Current Bionanotechnology (Discontinued) - Volume 1, Issue 2, 2015

Background: The use of DNA aptamers to replace antibodies in lateral flow (LF) test strip assays coupled to quantum dots (QDs) has demonstrated enhanced sensitivity for the rapid and portable detection of several foodborne pathogenic bacterial species with simple ultraviolet (UV) illumination and visual assessment (Bruno, Pathogens, 2014; 3: 341-355). Objectives: The present report extends and focuses development on detection of O157- specific lipopolysaccharide (LPS), intimin protein and Shiga toxin 1 (Stx 1) using the same aptamer-QD LF assay approach to aid in rapid and sensitive detection of E. coli O157:H7 and other pathogenic serotypes of E. coli. Methods: Numerous anti-O157 LPS, intimin and anti-Stx 1 aptamer DNA sequence candidates were developed and screened by enzyme-linked aptamer assay (ELASA) followed by further screening of the best candidates in less expensive aptamer- latex particle or aptamer-colloidal gold (CG) LF formats. The highest affinity and most specific aptamer candidates were incorporated into the aptamer-QD LF format. Results: Several high affinity and specific aptamers against intimin, Stx1, and O157 LPS were developed and found to work well in various capture-reporter conjugate combinations using latex particles, CG and QDs with detection limits as low as 100 E. coli O157:H7 bacterial cells and 10 ng of Stx 1 in buffer by visual assessment. Conclusion: The seminal work in this aptamer-QD LF area has been extended to sensitive detection of E. coli O157 cells as well as detection of other pathogenic E. coli serotypes by targeting intimin and Stx 1.

Background: The present study deals with new microwave assisted green, costeffectiveness, synthesis of metal nanoparticles using aqueous leaf extracts. The plant extracts with metal precursors were used to made eco-friendly/ biodegradable metal nanoparticles through conventional and microwave irradiated methods. The iron and silver nanoparticles are one of them. Methods: Synthesized AgNPs and FeNPs were primarily characterized by UV-Vis spectrophotometer and further evaluated with FTIR and Atomic Force Microscopy (AFM) techniques. AgNPs and FeNPs were studied for antimicrobial activity against various human pathogenic microorganisms E. coli, V. cholerae (Non-0319), Shigella flexneri (16), Klebsiella pneumoniae (BCH-271), V. Cholerae (Non-0319-CSK 6669), S. pneumoniae, S. aureus, Shigella dysenteriae using disc diffusion methods. Results: The synthesized silver nanoparticles using aqueous leaf extracts of P. guajava and Camellia sinensis showed maximum absorption peak at 420nm and 421nm respectively and iron nanoparticles synthesized using aqueous leaf extracts of Mangifera indica showed maximum absorption peak at 315nm. The AFM images of sample revealed the presence of metal nanoparticles and particle size was found as 42nm, synthesized under microwave in presence of plant extracts (P.guajava). 16nm and 52nm sized nanoparticles were synthesized under conventional methods using aqueous leaf extract of Camellia sinensis and Mangifera indica. Silver nanoparticles synthesized using aqueous leaf extracts of Camellia sinensis showed antibacterial activity against various pathogenic microorganisms E. coli (9mm), V. cholerae (Non-0319) (8mm), Shigella flexneri (16) (8mm), Klebsiella pneumoniae (BCH-271) (10mm), V. Cholerae (Non-0319- CSK 6669) (9mm), S. pneumoniae (6mm), S. aureus (No), Shigella dysenteriae (No). Silver nanoparticles and iron nanoparticles synthesized using aqueous leaf extract of P. guajava and Magnifera indica did not show any zone of inhibition against various pathogenic microorganisms. Conclusion: Aqueous leaf extracts are useful for the preparation of metal nanoparticles under microwave irradiation and conventional methods through green processes. The silver nanoparticles synthesized using aqueous leaf extract of Camellia sinensis showed antibacterial activity against various human pathogenic microorganisms.

Background: Biological applications such as imaging and targeted drug delivery and many others require non-toxic, stable, inert and biocompatible nanoparticles. Coating the nanoparticles with inert materials is one of the ways that is used to enhance the stability and biocompatibility of nanoparticles. In particular, gold coating is very attractive as it reduces the toxicity of the magnetic nanoparticles, enhances their stability in biological systems and provides stable and inert platforms for further functionalization. However, current methods for the fabrication of gold-coated magnetic nanoparticles are carried out in non-aqueous media with toxic organic solvents at very high temperatures. In this article, we demonstrate a novel procedure for the fabrication of gold-coated cobalt nanoparticles. Unlike previous procedures that are performed in nonaqueous conditions at elevated temperatures, this method can be carried out exclusively in aqueous media at ambient conditions. Methods: Cobalt nanoparticles were prepared by the reduction of a cobalt precursor and subsequently coated with gold by an electroless plating procedure. The Co NPs and the gold-coated Co NPs were characterized by ultraviolet-visible (UVVis) spectroscopy, X-ray diffraction (XRD), superconducting quantum interference device (SQUID) magnetometry, scanning electron (SEM) and transmission electron (TEM) microscopy. Results: Results from SEM and TEM imaging indicated that the resulting gold-coated Co NPs had a Co core and an Au shell, were well dispersed and within the nanoscale range in terms of size (45 ± 8 nm in diameter). EDX confirmed the presence of both Co and Au in the coated sample. XRD data demonstrated a pattern with diffraction peaks indicating presence of gold and cobalt. SQUID results indicated that the magnetic properties of the nanoparticles were maintained even after coating with gold. Conclusion: All these results confirmed the fabrication of stable, well dispersed magnetic gold-coated Co NPs in aqueous media making them potentially ideal for downstream applications such as in vivo imaging, therapeutics and biological applications.

