A sensitive gold-nanorods-based nanobiosensor for specific detection of Campylobacter jejuni and Campylobacter coli.

BACKGROUND: Campylobacteriosis is a zoonotic infectious disease that can be mostly undiagnosed or unreported due to fastidious Campylobacter species. The aim of this study was to develop a simple, sensitive, and quick assay for the detection of Campylobacter spp. and taking advantage of the great sensitivity of gold nanorods (GNRs) to trace changes in the local environment and interparticle distance. METHODS: Characterized GNRs were modified by specific ssDNA probes of cadF gene. First, the biosensor was evaluated using recombinant plasmid (pTG19-T/cadF) and synthetic single-stranded 95 bp gene, followed by a collection of the extracted DNAs of the stool samples. The sensing strategy was compared by culture, PCR, and real-time PCR. RESULTS AND DISCUSSION: Analysis of 283 specimens showed successful detection of Campylobacter spp. in 44 cases (16%), which was comparable to culture (7%), PCR (15%), and real-time PCR (18%). In comparison with real-time PCR, the sensitivity of the biosensor was reported 88%, while the specificity test for all assays was the same (100%). However, it was not able to detect Campylobacter in 6 positive clinical samples, as compared to real-time PCR. The limit of detection was calculated to be the same for the biosensor and real-time PCR (102 copy number/mL). CONCLUSIONS: Taking high speed and simplicity of this assay into consideration, the plasmonic nanobiosensor could pave the way in designing a new generation of diagnostic kits for detection of C. jejuni and C. coli species in clinical laboratories.

Synthesis and Characterization of Selenium Nanoparticles-Lysozyme Nanohybrid System with Synergistic Antibacterial Properties.

In the light of promising potency of selenium nanoparticles in biomedical applications, this is the first study to report the synergistic antibacterial activity of these nanoparticles and lysozyme. The nanohybrid system was prepared with various concentrations of each component. Resistance of Escherichia coli and Staphylococcus aureus was compared in the presence of individual Nano and Bio counterparts as well as the nanohybrid system. Upon interaction of SeNPs with Lysozyme, the nanohybrid system efficiently enhanced the antibacterial activity compared to the protein. Therefore, SeNPs play an important role in inhibition of bacterial growth at very low concentrations of protein; whereas very high amount of the protein is required to inhibit bacterial growth individually. On the other hand, lysozyme has also played a vital role in antibacterial property of SeNPs, inducing 100% inhibition at very low concentration of each component. Hence, presence of both nano and bio counterparts induced vital interplay in the Nanohybrid system. The aged samples also presented good stability of SeNPs both as the intact and complex form. Results of this effort highlight design of nanohybrid systems with synergistic antibacterial properties to overcome the emerging antibiotic resistance as well as to define fruitful applications in biomedicine and food safety.

Synergistic Antibacterial Activity and Wound Healing Properties of Selenium-Chitosan-Mupirocin Nanohybrid System: An in Vivo Study on Rat Diabetic Staphylococcus aureus Wound Infection Model.

The current study aimed to formulate Selenium-Chitosan-Mupirocin (M-SeNPs-CCH) complex. The nanohybrid system was prepared using chitosan-cetyltrimethylammonium bromide (CTAB)-based hydrogel (CCH) that entrapped mupirocin (M) and selenium nanoparticles (SeNPs). The in vitro studies were performed by evaluation of the antibacterial activity and toxicity on L929 mouse fibroblast cell line. The in vivo study was conducted on rat diabetic wound infection model that was infected by mupirocin-methicillin-resistant Staphylococcus aureus (MMRSA). The wounds were treated by M-SeNPs-CCH nanohybrid system with concentrations of M; 20 mg/ml, CCH; 2 mg/ml and SeNPs; 512 µg/ml in two times/day for 21 days. The therapeutic effect of this nanohybrid system was evaluated by monitoring wound contraction and histopathological changes. Evaluation of the average wound healing time showed a significant difference between the treatment and control groups (P≤0.05). The histopathological study indicated that the amount of wound healing was considerable in M-SeNPs-CCH nanohybrid system groups compared to the control and M groups. The M-SeNPs-CCH nanohybrid system formulated in this study was able to reduce 3-fold MIC of mupirocin with synergistic antibacterial activity as well as to play a significant role in wound contraction, angiogenesis, fibroblastosis, collagenesis, proliferation of hair follicle, and epidermis growth compared to the control group (P ≤ 0.05). This research suggests that this nanohybrid system might be a development for the treatment of diabetic wound infection at mild stage.

