A new method for non-labeling attomolar detection of diseases based on an individual gold nanorod immunosensor

Herein, we present the use of a single gold nanorod sensor for detection of diseases on an antibody-functionalized surface, based on antibody–antigen interaction and the localized surface plasmon resonance (LSPR) ?max shifts of the resonant Rayleigh light scattering spectra. By replacing the cetyltrimethylammonium bromide (CTAB), a tightly packed self-assembled monolayer of HS(CH2)11(OCH2CH2)6OCH2COOH(OEG6) has been successfully formed on the gold nanorod surface prior to the LSPR sensing, leading to the successful fabrication of individual gold nanorod immunosensors. Using prostate specific antigen (PSA) as a protein biomarker, the lowest concentration experimentally detected was as low as 111 aM, corresponding to a 2.79 nm LSPR ?max shift. These results indicate that the detection platform is very sensitive and outperforms detection limits of commercial tests for PSA so far. Correlatively, its detection limit can be equally compared to the assays based on DNA biobarcodes. This study shows that a gold nanorod has been used as a single nanobiosensor to detect antigens for the first time; and the detection method based on the resonant Rayleigh scattering spectrum of individual gold nanorods enables a simple, label-free detection with ultrahigh sensitivity.

Quantitative detection of DNA by autocatalytic enlargement of hybridized gold nanoprobes

Quantitative detection of specific viral DNA has become a pressing issue for the earlier clinical diagnosis of viral infectious diseases. Therefore, in this paper, we report a simple, sensitive, and inexpensive quantitative approach for DNA detection based on the autocatalytic Au deposition of gold nanoprobes via the surface reduction of AuCl4- to Au0 on their surface in the presence of ascorbic acid (AA) and cetyltrimethylammonium bromide (CTAB). On this basis, signal enhancements in the absorbance intensity and kinetic behavior of gold enlargement in the aqueous phase have been well investigated and explained for the selection of analytical parameters. To achieve high sensitivity, magnetic particles conjugated with capture probes (PMPs) were employed for the collection of gold nanoprobes. After denaturated by ion a pH 11 solution, the amplified signals of gold nanoprobes, which is proportional to the concentration of the target DNA, could easily be confirmed by a UV-vis scanning spectrophotometer. Limit of detection could be obtained as low as 1.0 fM by this simple method.

One-Pot, High-Yield Synthesis of Convex Au Octahedra by H2O2 - Mediated Process In Solution Phase

A simple, non-seeding and high-yield synthesis of convex gold octahedra with size of ca. 50 nm in aqueous solution is described. The octahedral nanoparticles were systematically prepared by reduction of HAuCl4 using ascorbic acid (AA) in the presence of cetyltrimethylammonium bromide (CTAB) as the stabilizing surfactant while concentrations of Au3+ were fixed. The synthesizing process is especially different to other wet synthesis of metallic nanoparticles because it is mediated by H2O2. Mechanism of the H2O2 – mediated process will be described in details. The gold octahedra were shown to be single crystals with all 8 faces belonging to {111} family. Moreover, the single crystalline particles also showed attractive optical properties towards LSPR that should find uses as labels for microscopic imaging, materials for colorimetric biosensings, or nanosensor developments.

Isotope dilution gas chromatography/mass spectrometry method for the determination of methionine sulfoxide in protein

We have developed a new technique for quantifying methionine sulfoxide (MetSO) in protein to assess levels of oxidative stress in physiological systems. In this procedure, samples are hydrolyzed with methanesulfonic acid (MSA) in order to avoid the conversion of MetSO to methionine (Met) that occurs during hydrolysis of protein in HCl. The hydrolysate is fractionated on a cation exchange column to remove the nonvolatile MSA from amino acids, and the amino acids are then derivatized as their trimethylsilyl esters for analysis by selected ion monitoring-gas chromatography/mass spectrometry. The limit of detection of the assay is 200 pmol of MetSO per analysis, and the interassay coefficient of variation is 5.8%. Compared to current methods, the SIM-GC/MS assay avoids the potential for conversion of Met to MetSO during sample preparation, requires less sample preparation time, has lower variability, and uses mass spectrometry for sensitive and specific analyte detection.

Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

The phase behaviour of 1-alkyl-3-methylimidazolium tetrafluoroborates; ionic liquids and ionic liquid crystals

Air- and water-stable 1-alkyl-3-methylimidazolium tetrafluoroborate salts with the general formula [C-mim][BF] (n = 0-18) have been prepared by metathesis from the corresponding chloride or bromide salts. The salts have been characterised by H NMR and IR spectroscopy, microanalysis, polarising optical microscopy and differential scanning calorimetry. Those with short alkyl chains (n = 2-10) are isotropic ionic liquids at room temperature and exhibit a wide liquid range, whereas the longer chain analogues are low melting mesomorphic crystalline solids which display an enantiotropic smectic A mesophase. The thermal range of the mesophase increases with increasing chain length and in the case of the longest chain salt prepared, [C-mim][BF], the mesophase range is ca. 150°C.

catena-poly[mercury(II)-di-mu-bromo-mu-pyridazine-kappa N-2 : N ']

The crystal structure of [HgBr2(Pyo)](n) (Pyo = pyridazine, C4H4N2) consists of strands of octahedrally coordinated mercuric centers asymmetrically bridged by bromide and connected by the two neighboring N atoms of pyridazine molecules to complete the octahedral coordination of mercury. The Hg atoms lie on inversion centers.

