Gold DNA-conjugates: ion specific self-assembly of gold nanoparticles via the dG-quartet

The Oxytricha telomere DNA hairpin 5'-d(G4T4G4) immobilized on 13 nm gold nanoparticles forms a supramolecular assembly via dGC-quartets, as determined by the color change and by SEM. The aggregation is ion-dependent and selective for sodium ions. K+ is less efficient while Li+ and Cs+ do not drive the aggregation. This work is the first effort exploring the use of secondary structures of DNA (quadruplexes) for producing self-assemblies of gold nanoparticles.     

Biosynthesis of gold nanoparticles by Azospirillum brasilense

Plant-associated nitrogen-fixing soil bacteria Azospirillum brasilense were shown to reduce the gold of chloroauric acid to elemental gold, resulting in formation of gold nanoparticles. Extracellular phenoloxidizing enzymes (laccases and Mn peroxidases) were shown to participate in reduction of Au⁺³ (HAuCl₄) to Au⁰. Transmission electron microscopy revealed accumulation of colloidal gold nanoparticles of diverse shape in the culture liquid of A. brasilense strains Sp245 and Sp7. The size of the electron-dense nanospheres was 5 to 50 nm, and the size of nanoprisms varied from 5 to 300 nm. The tentative mechanism responsible for formation of gold nanoparticles is discussed.     

Fungus mediated synthesis of gold nanoparticles and their conjugation with genomic DNA isolated from Escherichia coli and Staphylococcus aureus

Biological systems employing microorganisms have been used as an alternative to conventional chemical techniques for synthesizing gold nanoparticles. In the present study, gold nanoparticles have been synthesized from the supernatant broth (SB) and live cell filtrate (LCF) of the industrially important fungus Penicillium rugulosum. Additionally, potato dextrose broth (PDB) medium which is used for the growth of the fungus has also been able to synthesize gold nanoparticles. The size of the particles has been investigated by Bio-TEM before purification as well as after purification to find the difference in morphology pattern of the nanoparticles. Different characterization techniques like X-ray diffraction (XRD), infra-red (FTIR), X-ray photoelectron (XPS) and UV–vis spectroscopy have been used for analysis of the particles. SB of the fungus has yielded nanoparticles with better morphology and hence further optimization studies were conducted for controlling the size and shape of the above by altering pH and concentration of gold salt. A pH range of 4–6 has favored the synthesis process whereas increasing concentration of gold salt (beyond 2 mM) has resulted in the formation of bigger sized and aggregated nanoparticles. The optimized nanoparticles have been used to conjugate with isolated genomic DNA of bacteria Escherichia coli and Staphylococcus aureus. Visual observation of agarose gel electrophoresis images confirmed the binding of gold nanoparticles (4 μL and 6 μL) with isolated DNA (2 μL) fragments of both the organisms. The slight red shift of the surface plasmon (SP) band and minor aggregations noticed in Bio-TEM images for the DNA conjugated gold nanoparticles indicates that the genomic DNA could stabilize the particles against aggregation owing to negatively charged phosphate backbone.     

Single-Molecule TPM Studies on the Conversion of Human Telomeric DNA

Human telomere contains guanine-rich (G-rich) tandem repeats of single-stranded DNA sequences at its 3′ tail. The G-rich sequences can be folded into various secondary structures, termed G-quadruplexes (G4s), by Hoogsteen basepairing in the presence of monovalent cations (such as Na⁺ and K⁺). We developed a single-molecule tethered particle motion (TPM) method to investigate the unfolding process of G4s in the human telomeric sequence AGGG(TTAGGG)₃ in real time. The TPM method monitors the DNA tether length change caused by formation of the G4, thus allowing the unfolding process and structural conversion to be monitored at the single-molecule level. In the presence of its antisense sequence, the folded G4 structure can be disrupted and converted to the unfolded conformation, with apparent unfolding time constants of 82 s and 3152 s. We also observed that the stability of the G4 is greatly affected by different monovalent cations. The folding equilibrium constant of G4 is strongly dependent on the salt concentration, ranging from 1.75 at 5 mM Na⁺ to 3.40 at 15 mM Na⁺. Earlier spectral studies of Na⁺- and K⁺-folded states suggested that the spectral conversion between these two different folded structures may go through a structurally unfolded intermediate state. However, our single-molecule TPM experiments did not detect any totally unfolded intermediate within our experimental resolution when sodium-folded G4 DNA molecules were titrated with high-concentration, excess potassium ions. This observation suggests that a totally unfolding pathway is likely not the major pathway for spectral conversion on the timescale of minutes, and that interconversion among folded states can be achieved by the loop rearrangement. This study also demonstrates that TPM experiments can be used to study conformational changes in single-stranded DNA molecules.     

