A fluorescence Nano-biosensors immobilization on Iron (MNPs) and gold (AuNPs) nanoparticles for detection of Shigella spp.

The highly sensitive and specificity detection are very important in diagnosis of foodborne pathogens and prevention of spread diseases. Therefore, in the present study, a highly sensitive fluorescence Nano-biosensors was designed for detection of Shigella species. For achieved this purpose, DNA probes and gold nanoparticles (AuNPs) were designed and synthesized, respectively. Then, two DNA probes as signal reporter were immobilized on surface of AuNPs. On the other hand, Iron nanoparticles (MNPs) were synthesized and modified with SMCC (Sulfosuccinimidyl 4-Nmaleimidomethyl cyclohexane-1- carboxylate). The 3th DNA probe was immobilized on surface of MNPs for separation of target DNA. The MNP-DNA probe and DNA probe-AuNP-fluorescence DNA probe were added to target DNA. The MNP- DNA probe-target DNA-DNA probe-AuNP-fluorescence DNA probe complex was isolated by a magnet. The fluorescence DNA probe was released on surface of AuNPs and the fluorescence intensity was read by fluorescence spectrophotometry. Sensitivity and specificity of designed Nano-biosensor was determined. The results showed that the fluorescence intensity was increased with increasing of target DNA concentration. Linear related between target DNA and fluorescence intensity was observed in 2.3 × 102 up to 2.3 × 107 CFU mL-1. The linear equation and regression were Y = 1.8 X + 23.4 and R2 0.9953. Limit of detection (LOD) were determined 90 CFUmL-1. The specificity of Nano-biosensor in present of other bacteria was confirmed.

Bio-barcode technology for detection of Staphylococcus aureus protein A based on gold and iron nanoparticles.

S. aureus is one of important causes of disease, food poisoning in humans and animals. The generally methods for detection of S. aureus is time consuming. Therefore, a new method is necessary for rapid, sensitive and specific diagnosis of S. aureus. In the present study, two probes and a Bio-barcode DNA were designed for detection of S. aureus (Protein A). Firstly, magnetic nanoparticle (MNPs) and gold nanoparticle (AuNPs) were synthesized at 80 °C and 100 °C, respectively. The AuNPs and the MNPs were functionalized with probe1, Bio-barcode DNA and probe2, respectively. Target DNA was added into the nanomaterial's system containing bio-barcode DNA-AuNPs-probe1 and probe2-MNPs to formed bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex. The bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex was separated with magnetic field. Finally, the bio-barcode DNA was released from surface of complex using DTT (0.8 M) and there was isolated of nanoparticles by magnetic field and centrifuge. The fluorescence intensity of bio-barcode DNA was measured in different concentrations of S. aureus (101 to 108 CFU mL-1) by fluorescence spectrophotometry. The results showed that standard curve was linearly from 102 to 107 CFU mL-1. Limit of detection of bio-barcode assay for both PBS and real samples was 86 CFU mL-1.

The combinational effect of E6/E7 siRNA and anti-miR-182 on apoptosis induction in HPV16-positive cervical cells.

In the present research, we assumed that reducing the amounts of E6 and E7 oncoproteins by a specific siRNA sequence and recovering p53 and RB proteins, along with the recovery of the FOXO1 protein by applying anti-miR-182, would increase apoptosis and reduce proliferation rate in cancer cells. The HPV16-positive CaSki cervical cancer cell line was used. 48 hours after transfection of siRNA for targeting E6 and E7 oncoproteins and anti-miR-182, expression of its cellular targets p53, p21 and FOXO1 was assessed by real-time PCR, western blot analysis and immunocytofluorescence staining. In all treatments, apoptosis rate and viability were evaluated using Annexin-V-FITC apoptosis detection kits and MTT assays, respectively. Among the designed siRNAs, E6-1 and E7-2 proved the most effective in reducing E6 and E7 expressions by increasing the apoptotic rates to 12.4% and 16%, respectively, after 48 hours. Also, using anti-miR-182 increased apoptotic rate to 12.7% 48 hours after transfection of cervical cancer cells. The combinational use of either E6-1 or E7-2 siRNAs with anti-miR-182 resulted in a rise in apoptosis to 19.3% and 26%, respectively, higher than those obtained from the individual application of either without anti-miR-182. The simultaneous use of siRNA E6-1 and siRNA E7-2 with cisplatin increased sensitivity to cisplatin and reduced the viability of the cancer cells as compared to the use of cisplatin alone. The simultaneous use of cisplatin and anti-miR-182 had no considerable effect on viability or apoptosis rate compared to cisplatin alone.