Background: Storage stability of plasmid biopharmaceuticals is a critical issue that needs to be addressed during clinical and process development. Objectives: The goal of this work was to evaluate the ability of stabilizers to prolong the stability of plasmid DNA solutions and extend the duration of transgene expression of transfected cells. Methods: A plasmid harboring the GFP gene and Chinese Hamster Ovary (CHO) cells were used as models. Plasmid solutions were formulated with the stabilizer DNAstablePlusTM, 300 mM trehalose and 300 mM cellobiose. The biological activity was monitored by transfecting CHO cells with the preparations using Lipofectamine. Results: Protection against denaturation conferred by DNAstablePlusTM at 60 °C was outstanding, with 94% of the activity preserved after 7 days compared to 76% with trehalose, 70% with cellobiose and <10% without stabilizers. While plasmid DNA stored at room temperature lost 95% of its ability to express GFP in the first month, trehalose, cellobiose and DNAstablePlusTM were able to preserve it for 6, 8 and at least 12 months, respectively. The incorporation of trehalose, cellobiose and DNAstablePlusTM in lipoplexes also contributed to extend the expression of GFP in transfected cells. While a significant loss of GFP-expressing cells (~10%) was observed after 7days with no stabilizers, formulation with DNAstablePlusTM, cellobiose and trehalose increased the number of cells GFP-expressing cells to more than 50%. Conclusions: The biological activity of plasmid DNA solutions stored at room temperature was extended several fold by incorporating cellobiose, trehalose and DNAstablePlusTM in the formulations.

In conductometric enzymatic biosensors, enzymatic reaction is confined close to the interdigitated electrode surface, because enzyme is cross-linked in contact with this surface in the presence or absence of nanoparticles. The effect of the use of a new type of doubly-functionalized gold nanoparticles (PF-HEG-Au NPs) on the response of conductometric biosensor based on interdigitated electrodes (IDEs), for the detection of enzymatic substrates was studied. Gold nanoparticles (AuNPs) were first synthesized following the citrate process, with an average diameter of 14 nm. AuNPs were then functionalized with 11-mercaptoundecylhexaethyleneglycol (HEG) and then with 1H,1H,2H,2H-perfluorodecanethiol (PF). The doubly-functionalized AuNPs were characterized using TEM, UV-Vis spectrophotometry and FTIR spectroscopy. Urease, mixed with these doubly functionalized AuNPs, was then cross-linked with glutaraldhedyde vapor on the IDE surface. In the presence of urea, the conductometric response was measured in a differential mode. The best sensitivities for urea detection were obtained with PF-HEG-Au NPs (520 μS /mM and 0.5μM of detection limit), as compared to 284μS/mM and 2μM of detection limit with bare Au NPs, PF-AuNPs and HEG-AuNPs, and 1.07μS/mM and 100 μM of detection limit with urease directly crosslinked on IDEs.When stored in phosphate buffer (5 mM, pH 6.7) at 4 °C, the biosensor with PF-HEG-Au NPs showed good stability for more than 12 days.

Background: With the recent advances of nanotechnological interventions, nanoparticles have found immense importance in various sectors of water, energy, health, agriculture and environment. Concurrently, there has been an increasing concern about their toxicity on living systems. The bactericidal role of silver nanoparicle is well established. The present work explores the interaction of silver nanoparticles with halophilic bacteria Bacillus sp. EMB9. Given that halophiles thrive under saline/ hypersaline habitats and retain their structural and functional integrity under such high salt conditions, they present a new and interesting model system for understanding their interactions with metals in nanoparticulate form. Methods: Bacillus sp. EMB9 was incubated in the presence and absence of 1.0 mM silver nanoparticles and their growth profile monitored. Their responses towards the nano-stress environment were further evaluated using a proteomic approach. Result: Despite initial nanotoxic effects, Bacillus sp. EMB9 was able to resist silver mediated nanotoxicity and grow with a high specific growth rate. Proteomic analysis showed striking global changes in the intracellular bacterial proteome with almost a 50% reduction in the number of expressed proteins in the cells grown in presence of silver nanoparticles. Out of a total of 261 protein spots detected, 24 were newly expressed and expression of 132 spots was suppressed completely in the nanoparticle treated cells. Conclusion: The differential expression patterns are indicative of adaptive strategies being employed by the bacteria for functioning of the cellular machinery amidst nano-stress. A comprehensive understanding of the response of halophilic Bacillus sp. EMB9 to silver nanoparticles will provide significant insight into mechanistic interpretations of bacteriananoparticle interactions.

Biofilm development is a survival strategy for majority of bacteria to adapt to their living environment. These biofilms are prevalent on most surfaces in nature. Microbial cells in biofilm exhibit significantly enhanced tolerance and resistance to antimicrobial challenge and host defenses. Therefore, the control and eradication of biofilm-associated diseases present a great challenge. Recently, there is considerable biomedical incentive in the development of nanoparticles with self-antibacterial activity or as antibiotic carriers. The advantages of coatings incorporating different types of nano-agents for antimicrobial surface generation are here reviewed. Although the main coatings currently used or investigated and the associated synthesis processes are individually described, emphasis is made on silver-loaded mesoporous thin coatings. Recent developments suggest that mesoporous oxide thin films (especially titania) incorporating metal nanoparticles is becoming a prime candidate for antimicrobial coatings.