Annealing temperature plays an important role in the formation of an Au-Ga eutectic alloy. The effects of the annealing temperature on gold nanoparticles colloid and substrate surface were studied using AFM, FE-SEM and TEM. At 600oC, the layer of gold colloids particle formed an island in the state of molten eutectic alloy and absorbed evaporated metalorganics to formed nanowire (NW) underneath the alloy. Pit formed on the substrate surface due to the chemical reactions during the annealing process have an impact on the direction of growth of the NW. Without annealing, the NW formed vertically on the GaAs (100) surface. The growth direction depends on the original nucleation facets and surface energy when annealed. When annealed, the wire base is large and curved due to the migration of Ga atoms on the substrate surface towards the tip of the wire and the line tension between the substrate surface and gold particle.
Acanthamoeba keratitis is a serious infection with blinding consequences and often associated with contact lens wear. Early diagnosis, followed by aggressive topical application of drugs, is a prerequisite in successful treatment, but even then prognosis remains poor. Several drugs have shown promise, including chlorhexidine gluconate; however, host cell toxicity at physiologically relevant concentrations remains a challenge. Nanoparticles, subcolloidal structures ranging in size from 10 to 100 nm, are effective drug carriers for enhancing drug potency. The overall aim of the present study was to determine whether conjugation with gold nanoparticles enhances the antiacanthamoebic potential of chlorhexidine. Gold-conjugated chlorhexidine nanoparticles were synthesized. Briefly, gold solution was mixed with chlorhexidine and reduced by adding sodium borohydride, resulting in an intense deep red color, indicative of colloidal gold-conjugated chlorhexidine nanoparticles. The synthesis was confirmed using UV-visible spectrophotometry that shows a plasmon resonance peak of 500 to 550 nm, indicative of gold nanoparticles. Further characterization using matrix-assisted laser desorption ionization-mass spectrometry showed a gold-conjugated chlorhexidine complex at m/z 699 ranging in size from 20 to 100 nm, as determined using atomic force microscopy. To determine the amoebicidal and amoebistatic effects, amoebae were incubated with gold-conjugated chlorhexidine nanoparticles. For controls, amoebae also were incubated with gold and silver nanoparticles alone, chlorhexidine alone, neomycin-conjugated nanoparticles, and neomycin alone. The findings showed that gold-conjugated chlorhexidine nanoparticles exhibited significant amoebicidal and amoebistatic effects at 5 μM. Amoebicidal effects were observed by parasite viability testing using a Trypan blue exclusion assay and flow-cytometric analysis using propidium iodide, while amoebistatic effects were observed using growth assays. In contrast, chlorhexidine alone, at a similar concentration, showed limited effects. Notably, neomycin alone or conjugated with nanoparticles did not show amoebicidal or amoebistatic effects. Pretreatment of A. castellanii with gold-conjugated chlorhexidine nanoparticles reduced amoeba-mediated host cell cytotoxicity from 90% to 40% at 5 μM. In contrast, chlorhexidine alone, at similar concentrations, had no protective effects for the host cells. Similarly, amoebae treated with neomycin alone or neomycin-conjugated nanoparticles showed no protective effects. Overall, these findings suggest that gold-conjugated chlorhexidine nanoparticles hold promise in the improved treatment of A. castellanii keratitis.
The use of polyclonal antibody (IgG) has recently been applied to the detection of bacteria. We developed a lateral flow assay (LFA) strip using a specific IgG in combination with colloidal gold on a nitrocellulose membrane. A conjugate, gold-anti Escherichia coli (E. coli) O157:H7 IgG was developed in this study for the detection of E. coli O157:H7 in food. The 40 nm in size of colloidal gold nanoparticles was used to conjugate the anti-E. coli O157:H7 IgG. The optimal concentration, 12.0 µg/ml of the anti-E. coli O157:H7 IgG was determined by standard curve generated in titration method. The serially diluted of E. coli O157:H7 was detected and clearly visualized on the LFA strip as low as 106 CFU/ml (result not shown). The IgG raised in rabbit have shown specific binding capacity against E. coli O157:H7. No other genus of bacteria, including Salmonella typhimurium, Listeria monocytogenes and Campylobacter jejuni reacted to the IgG. The LFA strip could also detect E. coli O157:H7 in different food samples matrices after 18 h-enrichment and this result were in accordance with the results of the polymerase chain reaction (PCR) and colony count.