PDMS Nano-Modified Scaffolds for Improvement of Stem Cells Proliferation and Differentiation in Microfluidic Platform.

Microfluidics cell-based assays require strong cell-substrate adhesion for cell viability, proliferation, and differentiation. The intrinsic properties of PDMS, a commonly used polymer in microfluidics systems, regarding cell-substrate interactions have limited its application for microfluidics cell-based assays. Various attempts by previous researchers, such as chemical modification, plasma-treatment, and protein-coating of PDMS revealed some improvements. These strategies are often reversible, time-consuming, short-lived with either cell aggregates formation, not cost-effective as well as not user- and eco-friendly too. To address these challenges, cell-surface interaction has been tuned by the modification of PDMS doped with different biocompatible nanomaterials. Gold nanowires (AuNWs), superparamagnetic iron oxide nanoparticles (SPIONs), graphene oxide sheets (GO), and graphene quantum dot (GQD) have already been coupled to PDMS as an alternative biomaterial enabling easy and straightforward integration during microfluidic fabrication. The synthesized nanoparticles were characterized by corresponding methods. Physical cues of the nanostructured substrates such as Young's modulus, surface roughness, and nanotopology have been carried out using atomic force microscopy (AFM). Initial biocompatibility assessment of the nanocomposites using human amniotic mesenchymal stem cells (hAMSCs) showed comparable cell viabilities among all nanostructured PDMS composites. Finally, osteogenic stem cell differentiation demonstrated an improved differentiation rate inside microfluidic devices. The results revealed that the presence of nanomaterials affected a 5- to 10-fold increase in surface roughness. In addition, the results showed enhancement of cell proliferation from 30% (pristine PDMS) to 85% (nano-modified scaffolds containing AuNWs and SPIONs), calcification from 60% (pristine PDMS) to 95% (PDMS/AuNWs), and cell surface marker expression from 40% in PDMS to 77% in SPION- and AuNWs-PDMS scaffolds at 14 day. Our results suggest that nanostructured composites have a very high potential for stem cell studies and future therapies.

RGD-HK Peptide-Functionalized Gold Nanorods Emerge as Targeted Biocompatible Nanocarriers for Biomedical Applications.

Gold nanorods (GNRs) have been nominated as a promising candidate for a variety of biological applications; however, the cationic surfactant layer that surrounds a nanostructure places limits on its biological applicability. Herein, CTAB-GNRs were functionalized via a ligand exchange method using a (C(HK)4-mini PEG-RGD)-peptide to target the overexpressed αvß3 integrin in cancerous cells, increase the biocompatibility, and gain the ability of gene/drug delivery, simultaneously. To confirm an acceptable functionalization, UV-Visible, FTIR, and Raman spectroscopy, zeta potential, and transmission electron microscopy of nanostructures were done. MTT assay was applied to study the cytotoxicity of nanostructures on two cell lines, HeLa and MDA-MB-231, as positive and negative αvß3 integrin receptors, respectively. The cytotoxic effect of peptide-functionalized GNRs (peptide-f-GNRs) was less than that of CTAB-coated GNRs (CTAB-GNRs) for both cell lines. Uptake of peptide-f-GNRs and CTAB-GNRs was evaluated in two cell lines, using dark-field imaging and atomic absorption spectroscopy. Peptide-f-GNRs showed a proper cell uptake on the HeLa rather than MDA-MB-231 cell line according to the RGD (Arg-Gly-Asp) sequence in the peptide. The ability of peptide-f-GNRs to conjugate to antisense oligonucleotides (ASO) was also confirmed using zeta potential, which was due to the repeated HK (His-Lys) sequence inside the peptide. The result of these tests highlights the functionalization method as a convenient and cost-effective strategy for promising applications of targeted GNRs in the biological gene/drug delivery systems, and the repeated histidine-lysine pattern could be a useful carrier for negatively charged drug/gene delivery, too.

