Novel Microsynthesis of High-Yield Gold Nanoparticles to Accelerate Research in Biosensing and Other Bioapplications.

Gold nanoparticles (AuNPs) exhibit unique properties that make them appealing for applications in biosensing and other emerging fields. Despite the availability of numerous synthesis methods, important questions remain to be addressed regarding the volume effect on the synthesis yield and quality of AuNPs in the light of biosensing research. The present study addresses these issues by developing a novel microvolumetric citrate-reduction method to improve the synthesis of AuNPs, which were characterized by electronic microscopy, energy dispersive spectroscopy, zeta potential and colorimetric analysis. A comparison of the novel microsynthesis method with the standard Turkevich method demonstrated its superior performance in terms of yield, monodispersity, rapidity (in one step), reproducibility, and stability. The analytical behavior of AuNPs-based aptasensors prepared by microsynthesis was investigated using kanamycin detection and showed higher reproducibility and improved detection limits (3.4 times) compared to those of Turkevich AuNPs. Finally, the effect of pH was studied to demonstrate the suitability of the method for the screening of AuNP synthesis parameters that are of direct interest in biosensing research; the results showed an optimal pH range between 5.0 and 5.5. In summary, the approach described herein has the potential to improve research capabilities in biosensing, with the added benefits of lowering costs and minimizing waste generation in line with current trends in green nanotechnology.

A Novel Preanalytical Strategy Enabling Application of a Colorimetric Nanoaptasensor for On-Site Detection of AFB1 in Cattle Feed.

Aflatoxin contamination of cattle feed is responsible for serious adverse effects on animal and human health. A number of approaches have been reported to determine aflatoxin B1 (AFB1) in a variety of feed samples using aptasensors. However, rapid analysis of AFB1 in these matrices remains to be addressed in light of the complexity of the preanalytical process. Herein we describe an optimization on the preanalytical stage to minimize the sample processing steps required to perform semi-quantitative colorimetric detection of AFB1 in cattle feed using a gold nanoparticle-based aptasensor (nano-aptasensor). The optical behavior of the nano-aptasensor was characterized in different organics solvents, with acetonitrile showing the least interference on the activity of the nan-aptasensor. This solvent was selected as the extractant agent for AFB1-containing feed, allowing for the first time, direct colorimetric detection from the crude extract (detection limit of 5 µg/kg). Overall, these results lend support to the application of this technology for the on-site detection of AFB1 in the dairy sector.

A Simple Yet Effective Preanalytical Strategy Enabling the Application of Aptamer-Conjugated Gold Nanoparticles for the Colorimetric Detection of Antibiotic Residues in Raw Milk.

The misuse of antibiotics in the cattle sector can lead to milk contamination, with concomitant effects on the dairy industry and human health. Biosensors can be applied in this field; however, the influence of the milk matrix on their activity has been poorly studied in light of the preanalytical process. Herein, aptamer-conjugated gold nanoparticles (nanoaptasensors) were investigated for the colorimetric detection in raw milk of four antibiotics used in cattle. The effect of milk components on the colorimetric response of the nanoaptasensors was analyzed by following the selective aggregation of the nanoparticles, using the absorption ratio A520/A720. A preanalytical strategy was developed to apply the nanoaptasensors to antibiotic-contaminated raw milk samples, which involves a clarification step with Carrez reagents followed by the removal of cations through dilution, chelation (EDTA) or precipitation (NaHCO3). The colorimetric signals were detected in spiked samples at concentrations of antibiotics as low as 0.25-fold the maximum residue limits (MRLs) for kanamycin (37.5 µg/L), oxytetracycline (25 µg/L), sulfadimethoxine (6.25 µg/L) and ampicillin (1 µg/L), according to European and Chilean legislation. Overall, we conclude that this methodology holds potential for the semiquantitative analysis of antibiotic residues in raw milk obtained directly from dairy farms.

A Proteome-Wide Immunoinformatics Tool to Accelerate T-Cell Epitope Discovery and Vaccine Design in the Context of Emerging Infectious Diseases: An Ethnicity-Oriented Approach.

