens biofilms reduce Au³⁺ to AuNPs. From FTIR spectra, it appears that reduced sugars were involved in the bioreduction and synthesis of AuNPs and that amine groups acted as the major biomolecules involved in binding.     

Cytogenetic effects of silver and gold nanoparticles on Allium cepa roots

The present study evaluates the cytogenetic effects of both silver and gold nanoparticles on the root cells of Allium cepa. In this study, the root cells of Allium cepa were treated with both gold and silver nanoparticles of different concentrations (1?mg/L, 5?mg/L and 10?mg/L) along with control for 72?h. Experimental results revealed that after 72?h of exposure, a significant decrease in mitotic index (MI) from 68% (control) to 52.4% (1?mg/L), 47.3% (5?mg/L) and 41.4% (10?mg/L) for gold nanoparticles and 57.1% (1?mg/L), 53% (5?mg/l), 55.8% (10?mg/L) for silver nanoparticles. Through minute observation of the photograph, it was recorded that some specific chromosomal abnormalities such as stickiness of chromosome, chromosome breaks, nuclear notch, and clumped chromosome at different exposure conditions. Therefore, present results clearly suggest that Allium cepa root tip assay could be a viable path through which negative impact of both gold and silver nanoparticles can be demonstrated over a wide range of concentrations.     

Study of gold nanoparticles for mammography diagnostic and radiotherapy improvements

AimA study on the possibility to use gold nanoparticles in mammography, both for a better image diagnostics and radiotherapy, is presented and discussed. We evaluate quantitatively the increment of dose released to the tumor enriched with Au-NPs with respect to the near healthy tissues, finding that for X-rays the increase can reach two orders of greater intensity.BackgroundGold nanoparticles continue to be investigated for their potential to improve existing therapies and to develop novel therapies. They are simple to obtain, can be functionalized with different chemical approaches, are stable, non-toxic, non-immunogenic and have high permeability and retention effects in the tumor cells. The possibility to use these for breast calcified tumors to be better treated by radiotherapy is presented as a possible method to destroy the tumor.Materials and methodsThe nanoparticles can be generated in water using the top-down method, should have a size of the order of 10–20 nm and be treated to avoid their coalescence. Under diagnostic X-ray monitoring, the solution containing nanoparticles can be injected locally inside the tumor site avoiding injection in healthy tissues. The concentrations that can be used should be of the order of 10 mg/ml or higher.ResultsAn enhancement of the computerized tomography diagnostics using 80–150 keV energy is expected, due to the higher mass X-ray coefficient attenuation with respect to other contrast media. Due to the increment of the effective atomic number of the biological tissue containing the gold nanoparticles, also an improvement of the radiotherapy effect using about 30 keV X-ray energy is expected, due to the higher photoelectric cross sections involved.ConclusionsThe study carried out represents a feasibility proposal for the use of Au-nanoparticles for mammographic molecular imaging aimed at radiotherapy of tumor nodules but no clinical results are presented.     

DNAzyme self-assembled gold nanoparticles for determination of metal ions using fluorescence anisotropy assay

Gold nanoparticles can be exploited to facilitate a highly sensitive and selective metal ion detection based on fluorescence anisotropy assay with metal ion-dependent DNA-cleaving DNAzyme. This assay allows rapid and accurate determination of metal ions in aqueous medium at room temperature. The method has been demonstrated for determination of Cu²⁺ and Pb²⁺ ions. The detection sensitivity can be significantly improved to 1 nM by using a “nanoparticle enhancement” approach. Moreover, the assay was also tested in 384-well plates for high-throughput routine determination of toxic metal ions in environmental samples. The method showed distinct advantages over conventional methods in terms of its potential sensitivity, specificity, and ability for rapid response.     

In vitro and in vivo anticandidal efficacy of green synthesized gold nanoparticles using Spirulina maxima polysaccharide

This study, for the first time, demonstrated an unprecedented approach for the green synthesis of gold (Au) nanoparticles (NPs) using the polysaccharide of Spirulina maxima as a reducing agent. Time-kill kinetic analysis was used to evaluate the antifungal activity of the green synthesized Au NPs against the pathogenic Candida albicans (C. albicans). The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were found to be 32 μg/mL and 64 μg/mL, respectively. Ultra-structural analysis indicated prominent damage on cell wall of the C. albicans after Au NPs treatment, and suggested that the treatment could increase the membrane permeability and disintegration of cells leading to cellular death. The results of propidium iodide (PI) uptake assay showed the higher level of cell death in Au NPs treated C. albicans cells, further confirming the loss of plasma membrane integrity. Cytotoxicity analysis of Au NPs on HEK293T and A549 cells showed no cytotoxic effect up to 64 μg/mL of Au NPs concentration, indicating the potential use in in vivo studies. Also, the recovery of C. albicans infected zebrafish after Au NPs therapy suggest green synthesized Au NPs from S. maxima polysaccharide as a prospective anticandidal agent.     