Ultrahigh sensitive enhanced-electrochemiluminescence detection of cancer biomarkers using silica NPs/graphene oxide: A comparative study.

The increasing progress in using nano-biomaterials for medical purposes has opened new horizons toward researchers around the globe. To investigate the presence of these nanomaterials and the impacts they might have, a comparative enhanced-electrochemiluminescence immunosensing study has been designed. The effects of utilizing graphene oxide, silica, and gold nanoparticles in cancer diagnosis were evaluated during the quantification of two major cancer biomarkers (CEA and AFP) in different approaches. In other words, first and second approaches were designed to employ nanomaterials while third and fourth approaches were developed in absence of those. Accordingly, resulted LODs experienced dramatic amplification when nano-biomaterials were included in the immunosensor modification (for AFP: 1st and 3rd approaches: 1.36fg/ml in comparison with 0.39ng/ml, and for CEA: 2nd and 4th approaches: 1.90fg/ml versus 0.46ng/ml, respectively). Correspondingly, capability of nano-biomaterials for developing highly sensitive and more efficient immunosensors was validated through selectivity, stability, reproducibility, and feasibility examinations.

Affinity enhancement of nanobody binding to EGFR: in silico site-directed mutagenesis and molecular dynamics simulation approaches.

Epidermal growth factor receptor (EGFR), a transmembrane glycoprotein, is overexpressed in many cancers such as head-neck, breast, prostate, and skin cancers for this reason it is a good target in cancer therapy and diagnosis. In nanobody-based cancer diagnosis and treatment, nanobodies with high affinity toward receptor (e.g. EGFR) results in effective treatment or diagnosis of cancer. In this regard, the main aim of this study is to develop a method based on molecular dynamic (MD) simulations for designing of 7D12 based nanobody with high affinity compared with wild-type nanobody. By surveying electrostatic and desolvation interactions between different residues of 7D12 and EGFR, the critical residues of 7D12 that play the main role in the binding of 7D12 to EGFR were elucidated and based on these residues, five logical variants were designed. Following the 50 ns MD simulations, pull and umbrella sampling simulation were performed for 7D12 and all its variants in complex with EGFR. Binding free energy of 7D12 (and all its variants) with EGFR was obtained by weighted histogram analysis method. According to binding free energy results, GLY101 to GLU mutation showed the highest binding affinity but this variant is unstable after 50 ns MD simulations. ALA100 to GLU mutation shows suitable binding enhancement with acceptable structural stability. Suitable position and orientation of GLU in residue 100 of 7D12 against related amino acids of EGFR formed some extra hydrogen and electrostatic interactions which resulted in binding enhancement.

An enquiry on appropriate selection of polymers for preparation of polymeric nanosorbents and nanofiltration/ultrafiltration membranes for hormone micropollutants removal from water effluents.