Emerging infectious diseases (EIDs) caused by viruses are increasing in frequency, causing a high disease burden and mortality world-wide. The COVID-19 pandemic caused by the novel SARS-like coronavirus (SARS-CoV-2) underscores the need to innovate and accelerate the development of effective vaccination strategies against EIDs. Human leukocyte antigen (HLA) molecules play a central role in the immune system by determining the peptide repertoire displayed to the T-cell compartment. Genetic polymorphisms of the HLA system thus confer a strong variability in vaccine-induced immune responses and may complicate the selection of vaccine candidates, because the distribution and frequencies of HLA alleles are highly variable among different ethnic groups. Herein, we build on the emerging paradigm of rational epitope-based vaccine design, by describing an immunoinformatics tool (Predivac-3.0) for proteome-wide T-cell epitope discovery that accounts for ethnic-level variations in immune responsiveness. Predivac-3.0 implements both CD8+ and CD4+ T-cell epitope predictions based on HLA allele frequencies retrieved from the Allele Frequency Net Database. The tool was thoroughly assessed, proving comparable performances (AUC ~0.9) against four state-of-the-art pan-specific immunoinformatics methods capable of population-level analysis (NetMHCPan-4.0, Pickpocket, PSSMHCPan and SMM), as well as a strong accuracy on proteome-wide T-cell epitope predictions for HIV-specific immune responses in the Japanese population. The utility of the method was investigated for the COVID-19 pandemic, by performing in silico T-cell epitope mapping of the SARS-CoV-2 spike glycoprotein according to the ethnic context of the countries where the ChAdOx1 vaccine is currently initiating phase III clinical trials. Potentially immunodominant CD8+ and CD4+ T-cell epitopes and population coverages were predicted for each population (the Epitope Discovery mode), along with optimized sets of broadly recognized (promiscuous) T-cell epitopes maximizing coverage in the target populations (the Epitope Optimization mode). Population-specific epitope-rich regions (T-cell epitope clusters) were further predicted in protein antigens based on combined criteria of epitope density and population coverage. Overall, we conclude that Predivac-3.0 holds potential to contribute in the understanding of ethnic-level variations of vaccine-induced immune responsiveness and to guide the development of epitope-based next-generation vaccines against emerging pathogens, whose geographic distributions and populations in need of vaccinations are often well-defined for regional epidemics.

An ecofriendly nanocomposite of bacterial cellulose and hydroxyapatite efficiently removes lead from water.

An environmentally friendly nanocomposite adsorbent composed of two renewable biomaterials, bacterial cellulose (BC) nanofibrils and hydroxyapatite (HA) nanocrystals, was synthetized by an in situ wet chemical precipitation technique, using clam shell biowaste as feedstock. HA nanocrystals embedded in an ultrafine BC network were confirmed and characterized trough different instrumental techniques (SEM, FTIR, XRD, EDS, surface charge and BET analysis), describing its nanostructure, chemical composition and thermal stability. The adsorptive removal of lead ions by the nanocomposite was investigated through batch experiments conducted under different pH, contact times and Pb(II) initial concentrations, proving that the process was highly favorable according to the Langmuir isotherm model (monolayer adsorption) with chemisorption as the main mechanism and kinetic data obeying a nonlinear pseudo-second order kinetic model. The developed nanocomposite showed a strong removal capacity of Pb(II) both in batch experiments (192 mg/g) and packed-bed column systems (188 mg/g), placing this new nanocomposite among top-performing BC-based biomaterials for lead removal.

Improved Antibiotic Detection in Raw Milk Using Machine Learning Tools over the Absorption Spectra of a Problem-Specific Nanobiosensor.