Biosynthesis of silver and gold nanoparticles using thermophilic bacterium Geobacillus stearothermophilus

Nanomaterials have assumed a great deal of importance as they often display unique and considerably modified physical, chemical and biological properties as compared to their counterparts of the macroscale. In this study, biogenic synthesis of silver and gold nanoparticles by Geobacillus stearothermophilus has been attempted. The exposure of G. stearothermophilus cell free extract to the metal salts leads to the formation of stable silver and gold nanoparticles in the solution. These nanoparticles were characterized by UV–Vis spectra, FTIR, TEM, and XRD. The silver and gold nanoparticles have absorption maxima at 423 nm and 522 nm respectively. The TEM micrograph revealed the formation of polydispersed particles in the case of silver nanoparticles and monodispersed particles with respect to the gold nanoparticles. High stability of the nanoparticle solution could be attributed to the secretion of certain capping proteins by the bacterium in the reaction mixture. The involvement of these proteins was confirmed by FTIR and SDS PAGE.     

Enhanced cytotoxicity of gold porphyrin complexes after inclusion in cyclodextrin scaffolds adsorbed on polyethyleneimine-coated gold nanoparticles

A new gold nanoparticle-based construct has been designed to hydrophobic drugs delivery into cancer cells. Cyclodextrin scaffolds adsorbed on polyethyleneimine-coated gold nanoparticles (AuNP@PEI@CD) have been used to encapsulate hydrophobic tetrapyrrolic compounds consisting of gold complexes of 5,10,15,20-tetraphenyl porphyrin (AuTPPCl) and 5-(4-acetoxyphenyl)-10,15,20-triphenyl porphyrin (AuTPPOAcCl). These two nanoparticles have been tested for their cytotoxic activities against the two colorectal cancer cell lines HT-29 and HCT-116 and have shown significant increases in toxicity when compared to the corresponding non-vectorized tetrapyrrolic macrocycles.     

微生物介导的金纳米颗粒合成

金纳米颗粒凭借其独特的光学和电化学特性,广泛应用于信息存储、化学传感、医学成像、药物传输以及生物标记等领域。近年来,生物法合成金纳米颗粒因其环境友好、绿色低毒等特点引起研究者的广泛关注。研究表明,多种微生物包括细菌、放线菌、真菌和病毒等均具有合成金纳米颗粒的能力。本文综述了微生物介导合成金纳米颗粒的特性、机制及应用,并对未来发展趋势进行了展望。     

Fungal surface protein mediated one-pot synthesis of stable and hemocompatible gold nanoparticles

Despite their large secretome and wide applications in bioprocesses, fungi remain underexplored in metal nanoparticles (MNP) biosynthesis. Previous studies have shown that cell surface proteins of Rhizopus oryzae play a crucial role in biomineralization of Au(III) to produce gold nanoparticles (AuNPs). Therefore, it is hypothesized that purified cell surface protein may produce in vitro AuNPs with narrow size distribution for biomedical and biocatalytic applications. However, different protein extraction methods might affect protein stability and the AuNP biosynthesis process. Herein, we have explored the extraction of cell surface proteins from R. oryzae using common detergents and reducing agent (sodium dodecyl sulfate (SDS) Triton X-100, and 1,4-dithiothreitol (DTT)) and their effect on the size and shape of the biosynthetic AuNPs. The surface proteins extracted with reducing agent (DTT) and non-ionic detergent (Triton X-100) produce spherical AuNPs with a mean particle size of 16 ± 7 nm, and 19 ± 4 nm, respectively, while the AuNPs produced by the surface protein extracted by ionic detergent (SDS) are flower-like AuNPs with broader size distribution of 43 ± 19 nm. This synthetic approach does not require use of any harsh chemicals, multistep preparation and separation process, favouring environmental sustainability. The biosynthetic AuNPs thus formed, are stable in different physiological buffers and hemocompatible, making them suitable for biomedical applications.     