Selective detection of silver ions using mushroom-like polyaniline and gold nanoparticle nanocomposite-based electrochemical DNA sensor

A highly sensitive electrochemical DNA biosensor made of polyaniline (PANI) and gold nanoparticles (AuNPs) nanocomposite (AuNPs@PANI) has been used for the detection of trace concentration of Ag⁺. In the presence of Ag⁺, with the interaction of cytosine–Ag⁺–cytosine (C–Ag⁺–C), cytosine-rich DNA sequence immobilized onto the surface of AuNPs@PANI has a self-hybridization and then forms a duplex-like structure. The whole detection procedure of Ag⁺ based on the developed biosensor was evaluated by electrochemical impedance spectroscopy. On semi-logarithmic plots of the log Ag⁺ concentration versus peak current, the results show that the prepared biosensor can detect silver ions at a wide linear range of 0.01–100 nM (R = 0.9828) with a detection limit of 10 pM (signal/noise = 3). Moreover, the fabricated sensor exhibits good selectivity and repeatability. The detection of Ag⁺ was determined by Ag⁺ self-induced conformational change of DNA scaffold that involved only one oligonucleotide, showing its convenience and availability.     

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl₄ with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl₄ to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na⁺[Au(OH_4-x)Cl_x]^-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated >300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.     

Gold nanoparticles disturb nuclear chromatin decondensation in mouse sperm in vitro

The effect of gold nanoparticles on mouse epididymal sperm has been studied using the model system of nuclear chromatin decondensation in vitro. It is shown that the treatment of gametes, preliminary membrane-freed by sodium dodecyl sulfate, in the mediums containing gold nanoparticles (with diameter ∼2.5 nm) in concentrations 1.0 × 10¹⁵ or 0.5 × 10¹⁵ particles/ml and following incubation in dithiothreitol solution (DTT) resulted in failure of chromatin decondensation process and nucleus structure. We conclude that gold nanoparticles possess spermatotoxicity. The mechanism of cytotoxic effect of gold nanoparticles may be related with their interaction with molecules of double-helix DNA. The model system studied in this research is applicable for further investigations of cytotoxic effects of nanoparticles of different origin and made of different metals.     

Fluorescence‐based logic gate for sensing of Ca2+ and F− ions using PVP crowned chrysene nanoparticles in aqueous medium

Polyvinyl pyrrolidone (PVP) crowned chrysene nanoparticles (CHYNPs) were prepared by using a reprecipitation method. Dynamic light scattering (DLS) and scanning electron microscope (SEM) studies indicate that the monodispersed spherical nanoparticles bear a negative charge on their surfaces. The bathochromic spectral shift in the UV–visible and fluorescence spectrum of CHYNPs from chrysene (CHY) in acetone solution supports the J‐ type aggregation of nanoparticles. The aggregation‐induced enhanced emission of CHYNPs at 486 and 522 nm decreases by increasing the concentration of the Ca²⁺ ion solution. It can display an ON–OFF type fluorescence response with high selectivity towards Ca²⁺ ions aqueous medium. Furthermore, the in situ generated PVP–CHYNPs–Ca²⁺ ensemble could recover the quenched fluorescence upon the addition of fluoride anions resulting in an OFF–ON type sensor. The present method has a correlation coefficient R² = 0.988 with a detection limit of 1.22 μg/mL for Ca²⁺ in the aqueous medium. The fluorescence changes of PVP crowned CHYNPs upon the addition of Ca²⁺ and F^? can be utilized as an INHIBIT logic gate at the molecular level, using Ca²⁺ and F^? chemical inputs and the fluorescence intensity signal as output.     

Naked Eye Detection of Cr3+ and Co2+ Ions by Gold Nanoparticle Modified with Azomethine

This paper reports the synthesis of azomethine-modified gold nanoparticles with azomethine (azomethine-AuNPs) in aqueous media, which were characterized by FT-IR spectroscopy, ultraviolet–visible spectroscopy (UV-Vis), dynamic light scattering (DLS), thermogravimetric analysis (TGA), and transmission electron microscopy (TEM). The azomethine-AuNPs were employed as colorimetric for Cr³⁺ and Co²⁺ ions at pH 6.2–7.5 and 8.1–9.1, at room temperature in aqueous solution. In the presence of Cr³⁺ and Co²⁺, the azomethine-AuNPs induce aggregation of the nanoparticles. Upon aggregation, the surface plasmon absorption band red-shifts so that the nanoparticle solution appears a blue color. The sensitivity of azomethine-AuNPs towards other metal ions, Mg²⁺, Mn²⁺, Cr⁶⁺, Na⁺, Ni²⁺, Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, Cd²⁺, K⁺, Co³⁺, Ni²⁺, Pb²⁺, and Zn²⁺ are negligible. This highly selective sensor allows a direct quantitative assay of Co²⁺ and Cr³⁺ with colorimetric detection limits of 83.22 and 108 nM, respectively.