In this article we present the development of a biosensor system that integrates nanotechnology, optomechanics and a spectral detection algorithm for sensitive quantification of antibiotic residues in raw milk of cow. Firstly, nanobiosensors were designed and synthesized by chemically bonding gold nanoparticles (AuNPs) with aptamer bioreceptors highly selective for four widely used antibiotics in the field of veterinary medicine, namely, Kanamycin, Ampicillin, Oxytetracycline and Sulfadimethoxine. When molecules of the antibiotics are present in the milk sample, the interaction with the aptamers induces random AuNP aggregation. This phenomenon modifies the initial absorption spectrum of the milk sample without antibiotics, producing spectral features that indicate both the presence of antibiotics and, to some extent, its concentration. Secondly, we designed and constructed an electro-opto-mechanic device that performs automatic high-resolution spectral data acquisition in a wavelength range of 400 to 800 nm. Thirdly, the acquired spectra were processed by a machine-learning algorithm that is embedded into the acquisition hardware to determine the presence and concentration ranges of the antibiotics. Our approach outperformed state-of-the-art standardized techniques (based on the 520/620 nm ratio) for antibiotic detection, both in speed and in sensitivity.

Generation of engineered core-shell antibiotic nanoparticles.

Well-defined nanocomposite structures have received significant attention due to their superior combinatorial properties. Rational tuning of the core and shell of the nanostructure(s) can offer potent antibacterial activity. Such advanced core-shell nanocomposite methodologies allow not only the incorporation of antibacterial agents on the shell but also provide its stability and nurture antibacterial activity. Herein, antibiotic zinc oxide-curcumin (ZnO-Cum) core-shell nanoparticles for antibacterial application were synthesised. The ZnO-Cum core-shell nanoparticles were prepared by curcumin nanolayer deposition on zinc oxide nanoparticles via a sonication process. The resulting ZnO-Cum core-shell nanoparticles were spiracle in shape with a ∼45 nm ZnO core and ∼12 nm curcumin shell layer size, respectively, determined by transmission electron microscopy. X-ray diffraction analysis confirmed the formation of a core-shell crystal structure. Additionally, UV-DRS and ATR-FTIR spectral analysis support the existence of ZnO and curcumin in a core-shell nanocomposite. The antibacterial activities of nanoparticles developed were studied against Staphylococcus aureus and Streptococcus pneumoniae and Escherichia coli and Shigella dysenteriae bacterial stains using the diffusion method. A greater inhibition of the growth of Gram positive and negative bacteria was noticed upon treatment with core-shell ZnO and curcumin nanoparticles than the commercial antibiotic amoxicillin which indicates their antibacterial property. The findings of this study provide evidence that the zinc oxide-curcumin core-shell nanoparticles may be highly promising for antibacterial and biomedical applications.

Presence and characterization of microplastics in fish of commercial importance from the Biobío region in central Chile.

In this study we have identified and characterized microplastic particles (MPs) found in six fish species of commercial importance in central Chile. The fish species belong to different trophic levels and were obtained from the oceanic and coastal habitats. To analyze MPs, the fish gastrointestinal content was extracted, analyzed and characterized using a microscopy equipped with Fourier-transform infrared spectroscopy (FT-IR). The MPs found in fish samples were mainly constituted by red microfibers (70-100%) with sizes ranging between 176 and 2842 µm. Polyester, polyethylene (PE) and polyethylene terephthalate (PET) were identified as the prevalent polymers detected. The coastal species showed the presence of microfibers with a higher size and abundance (71%) compared to oceanic species (29%), suggesting there is a greater exposure risk. These findings are consistent with results found in other investigations worldwide. However, further research is still needed to accurately establish the potential exposure risk for the public consuming these fish and the impact of MPs in the Chilean fishery activities.

Trickling filter technology for biotreatment of nitrogenous compounds emitted in exhaust gases from fishmeal plants.