Effect of laser treatment on the attachment and viability of mesenchymal stem cell responses on shape memory NiTi alloy

The objectives of this study were to investigate the effect of laser-induced surface features on the morphology, attachment and viability of mesenchymal stem cells (MSCs) at different periods of time, and to evaluate the biocompatibility of different zones: laser-melted zone (MZ), heat-affected zone (HAZ) and base metal (BM) in laser-treated NiTi alloy. The surface morphology and composition were studied by scanning electron microscope (SEM) and X-ray photoemission spectroscopy (XPS), respectively. The cell morphology was examined by SEM while the cell counting and viability measurements were done by haemocytometer and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The results indicated that the laser-induced surface features, such as surface roughening, presence of anisotropic dendritic pattern and complete surface Ni oxidation were beneficial to improve the biocompatibility of NiTi as evidenced by the highest cell attachment (4 days of culture) and viability (7 days of culture) found in the MZ. The biocompatibility of the MZ was the best, followed by the BM with the HAZ being the worst. The defective and porous oxide layer as well as the coarse grained structure might attribute to the inferior cell attachment (4 days of culture) and viability (7 days of culture) on the HAZ compared with the BM which has similar surface morphology.

High content analysis: A sensitive tool to detect and quantify the cytotoxic, synergistic and antagonistic effects of chemical contaminants in foods

Aflatoxin B1 (AFB1), ochratoxin A (OTA) and fumonisin B1 (FB1) are important mycotoxins in terms of
human exposure via food, their toxicity and regulatory limits that exist worldwide. Mixtures of toxins can frequently be present in foods, however due to the complications of determining their combined toxicity,
legal limits of exposure are determined for single compounds, based on long standing toxicological
techniques. High content analysis (HCA) may be a useful tool to determine total toxicity of complex
mixtures of mycotoxins. Endpoints including cell number (CN), nuclear intensity (NI), nuclear area (NA),
plasma membrane permeability (PMP), mitochondrial membrane potential (MMP) and mitochondrial
mass (MM) were compared to the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium
bromide (MTT) and neutral red (NR) endpoints in MDBK cells. Individual concentrations of each
mycotoxin (OTA 3mg/ml, FB1 8mg/ml and AFB11.28mg/ml) revealed no cytotoxicity with MTTor NR but
HCA showed significant cytotoxic effects up to 41.6% (p0.001) and 10.1% (p0.05) for OTA and AFB1,
respectively. The tertiary mixture (OTA 3mg/ml, FB1 8mg/ml and AFB1 1.28mg/ml) detected up to 37.3%
and 49.8% more cytotoxicity using HCA over MTT and NR, respectively. Whilst binary combinations of
OTA (3mg/ml) and FB1 (8mg/ml) revealed synergistic interactions using HCA (MMP, MM, NI endpoints)
not detected using MTT or NR. HCA is a highly novel and sensitive tool that could substantially help
determine future regulatory limits, for single and combined toxins present in food, ensuring legislation is based on true risks to human health exposure.

3-Methylpiperidinium ionic liquids

A wide range of room temperature ionic liquids based on the 3-methylpiperdinium cation core were produced from 3-methylpiperidine, which is a derivative of DYTEK® A amine. First, reaction with 1-bromoalkanes or 1-bromoalkoxyalkanes generated the corresponding tertiary amines (Rmβpip, R = alkyl or alkoxyalkyl); further quaternisation reactions with the appropriate methylating agents yielded the quaternary [Rmmβpip]X salts (X(-) = I(-), [CF3CO2](-) or [OTf](-); Tf = -SO2CF3), and [Rmmβpip][NTf2] were prepared by anion metathesis from the corresponding iodides. All [NTf2](-) salts are liquids at room temperature. [Rmmβpip]X (X(-) = I(-), [CF3CO2](-) or [OTf](-)) are low-melting solids when R = alkyl, but room temperature liquids upon introduction of ether functionalities on R. Neither of the 3-methylpiperdinium ionic liquids showed any signs of crystallisation, even well below 0 °C. Some related non-C-substituted piperidinium and pyrrolidinium analogues were prepared and studied for comparison. Crystal structures of 1-hexyl-1,3-dimethylpiperidinium tetraphenylborate, 1-butyl-3-methylpiperidinium bromide, 1-(2-methoxyethyl)-1-methylpiperidinium chloride and 1-(2-methoxyethyl)-1-methylpyrrolidinium bromide are reported. Extensive structural and physical data are collected and compared to literature data, with special emphasis on the systematic study of the cation ring size and/or asymmetry effects on density, viscosity and ionic conductivity, allowing general trends to be outlined. Cyclic voltammetry shows that 3-methylpiperidinium ionic liquids, similarly to azepanium, piperidinium or pyrrolidinium counterparts, are extremely electrochemically stable; the portfolio of useful alternatives for safe and high-performing electrolytes is thus greatly extended.