In Vitro Cytotoxicity of Folate-Silica-Gold Nanorods on Mouse Acute Lymphoblastic Leukemia and Spermatogonial Cells.

OBJECTIVE: The purpose of this study was to evaluate in vitro cytotoxicity of gold nanorods (GNRs) on the viability of spermatogonial cells (SSCs) and mouse acute lymphoblastic leukemia cells (EL4s). MATERIALS AND METHODS: In this experimental study, SSCs were isolated from the neonate mice, following enzymatic digestion and differential plating. GNRs were synthesized, then modified by silica and finally conjugated with folic acid to form F-Si-GNRs. Different doses of F-Si-GNRs (25, 50, 75, 100, 125 and 140 µM) were used on SSCs and EL4s. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay was performed to examine the GNRs toxicity. Flow cytometry was used to confirm the identity of the EL4s and SSCs. Also, the identity and functionality of SSCs were determined by the expression of specific spermatogonial genes and transplantation into recipient testes. Apoptosis was determined by flow cytometry using an annexin V/propidium iodide (PI) kit. RESULTS: Flow cytometry showed that SSCs and EL4s were positive for Plzf and H-2kb, respectively. The viability percentage of SSCs and EL4s that were treated with 25, 50, 75, 100, 125 and 140 µM of F-Si-GNRs was 65.33 ± 3.51%, 60 ± 3.6%, 51.33 ± 3.51%, 49 ± 3%, 30.66 ± 2.08% and 16.33 ± 2.51% for SSCs and 57.66 ± 0.57%, 54.66 ± 1.5%, 39.66 ± 1.52%, 12.33 ± 2.51%, 10 ± 1% and 5.66 ± 1.15% for EL4s respectively. The results of the MTT assay indicated that 100 µM is the optimal dose to reach the highest and lowest level of cell death in EL4s and in SSCs, respectively. CONCLUSION: Cell death increased with increasing concentrations of F-Si-GNRs. Following utilization of F-Si-GNRs, there was a significant difference in the extent of apoptosis between cancer cells and SSCs.

Assembly of Gold Nanorods on HSA Amyloid Fibrils to Develop a Conductive Nanoscaffold for Potential Biomedical and Biosensing Applications.

Today, Gold Nanorods have promised variety of applications in conjugation with biomolecules of interest. Discovery of functional amyloids has also been highlighted with possible use in designing high performance materials. To exploit dual properties of both Nano and Bio counterparts in new functional materials, this effort has focused on synthesis of a potential hybrid system of Gold nanorods (GNRs) and HSA amyloid fibrils to develop a conductive nanoscaffold. UV-Vis spectroscopy, Thioflavin T (ThT) assay, Far-UV Circular Dichroism (CD) spectropolarimetry, fluorescence and Transmission Electron microscopy were used to characterize formation of the nanostructures and amyloid fibrils. Surface plasmon resonance of GNRs was also monitored upon interaction with HSA amyloid fibrils, showing that the plasmonic component of the hybrid system has maintained its characteristic rod morphology without any perturbations. Analysis of Nyquist plots for the hybrid nanoscaffold showed that the electronic behavior of the hybrid system has been enhanced due to the presence of the assembled GNRs. Results of this investigation highlight the possibility of fabricating hybrid nano-bioscaffolds as promising candidates in versatile biomedical and biosensing applications.