Odour emissions are a major environmental issue associated with fishmeal production. Laboratory-scale biotrickling filters (BTFs) were inoculated with microbial consortia derived from sewage sludge, with the goal to study the biotreatment of low-loads of methylamines and ammonia that are main components of odorous exhaust gases produced by fishmeal processing plants. A BTF packed with ceramic rings was subjected to a real fishmeal plant emission containing trimethylamine (TMA), dimethylamine (DMA) and monomethylamine (MMA). The highest elimination capacities (ECs) obtained were 372 mg TMA m-3 h-1, 5.518 mg DMA m-3 h-1 and 1.038 mg MMA m-3 h-1, with maximal removal efficiencies of 92% (TMA), 83% (DMA) and 95% (MMA) after 30 days operation. In a different experiment, a polyurethane foam packing was employed to treat ammonia (NH3) at low inlet loads, reaching an EC of 47.19 mg N m-3 h-1 with 99.8% efficiency (inlet load of 47.27 mg N m-3 h-1). Likewise, the microbial community of the polyurethane-associated biofilm was diverse and stable during operation. These results suggested that elimination of volatile amino-compounds using BTFs inoculated with a methylotrophic microbial consortium holds potential for odour removal. In addition, sequencing analysis of 16S rDNA gene fragments allowed the identification of heterotrophic ammonia-oxidizing bacteria that are promising candidates to effectively maintain ammonia elimination in a biotreatment operation of nitrogenous compounds present in exhaust gases from fishmeal facilities.

Heavy metal removal from aqueous systems using hydroxyapatite nanocrystals derived from clam shells.

Hydroxyapatite (HA) was synthesized by wet chemical precipitation, using clam shell (CS) waste as feedstock. SEM and TEM observation of the produced hydroxyapatite revealed the presence of rod-shaped nanocrystals, while XRD and EDS analyses confirmed the characteristic patterns of hydroxyapatite molecules. This material was subsequently employed as a sorbent for heavy metal removal from aqueous solutions, both in batch and column equilibrium procedures. In batch studies, higher sorption efficiencies were obtained at pH 5, with the highest adsorption capacities of 265, 64, and 55 mg g-1 for Pb(ii), Cd(ii), and Cu(ii), respectively. In addition, an adsorption capacity of 42.5 mg g-1 was determined using a CS-HA packed bed column fed with a solution of Pb(ii). Finally, the breakthrough curve was fitted with Thomas model in order to predict column behavior and scaling up.

Slight pH Fluctuations in the Gold Nanoparticle Synthesis Process Influence the Performance of the Citrate Reduction Method.

Gold nanoparticles (AuNPs) are currently under intense investigation for biomedical and biotechnology applications, thanks to their ease in preparation, stability, biocompatibility, multiple surface functionalities, and size-dependent optical properties. The most commonly used method for AuNP synthesis in aqueous solution is the reduction of tetrachloroauric acid (HAuCl4) with trisodium citrate. We have observed variations in the pH and in the concentration of the gold colloidal suspension synthesized under standard conditions, verifying a reduction in the reaction yield by around 46% from pH 5.3 (2.4 nM) to pH 4.7 (1.29 nM). Citrate-capped AuNPs were characterized by UV-visible spectroscopy, TEM, EDS, and zeta-potential measurements, revealing a linear correlation between pH and the concentration of the generated AuNPs. This result can be attributed to the adverse effect of protons both on citrate oxidation and on citrate adsorption onto the gold surface, which is required to form the stabilization layer. Overall, this study provides insight into the effect of the pH over the synthesis performance of the method, which would be of particular interest from the point of view of large-scale manufacturing processes.

Nano-hydroxyapatite polymeric hydrogels for dye removal.

Herein, two kinds of nano-hydroxyapatite were synthesized from Clam and Magellan shell by wet chemical precipitation method. Mainly, carboxymethyl cellulose/acrylamide/nano-hydroxyapatite composite hydrogels were developed via a free-radical polymerization process and investigated as a sorbent for Acid Blue 113 (AB) from aqueous AB solution. The swelled and kinetic behaviours of hydrogels were investigated using a gravimetric method. The swelling properties of the CMC-AM-hydrogels were influenced by the calcium electrolytes (Ca2+) content in nano-hydroxyapatites. The diffusion coefficient value increased with the increase of nano-hydroxyapatite content in the CMC-AM/nHA-CS (0.22353-0.27681 cm2 s-1) and CMC-AM/nHA-MS (0.22377-0.29737 cm2 s-1) hydrogels. The mechanism of water diffusion was found to be anomalous transport. The CMC-AM/nHA-MS hydrogels showed high AB absorption efficiency and adsorption capacities. These results explained that the nano-hydroxyapatites of Magellan shells based hydrogels are attractive nanocomposite hydrogels for the adsorption of dye in the water purification applications.