     

Plasmonic Spectral Detection of Carcinoembryonic Antigen by Preventing the Direct Binding of Rhodamine 6G with Au Nanoparticles

Carcinoembryonic antigen (CEA) was used as a separator to prevent the Rhodamine 6G (R6G)-induced aggregation of colloidal gold nanoparticles. The destroyed aggregation has been monitored by measuring the absorption and resonance light scattering peaks corresponding to the longitudinal surface plasmon resonance (SPR) of the chain-like aggregated gold nanoparticles (AuNPs). It was found that the pre-adding of CEA with different concentrations to the gold colloids before mixing them with R6G could lead to the longitudinal SPR peak decrease and blue shift. By analysing the intensity changing and wavelength shifting of the absorption spectra, CEA could be detected in a linear range from 0.2 to 4 ng/mL, and the limit of detection reaches to 0.1 ng/mL. The sensitivity of the CEA concentration dependent shifting and quenching of the plasmonic absorption and scattering corresponding to the AuNPs aggregation presents a well potential application of biologic spectral sensing.     

Monomolecular condensation of lambda-DNA induced by cobalt hexamine

Measurements of static and dynamic light scattering have been used to distinguish between monomolecular DNA condensation and aggregation of condensed molecules. In low salt, using Co³⁺(NH₃)₆ as the condensing agent, and at λ-DNA concentrations below 0.2 μg/mL, the transition curves for monomolecular condensation and aggregation are well separated for times of 16 h. In these conditions, the intensity of scattered light (90°) and also the diffusion coefficient of the condensed DNA show reasonable values for monomolecular condensation that are independent of DNA concentration and also of Na⁺ Co³⁺(NH₃)₆ concentrations for which monomolecular condensation is complete. At higher Co³⁺(NH₃)₆ concentrations, which produce aggregation (as judged by the intensity of scattered light), the diffusion coefficient decreases sharply. The transition curve for monomolecular condensation is independent of DNA concentration but shows a hysteresis loop. The kinetics of condensation are slow in the forward direction and fast in the reverse direction, indicating that the actual transition curve is measured closely by reversal experiments. Aggregation is blocked kinetically in both the forward and reverse directions when Co³⁺(NH₃)₆ is the condensing agent at low Na⁺ concentrations. When spermine or spermidine is the condensing agent and observations are made at 16 h, it is not possible to separate the transition curves for monomolecular condensation and for aggregation in conditions that are successful with Co³⁺(NH₃)₆. Some interesting properties of monomolecular condensation are noted. (1) The transition is not a two-state reaction, as judged by measurements of the diffusion coefficient through the transition zone. (2) The transition for monomolecular condensation is diffuse. (3) The dimensions of the monomolecular condensates have been calculated from the translational diffusion coefficient for an assumed toroidal shape by the formula derived by Allison and coworkers [(1981) Biopolymers 20 , 469–488]. These dimensions are in reasonable agreement with ones deduced from electron microscopy by Chattoraj and coworkers [(1978) J. Mol. Biol. 121 , 327–337]. (4) The phase diagram relating the Na⁺ to the Co³⁺(NH₃)₆ concentrations needed for condensation has a slope of 0.6 in a log–log plot. According to numerical solutions of Manning's theory for the atmospheric binding of competing cations to DNA, this means that condensation occurs at a late stage in the replacement of Na⁺ by Co³⁺(NH₃)₆ around the DNA. The fraction of DNA phosphate charge neutralized at condensation is computed to be in the neighborhood of 0.9, as found by Wilson and Bloomfield [(1979) Biochemistry 18 , 2192–2196], but to vary with the Na⁺ concentration.     

Ion-specific aggregation of gold-DNA nanoparticles using the dG quartet hairpin 5'-d(G4T4G4)

Ion-selective self-assembly of Au nanoparticles is described. The Oxytricha telomere DNA hairpin 5'-d(G4T4G4) immobilized on 13-nm Au nanoparticles forms a supramolecular assembly via dG-quartets, as determined by the color change and by scanning electron microscopy (SEM). The aggregation is ion-dependent and selective for Na+ ions. K+ is less efficient, while Li+ and Cs+ do not drive the aggregation. This work is the first effort to explore the use of secondary structures of DNA (quadruplexes) for producing self-assemblies of Au nanoparticles. It can be utilized to generate novel devices and materials, potentially useful for sensing and other applications in medicinal or engineering science.     