This study investigated the applications of recombinant monoclonal antibodies (rmAbs) produced against two recombinant filarial proteins of diagnostic value. Ab5B and Ab3A were produced against recombinant BmSXP, and Ab4 and Ab4-fragment crystallizable (Fc) against recombinant BmR1. Ab5B and Ab4-Fc were found to be useful as quality control (QC) reagents for two commercial rapid test kits, such as Brugia RapidTM and BLF Rapid® (Reszon Diagnostics International Sdn. Bhd., 47600 Subang Jaya, Selangor, Malaysia), respectively. The two rmAbs reacted positively with the corresponding recombinant proteins lined on the nitrocellulose strips of the cassette tests, thus may replace or reduce the need for patient serum samples as positive controls for QC of the commercial kits. They were also successfully conjugated to gold nanoparticles and reacted positively with the test lines containing the corresponding recombinant proteins when directly applied to the cassette tests. The gold-conjugated reagents can be used to confirm the antigenicity of test lines after the storage of the rapid tests for a prolonged period or under unfavorable conditions. Furthermore, Ab5B and Ab3A were shown to be able to capture the target recombinant proteins through immunoaffinity purification, enabling their use for applications that need very highly purified proteins. In conclusion, this study demonstrated several potential uses of rmAb proteins produced against recombinant filarial proteins.
Pseudorabies (Aujeszky's disease) is an economically significant disease of swine known to cause central nervous disorders, respiratory disease, reproductive failure and mortality in infected pigs. In attempts to eradicate the disease from becoming endemic, early detection is important to prevent further economic losses and to allow for detection and removal of infected pigs in domestic herds. Thus, a rapid and sensitive technique is necessary for the detection of the virus. For rapid and simple examination, an immuno - chromatographic lateral - flow assay system based on immunologic recognition of specific pseudorabies virus antigen was developed by utilising, as signal generator, colloidal gold conjugated to secondary antibody to detect primary or sample antibody in the sera of pseudorabies infected animals. The pseudorabies virus used as a capture antigen in the test strip was first cultivated in VERO cell culture and then purified by sucrose gradient separation to produce the viral protein concentration of 3.8 mg/ml. The standard pseudorabies antigens reacted well with the hyperimmune serum (HIS). The antibody detection system is basically composed of colloidal gold - labelled antibodies fixed on a conjugate pad, and the complementary pseudorabies antigen immobilised onto a nitrocellulose membrane forming capture zone. If the target antibody is present in a specimen, the colloidal gold-labelled antibody will form a complex with the antibody sample. Subsequently, the formed complex will migrate to the capture zone and is then bound to the solid phase via antigen - antibody interaction. As a result, a signal marker is generated by the accumulation of colloidal gold for detection confirmation. The results obtained demonstrated that the optimum combination of pseudorabies antigen needed as the capture reagent and gold conjugate as secondary antibody recognition marker was at a concentration of 0.38mg/ml and at 1:10 dilution factor respectively. The sensitivity of the solid - based test strip towards pseudorabies antibodies was high with a detection limit of 1 to 10,000 - dilution factor. The specificity of the assay was 100% with no cross - reaction being observed with other sera or antibodies. Accurate reading time needed for confirmation of the assay can be completed in 5 min with a whole blood sample of 25 microl. The colloidal gold - labelled antibody is stable at room temperature for 6 months or more (data not shown). Findings from this study indicated that the solid - based test strip assay system provided high sensitivity and specificity for the detection of pseudorabies at low levels of antibody concentration. The assay was rapid, simple, cheap, and does not require any sophisticated equipment. Thus, the solid based test strip will be a useful serological screening technique or for rapid diagnosis of an infectious disease in target populations of animals characterised by heterogeneous antibody responses.