In Silico Analysis of the cadF Gene and Development of a Duplex Polymerase Chain Reaction for Species-Specific Identification of Campylobacter jejuni and Campylobacter coli.

BACKGROUND: Campylobacteriosis is a zoonotic infectious disease caused by Campylobacter jejuni and C. coli. The cadF gene is considered as a genus-specific gene while other genes are mainly used for discrimination at the species level. OBJECTIVES: This study aimed to analyze the cadF gene and to develop a duplex PCR assay for simultaneous detection of C. coli and C. jejuni, the two commonly encountered species. MATERIALS AND METHODS: In silico analysis of the cadF gene was carried out by several software and available online tools. A duplex PCR optimized with specific primers was used for detection and differentiation of both species. To evaluate specificity and sensitivity of the test, a panel of different Campylobacter spp. together with several intestinal bacterial pathogens was tested. The limit of detection (LOD) of method was determined using serial dilutions of standard genomes. RESULTS: The analysis of the full size cadF gene indicated variations in this gene, which can be used to differentiate C. jejuni and C. coli. The duplex PCR designed in this study showed that it could simultaneously detect and differentiate both C. jejuni and C. coli with product sizes of 737 bp and 461 bp, respectively. This assay, with 100% specificity and sensitivity, had a limit of detection (LOD) of about 14 and 0.7 µg/mL for C. jejuni and C. coli, respectively. CONCLUSIONS: In silico analysis of the cadF full-gene showed variations between the two species that can be used as a molecular target for differentiating C. jejuni and C. coli in a single-step duplex-PCR assay with high specificity and sensitivity.

Heat induced aggregation of gold nanorods for rapid visual detection of lysozyme.

Gold nanorods have been nominated as propitious candidates for nanobiodiagnostic applications. Herein, a technique has been introduced for rapid visual detection of lysozyme, as its high level of excretion in biological fluids is a characteristic sign of leukemia and kidney disorders. Gold nanorods were biofunctionalized with lysozyme aptamer and characterized with UV-Visible and FTIR spectroscopy, zeta potential analyzer and transmission electron microscopy. Exposure of the nanoprobe to nano molar levels of lysozyme (20 nmol l(-1)) lead to dictated aggregation of the nanostructures at ambient temperature; which was significantly improved by heat induced morphological perturbations and rapid detection by the naked eye (down to pico molar level). Qualitative analysis of Acute myeloid leukemia, Acute lymphocytic leukemia and Lymphoma blood serums showed sensitivity and specificity of the fabricated aptasensor under both temperature conditions. This report encourages utilization of heat-induced aggregation of gold nanorods as a promising nanodiagnostic technique for the emerging nanotechnologies.

Functional motions of Candida antarctica lipase B: a survey through open-close conformations.

Candida antarctica lipase B (CALB) belongs to psychrophilic lipases which hydrolyze carboxyl ester bonds at low temperatures. There have been some features reported about cold-activity of the enzyme through experimental methods, whereas there is no detailed information on its mechanism of action at molecular level. Herein, a comparative molecular dynamics simulation and essential dynamics analysis have been carried out at three temperatures (5, 35 and 50 °C) to trace the dominant factors in the psychrophilic properties of CALB under cold condition. The results clearly describe the effect of temperature on CALB with meaningful differences in the flexibility of the lid region (α5 helix), covering residues 141-147. Open- closed conformations have been obtained from different sets of long-term simulations (60 ns) at 5 °C gave two reproducible distinct forms of CALB. The starting open conformation became closed immediately at 35 and 50 °C during 60 ns of simulation, while a sequential open-closed form was observed at 5 °C. These structural alterations were resulted from α5 helical movements, where the closed conformation of active site cleft was formed by displacement of both helix and its side chains. Analysis of normal mode showed concerted motions that are involved in the movement of both α5 and α10 helices. It is suggested that the functional motions needed for lypolytic activity of CALB is constructed from short-range movement of α5, accompanied by long-range movement of the domains connected to the lid region.