One-step homogeneous non-stripping chemiluminescence metal immunoassay based on catalytic activity of gold nanoparticles

The catalytic activity of gold nanoparticles (AuNPs) on a luminol–H₂O₂ chemiluminescence (CL) system is found to be greatly enhanced after its crosslinking aggregation induced by immunoreaction. Based on this observation, a one-step homogeneous non-stripping CL metalloimmunoassay was designed. In the presence of corresponding antigen (Ag), the immunoreaction caused the aggregation of antibody (Ab)-modified AuNPs, and these crosslinking aggregated AuNPs could catalyze luminol–H₂O₂ CL reaction to produce a much stronger CL signal than dispersed Ab-modified AuNPs. The assay, including immunoreaction and detection, can be accomplished in homogeneous solution. In the assay, no tedious and strict stripping of metal nanoparticles, difficult synthesis of labels, multiple steps of immunoreactions and washings, and complicated magnetic separation process were required. The detection limit of human immunoglobulin G (IgG, 3σ) was estimated to be as low as 3.2 × 10⁻¹¹ g ml⁻¹. The sensitivity was increased by two orders of magnitude over that of other AuNP-based CL immunoassay. The current CL metalloimmunoassay offers the advantages of being simple, cheap, rapid, and sensitive.     

Structural basis of sodium–potassium exchange of a human telomeric DNA quadruplex without topological conversion

Understanding the mechanism of Na⁺/K⁺-dependent spectral conversion of human telomeric G-quadruplex (G4) sequences has been limited not only because of the structural polymorphism but also the lack of sufficient structural information at different stages along the conversion process for one given oligonucleotide. In this work, we have determined the topology of the Na⁺ form of Tel23 G4, which is the same hybrid form as the K⁺ form of Tel23 G4 despite the distinct spectral patterns in their respective nuclear magnetic resonance (NMR) and circular dichroism spectra. The spectral difference, particularly the well-resolved imino proton NMR signals, allows us to monitor the structural conversion from Na⁺ form to K⁺ form during Na⁺/K⁺ exchange. Time-resolved NMR experiments of hydrogen–deuterium exchange and hybridization clearly exclude involvement of the global unfolding for the fast Na⁺/K⁺ spectral conversion. In addition, the K⁺ titration monitored by NMR reveals that the Na⁺/K⁺ exchange in Tel23 G4 is a two-step process. The addition of K⁺ significantly stabilizes the unfolding kinetics of Tel23 G4. These results offer a possible explanation of rapid spectral conversion of Na⁺/K⁺ exchange and insight into the mechanism of Na⁺/K⁺ structural conversion in human telomeric G4s.     

Fluorescein-labeled “arch-like” DNA probes for electrochemical detection of DNA on gold nanoparticle-modified gold electrodes

In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0 × 10⁻⁹ M to 2.0 × 10⁻⁸ M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10 × 10⁻¹⁰ M. Additionally, the specificity and hybridization response of this simple sensor were investigated.     

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.     

Whole serum BSA antibody screening using a label-free biophotonic nanoparticle array

Bovine serum albumin antibodies (aBSA) have been screened from whole leporine anti serum on a biophotonic array. The array was initially printed with seed gold nanoparticles into a 96-spot configuration, and 130-nm gold nanoparticles were synthesised in situ on the surface of each spot. The gold nanoparticle surface was then functionalized with the proteins bovine serum albumin (BSA), fibrinogen, and immunoglobulin G (IgG) and with the amino acid glycine. The concentration of aBSA in the whole serum was determined using a kinetic analysis of the time-dependent light scattering from the nanoparticles. The aBSA-BSA kinetic parameters derived from the array are k_a = (1.3 ± 0.3) × 10⁵ M⁻¹ s⁻¹, k_d = (4 ± 2) × 10⁻⁴ s⁻¹, and K_D = 3 nM, which compare favorably with those from continuous gold surfaces. The ultimate sensitivity of the array reader to the bulk refractive index (RI) is 1 × 10⁻⁴ refractive index units (RIU), corresponding to 1 μg ml⁻¹ for aBSA. The nanoparticles appear to be more sensitive than the continuous gold surface to the aBSA binding event from whole serum, and this is interpreted in terms of the difference in RI contrast in the plasmon fields.     

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.     

Surface-functionalized gold nanoparticles mediate bacterial transformation: a nanobiotechnological approach

Transformation of bacteria is an important step in molecular biology. Viral and non-virus-based gene delivery techniques, including chemical/biological and physical approaches, have been applied to bacterial, mammalian and plant cells. E. coli is not competent to take up DNA; hence, different methods are used to incorporate plasmid DNA. A novel method has been developed using glutathione-functionalized gold nanoparticles to mediate transformation of plasmid DNA (pUC19) into E. coli DH5α that does not require the preparation of competent cells. The glutathione-functionalized gold nanoparticles acted as a vector and facilitated the entry of DNA into the host cell. The method also gave a higher transformation efficiency (4.2 × 10⁷/μg DNA) compared to 2.3 × 10⁵/μg DNA using the conventional CaCl₂-mediated method. It was also non-toxic to the bacterium making it suitable for biotechnological applications.