Simple preparation of positively charged silver nanoparticles for detection of anions by surface-enhanced Raman spectroscopy

Modification of citrate and hydroxylamine reduced Ag colloids with thiocholine bromide, a thiol functionalized quaternary ammonium salt, creates particles where the zeta potential is switched from the normal values of ca. -50 mV to ca. + 50 mV. These colloids are stable but can be aggregated with metal salts in much the same way as the parent colloids. They are excellent SERS substrates for detection of anionic targets since their positive zeta potentials promote adsorption of negatively charged ions. This is important because the vast majority of published SERS studies involve cationic or neutral targets. Moreover, the fact that the modifier is a quaternary ammonium ion means that the negative surface charge is maintained even at alkaline pH. The modified colloids can be used to detect compounds which cannot be detected using conventional negatively-charged citrate or hydroxylamine reduced metal nanoparticles, for example the detection limit was 5.0 x 10(-5) M for perchlorate and

The pyrrolo-1,5-benzoxazepine, PBOX-6, inhibits the growth of breast cancer cells in vitro independent of estrogen receptor status and inhibits breast tumour growth in vivo

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.

Effects of tobacco products on the attachment and growth of periodontal ligament fibroblasts

BACKGROUND: Cigarette smoking is one of the most significant risk factors in the development and further advancement of inflammatory periodontal disease, however, the role of either nicotine or its primary metabolite cotinine in the progression of periodontitis is unclear. This study aimed to investigate the effects of nicotine and cotinine on the attachment and growth of fibroblasts derived from human periodontal ligament (PDL).

METHODS: Primary cultures were prepared from the roots of extracted premolar teeth. Cells were used at both low (P3 to P5) and high (P11 to P13) passage. Cell numbers were determined over 14 days using either the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay or with a Coulter counter. Cultures were exposed to culture medium supplemented with 1) 15% fetal calf serum (FCS) only; 2) 1% FCS only; 3) 1% FCS and nicotine (concentration range 5 ng/ml to 10 mg/ml); or 4) 1% FCS and cotinine (concentration range 0.5 ng/ml to 10 microg/ml).

RESULTS: Nicotine significantly (P <0.05, by ANOVA) inhibits attachment and growth of low passage cells at concentrations >1 mg/ml and high passage PDL fibroblasts at concentrations >0.5 mg/ml. Cotinine, at the highest concentration used (10 microg/ml), appeared to inhibit attachment and growth of both low and high passage fibroblasts but this was not statistically significant (P >0.05, by ANOVA).

CONCLUSIONS: Tobacco products inhibit attachment and growth of human PDL fibroblasts. This may partly explain the role of these substances in the progression of periodontitis.

Antibacterial Activity of Peptides from the African Volcano Frog

Introduction: Cationic, α- helical antimicrobial peptides found in skin secretions of the African Volcano Frog, Xenopus amieti include magainin-AM1, peptide glycine-leucine-amide (PGLa-AM1) and caerulein-precursor fragment (CPF-AM1). Objectives: The principle objective of this study was to determine the antibacterial activity of these peptides against a range of aerobic and anaerobic and oral pathogens. Secondary objectives were to establish their lipopolysaccharide (LPS) binding activity and determine potential cytotoxic effects against host cells. Methods: Magainin-AM1, PGLa-AM1 and CPF-AM1 were assessed for their antimicrobial activity against Fusobacteriim nucleatum, Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis and Streptococcus milleri using a double layer radial diffusion assay. The propensity for each peptide to bind LPS was determined using an indirect ELISA. The potential cytotoxicity of the peptides against human pulp cells in vitro was determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Magainin-AM1, PGLa-AM1 and CPF-AM1 displayed potent antimicrobial activity against all the bacterial pathogens tested, with Magainin-AM1 being the least effective. PGLa-AM1 was most potent against S. mutans, with a minimum inhibitory concentration (MIC) of 1.2 μM. PGLa-AM1 and CPF-AM1 were both very active against F. nucleatum with MIC values of 1.5 μM and 2.2 μM respectively. The LPS binding ability of the peptides varied depending on the bacterial source of the LPS, with PGLa-AM-1 being the most effective at binding LPS. Cytotoxicity studies revealed all three peptides lacked cytotoxic effects at the concentrations tested. Conclusions: The peptides magainin-AM1, PGLa-AM1 and CPF-AM1 from the African Volcano Frog, Xenopus amieti displayed potent antimicrobial activity and LPS binding activity against a range of oral pathogens with little cytotoxic effects. These peptides merit further studies for the development of novel therapeutics to combat common oral bacterial infections.