To analyze polymeric nanosorbents and nanofiltration/ultrafiltration membranes for hormone micropollutants removal from water effluents, here an in-through investigation on the suitability and compatibility of various polymers has been carried out. For this work, estradiol, estrone, testosterone, progesterone, estriol, mestranol, and ethinylestradiol were considered. A total number of 452 polymers were analyzed and initially screened using Hansen solubility parameters. The identified good pairs of hormones and polymers then were examined to obtain the equilibrium capacity of hormones removal from water effluents using a modified Flory-Huggins model. A distribution coefficient was defined as the ratio of hormones in water effluent phase and polymer phase. For removal of mestranol, estradiol and ethinylestradiol, no compatible polymer was identified based on initial screening of collected database. Three compatible polymers were identified for estriol. For progesterone, a wide variety of polymers was identified as good matching of polar, dispersion and hydrogen forces contributions can be observed for these pairs. For estrone, only two polymers can be proposed due to the mismatch observed between polar, dispersion and hydrogen forces contributions of other polymers and this hormone. The phase calculations showed that not all the identified good pairs could be used for practical separation applications. The domain of applicability of each good pair was investigated and potential polymers for practical micropollutants removal together with their removal capacity were represented in terms of phase envelops. The theoretical approach follows fundamental chemical thermodynamic equations and then can be simply applied for any system of interest.

Osteoblast differentiation of mesenchymal stem cells on modified PES-PEG electrospun fibrous composites loaded with Zn2SiO4 bioceramic nanoparticles.

Tissue engineering has attracted a great deal of interest by combining fibrous scaffolds and stem cells regarding bone regeneration applications. In the present study, polymeric fibrous polyethersulphone-polyethylene glycol (PES-PEG) was fabricated by electrospinning. It was then treated with NH3 plasma to enhance surface hydrophilicity, cell attachment, growth and differentiation potential. X-ray photoelectron spectroscopy (XPS) measurements were used to evaluate the modification of the scaffold's surface chemistry. Electrospun scaffolds were coated with willemite (Zn2SiO4) bioceramic nanoparticles. Scaffold characterization was done by scanning electron microscope (SEM), differential scanning calorimetry (DSC), contact angle measurements and tensile analysis. MTT assay was used to assess the biocompatibility of fibrous scaffolds loaded with Zn2SiO4 regarding proliferation support. Osteogenic differentiation of cultured human mesenchymal stem cells (hMSCs) on fibers was evaluated using common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium mineral deposition, quantitative real-time PCR (qPCR) and immunocytochemical analysis (ICC). According to the results, proliferation and osteogenic differentiation of hMSCs were significantly enhanced after coating Zn2SiO4 on fibrous scaffolds. These results were detected by higher ALP activity, biomineralization and expression of osteogenic related genes and proteins in differentiated hMSCs. In conclusion, our results indicated that the combination of Zn2SiO4 nanoparticles and electrospun fibers is able to provide a new, suitable and more efficient matrix to support stem cells differentiation for bone tissue engineering applications.

Bcr-abl silencing by specific small-interference RNA expression vector as a potential treatment for chronic myeloid leukemia.

BACKGROUND: RNA interference (RNAi) is the mechanism of gene silencing-mediated messenger RNA degradation by small interference RNA (siRNA), which becomes a powerful tool for in vivo research, especially in the areas of cancer. In this research, the potential use of an expression vector as a specific siRNA producing tool for silencing of Bcr-abl in K562 cell line has been investigated. METHODS: siRNA specific for Bcr-abl as short hairpin RNA (shRNA) was designed and cloned in expression vector (pRNAH1.1/Neo). K562 cells were cultured in RPMI media and transfected with shRNA expressing vector using lipofectamin 2000. Successful transfection was confirmed by significant increase of enhanced green fluorescent protein (EGFP) levels in K562-treated cells with expression vector (pEGFP-C1). In vitro studies in human K562 cell line entailed modulation of endogenous Bcr-abl mRNA levels which induced apoptosis. Effects of siRNA treatment on K562 cells were measured by ELISA. RESULTS: Successful expression of siRNA was confirmed by significant reduction of Bcr-abl mRNA levels in K562 cells treated with expression vector (pRNAH1.1/Neo). siRNA directed against Bcr-abl effectively induced apoptosis and reduced viability in human K562 cell lines. CONCLUSION: Expression vector of siRNA can be used in vitro to target specific RNA and to reduce the levels of the specific gene product in the targeted cells. Results of this work suggest that RNAi has potential application for the treatment of a variety of diseases, including those involving abnormal gene expression and viral contamination.