A novel "in-feed" delivery platform applied for oral DNA vaccination against IPNV enables high protection in Atlantic salmon (Salmon salar).

BACKGROUND: DNA vaccination has emerged as a promising tool against infectious diseases of farmed fish. Oral delivery allows stress-free administration that is ideal for mass immunization and of paramount importance for infectious pancreatic necrosis (IPN) and other viral disease that affect young salmonids and cause economic losses in aquaculture worldwide. METHODS: We describe the development and in vivo assessment of an "in-feed" formulation strategy for oral immunization with liposomal DNA vaccines, by delivering a vaccine construct coding for an immunogenic region of the VP2 capsid protein. A challenge against IPNV was carried out to determine the vaccine efficacy, by comparing the mortality of pre-smolt Atlantic salmons immunized and non-immunized with the oral vaccine. The antibody response (ELISA) and hematological parameters after immunization were examined, as well as the vaccine effect on the growth and internal structures of fry salmons (histological analysis). The vaccine distribution in the experimental tank after oral administration was investigated by HPLC and PCR amplification. RESULTS: The oral vaccine induced detectable levels of VP2-specific antibodies and conferred significant protection following IPNV challenge, with relative percent survivals (RPS) of 58.2%, for single dose (1mgpDNA/kgfish⋅d), and 66% for double dose (2mgpDNA/kgfish⋅d). We further provide evidence in favour of the vaccine safety to fish and demonstrated absence of pDNA in the tank water, but presence of vaccine residues in faeces and unconsumed feed sediments (solid wastes). CONCLUSION: The delivery platform for liposomal DNA vaccination via feed was successfully proved against IPNV in Atlantic salmon, showing the oral vaccine to be immunogenic and safe for fish, and providing significant protection after oral administration. The "in-feed" technology for oral DNA vaccination holds potential to be applied against IPNV and other pathogens that currently threaten the aquaculture worldwide.

Recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production.

Novel vaccination approaches based on rational design of B- and T-cell epitopes - epitope-based vaccines - are making progress in the clinical trial pipeline. The epitope-focused recombinant protein-based malaria vaccine (termed RTS,S) is a next-generation approach that successfully reached phase-III trials, and will potentially become the first commercial vaccine against a human parasitic disease. Progress made on methods such as recombinant DNA technology, advanced cell-culture techniques, immunoinformatics and rational design of immunogens are driving the development of these novel concepts. Synthetic recombinant proteins comprising both B- and T-cell epitopes can be efficiently produced through modern biotechnology and bioprocessing methods, and can enable the induction of large repertoires of immune specificities. In particular, the inclusion of appropriate CD4+ T-cell epitopes is increasingly considered a key vaccine component to elicit robust immune responses, as suggested by results coming from HIV-1 clinical trials. In silico strategies for vaccine design are under active development to address genetic variation in pathogens and several broadly protective "universal" influenza and HIV-1 vaccines are currently at different stages of clinical trials. Other methods focus on improving population coverage in target populations by rationally considering specificity and prevalence of the HLA proteins, though a proof-of-concept in humans has not been demonstrated yet. Overall, we expect immunoinformatics and bioprocessing methods to become a central part of the next-generation epitope-based vaccine development and production process.

A bioinformatics tool for epitope-based vaccine design that accounts for human ethnic diversity: application to emerging infectious diseases.