Unveiling the anticancer and antimycobacterial potentials of bioengineered gold nanoparticles

Synthesis of gold nanoparticles was carried out using Pongammia pinnata (pongam) leaf extract and their anticancer and antimycobacterial activities were studied. Gold nanoparticle formation was confirmed by UV–vis, XRD and HR-TEM. The anticancer efficacies of the biogenic gold nanoparticles were analyzed using cytotoxicity, cell morphology analysis, oxidative DNA damage, apoptosis detection and toxicity studies. Biogenic gold nanoparticles inhibited breast cancer cell line (MCF-7) proliferation with an efficacy of IC₅₀ of 1.85 μg/mL. The antimycobacterial potential of the biogenic gold nanoparticles was screened against M. tuberculosis by Luciferase Reporter Phage (LRP) assay. The gold nanoparticles showed inhibition against sensitive M. tuberculosis with the minimum inhibitory concentration (MIC) of 10 μg/mL whereas no inhibition was found against the rifampicin resistant M. tuberculosis.     

Hyper-Rayleigh scattering of protein-modified gold nanoparticles

The nonlinear optical properties of protein-modified gold nanoparticles has been studied by the hyper-Rayleigh scattering (HRS) technique. HRS signals from the nanoparticles coated with goat-anti-human IgG have been obtained when pumped with a laser pulse with a wavelength of 1064 nm. The HRS signals of gold nanoparticles with IgG were larger than those of bare gold nanoparticles. This can be explained by a noncentrosymmetric effect. It was also found that the HRS signals from the IgG-coated gold nanoparticles could be greatly increased when the antigen was added due to gold nanoparticle aggregation. Our experiment found that the HRS method could produce a measurable signal with 10 microg/ml antigen added, while the colorimetric method using UV spectrum detection required 100 microg/ml of added antigen. The results show that the HRS measurement of immunogold nanoparticles could become a potential immunoassay in determining small levels of antigen in aqueous samples.     

Environmentally benign rapid biosynthesis of extracellular gold nanoparticles using Aspergillus flavus and their cytotoxic and catalytic activities

In this study, the rapid biosynthesis of gold nanoparticles (AuNPs) by Aspergillus flavus culture supernatant was achieved by reducing 1 mM of chloroauric acid (HAuCl₄) within 2 min at pH 7 and 30 °C. The biosynthesized nanoparticles exhibited maximum absorbance at 545 nm in UVvis spectroscopy. Transmission electron microscopy exhibited that AuNPs tend to take nearly spherical shapes with an average size of 12 nm. Fourier transform infrared analysis indicated that carboxyl, amine, and hydroxyl groups may participate in the biosynthesis and stabilization of AuNPs. Its zeta potential was found to be -33.01 mV. Energy dispersive X-rays showed a strong and typical beak of gold nanocrystallites with 80.84 % of analyzed sample. X-Ray diffraction spectrum displayed Bragg reflections identical to the gold nanocrystals. The results confirmed that biosynthesized AuNPs are a potent anticancer agent against A549, HepG2 and MCF7 cell lines with IC₅₀ value 53.5, 60.7 and 100 μg/mL, respectively. Crystal violet assay confirmed the cytopathic effects of AuNPs on HepG2 and A549 cell lines. Annexin-V FITC assay and cell cycle confirmed the apoptotic effect and cell cycle arrest in G2/M phase, respectively for A549 cell line. Moreover, the results showed a degradation efficiency of AuNPs to 4-nitrophenol within 16 min.     

Determination of nanomolar uric and ascorbic acids using enlarged gold nanoparticles modified electrode

Individual and simultaneous determination of 50 nM uric acid (UA) and ascorbic acid (AA) using enlarged, citrate-stabilized gold nanoparticles (AuNPs) self-assembled to 2,5-dimercapto-1,3,4-thiadiazole (DMT) monolayer modified Au (Au/DMT) electrode by an amperometric method is described for the first time. Self-assembly of AuNPs on the electrode surface was confirmed by atomic force microscopy (AFM), attenuated total reflectance FT-IR and diffuse reflectance spectral measurements. The electron transfer reaction (ETR) of [Fe(CN)₆]^3−/4− was blocked at Au/DMT electrode, whereas it was restored with a peak separation of 200 mV after the attachment of AuNPs on the Au/DMT (Au/DMT/AuNPs) electrode, which was confirmed from the ETR of the [Fe(CN)₆]^3−/4− redox couple. When the self-assembled AuNPs were enlarged by hydroxylamine seeding, the ETR of [Fe(CN)₆]^3−/4− was improved significantly with a peak separation of 100 mV. Tapping mode AFM showed that the average size of the enlarged-AuNPs (E-AuNPs) was 50-70 nm. The E-AuNPs modified electrode catalyzes the oxidation of AA and UA, separates their voltammetric signals by 200 mV, and has excellent sensitivity towards AA and UA with a detection limit of 50 nM. The practical application of the modified electrode was demonstrated by measuring the concentration of UA in blood serum and urine.