BACKGROUND: Peptide vaccination based on multiple T-cell epitopes can be used to target well-defined ethnic populations. Because the response to T-cell epitopes is restricted by HLA proteins, the HLA specificity of T-cell epitopes becomes a major consideration for epitope-based vaccine design. We have previously shown that CD4+ T-cell epitopes restricted by 95% of human MHC class II proteins can be predicted with high-specificity. METHODS: We describe here the integration of epitope prediction with population coverage and epitope selection algorithms. The population coverage assessment makes use of the Allele Frequency Net Database. We present the computational platform Predivac-2.0 for HLA class II-restricted epitope-based vaccine design, which accounts comprehensively for human genetic diversity. RESULTS: We validated the performance of the tool on the identification of promiscuous and immunodominant CD4+ T-cell epitopes from the human immunodeficiency virus (HIV) protein Gag. We further describe an application for epitope-based vaccine design in the context of emerging infectious diseases associated with Lassa, Nipah and Hendra viruses. Putative CD4+ T-cell epitopes were mapped on the surface glycoproteins of these pathogens and are good candidates to be experimentally tested, as they hold potential to provide cognate help in vaccination settings in their respective target populations. CONCLUSION: Predivac-2.0 is a novel approach in epitope-based vaccine design, particularly suited to be applied to virus-related emerging infectious diseases, because the geographic distributions of the viruses are well defined and ethnic populations in need of vaccination can be determined ("ethnicity-oriented approach"). Predivac-2.0 is accessible through the website http://predivac.biosci.uq.edu.au/.

PREDIVAC: CD4+ T-cell epitope prediction for vaccine design that covers 95% of HLA class II DR protein diversity.

BACKGROUND: CD4+ T-cell epitopes play a crucial role in eliciting vigorous protective immune responses during peptide (epitope)-based vaccination. The prediction of these epitopes focuses on the peptide binding process by MHC class II proteins. The ability to account for MHC class II polymorphism is critical for epitope-based vaccine design tools, as different allelic variants can have different peptide repertoires. In addition, the specificity of CD4+ T-cells is often directed to a very limited set of immunodominant peptides in pathogen proteins. The ability to predict what epitopes are most likely to dominate an immune response remains a challenge. RESULTS: We developed the computational tool Predivac to predict CD4+ T-cell epitopes. Predivac can make predictions for 95% of all MHC class II protein variants (allotypes), a substantial advance over other available methods. Predivac bases its prediction on the concept of specificity-determining residues. The performance of the method was assessed both for high-affinity HLA class II peptide binding and CD4+ T-cell epitope prediction. In terms of epitope prediction, Predivac outperformed three available pan-specific approaches (delivering the highest specificity). A central finding was the high accuracy delivered by the method in the identification of immunodominant and promiscuous CD4+ T-cell epitopes, which play an essential role in epitope-based vaccine design. CONCLUSIONS: The comprehensive HLA class II allele coverage along with the high specificity in identifying immunodominant CD4+ T-cell epitopes makes Predivac a valuable tool to aid epitope-based vaccine design in the context of a genetically heterogeneous human population.The tool is available at: http://predivac.biosci.uq.edu.au/.

Comparison on the removal of hydrogen sulfide in biotrickling filters inoculated with Thiobacillus thioparus and Acidithiobacillus thiooxidans

Emissions of hydrogen sulfide (H2S) by industrial activities is frequent cause of corrosion and unpleasant odours. Treatment of gaseous emissions contaminated with H2S by biotrickling filters inoculated with single cultures of sulfur oxidizer bacteria exhibit several advantages over physicochemical methods, such as shorter adaptation times and higher removal ability. Biofilms of Thiobacillus thioparus and Acidithiobacillus thiooxidans have proved to exhibit high removal capacities, yet no comparative studies between them have been reported. This article reports the efficiency of biotrickling filters inoculated with T. thioparus and A. thiooxidans under similar conditions excepting the pH, that was the optimal for the bacterial growth, for the removal of H2S. The support was selected by determining the respirometric coefficients of the biomass. The maximum removal capacity of the biofilter inoculated with T. thioparus, operating within the range of pH (5.5-7.0) was 14 gS m-3 h-1, lower the value obtained for the biotrickling filter inoculated with A. thiooxidans; 370 gS m-3 h-1. Therefore, it is concluded that acid biotrickling filter inoculated with A. thiooxidans constitute the best strategy to remove H2S, with the advantage that the system not require an exhaustive pH control of the liquid media.