Au@Ag Core-Shell Nanoparticles for Colorimetric and Surface-Enhanced Raman-Scattering-Based Multiplex Competitive Lateral Flow Immunoassay for the Simultaneous Detection of Histamine and Parvalbumin in Fish.

Foodborne allergies and illnesses represent a major global health concern. In particular, fish can trigger life-threatening food allergic reactions and poisoning effects, mainly caused by the ingestion of parvalbumin toxin. Additionally, preformed histamine in less-than-fresh fish serves as a toxicological alert. Consequently, the analytical assessment of parvalbumin and histamine levels in fish becomes a critical public health safety measure. The multiplex detection of both analytes has emerged as an important issue. The analytical detection of parvalbumin and histamine requires different assays; while the determination of parvalbumin is commonly carried out by enzyme-linked immunosorbent assay, histamine is analyzed by high-performance liquid chromatography. In this study, we present an approach for multiplexing detection and quantification of trace amounts of parvalbumin and histamine in canned fish. This is achieved through a colorimetric and surface-enhanced Raman-scattering-based competitive lateral flow assay (SERS-LFIA) employing plasmonic nanoparticles. Two distinct SERS nanotags tailored for histamine or ß-parvalbumin detection were synthesized. Initially, spherical 50 nm Au@Ag core-shell nanoparticles (Au@Ag NPs) were encoded with either rhodamine B isothiocyanate (RBITC) or malachite green isothiocyanate (MGITC). Subsequently, these nanoparticles were bioconjugated with anti-ß-parvalbumin and antihistamine, forming the basis for our detection and quantification methodology. Additionally, our approach demonstrates the use of SERS-LFIA for the sensitive and multiplexed detection of parvalbumin and histamine on a single test line, paving the way for on-site detection employing portable Raman instruments.

Rapid and sensitive detection of E. coli O157:H7 by lateral flow immunoassay and silver enhancement.

The aim of this study was to develop a sensitive lateral flow immunoassay (LFIA) for the rapid detection of Escherichia coli (E. coli) O157:H7, a pathogen contributor to diseases and fatalities worldwide. Au nanoparticles with high stability, uniform size, and shape were synthesized and coated with heterobifunctional PEG polymer with carboxyl groups, and they were bioconjugated to be used as label in sandwich-LFIA. Then, a silver enhancement strategy was developed as an accessible, rapid, and cost-effective approach for signal amplification to reduce the limit of detection (LOD). The optimal results were achieved when a solution of silver nitrate and hydroquinone/citrate buffer was added to the strips for 4 min. This led to a decrease in the visual LOD from 2 × 106 (CFU mL-1) to 2 × 103 (CFU mL-1), resulting in a threefold improvement in sensitivity compared to the conventional LFIA system. The specificity of the system was evaluated by using non-target bacteria (E. coli BL21 and E. coli T515) and its reliability was determined by testing commercial food samples (milk, tap water, and orange juice), demonstrating its effectiveness for quickly detecting pathogenic bacteria in food products.

Fe3O4@Au Core-Shell Magnetic Nanoparticles for the Rapid Analysis of E. coli O157:H7 in an Electrochemical Immunoassay.

Escherichia coli (E. coli) O157:H7 is a pathogenic bacterium that causes serious toxic effects in the human gastrointestinal tract. In this paper, a method for its effective analytical control in a milk sample was developed. To perform rapid (1 h) and accurate analysis, monodisperse Fe3O4@Au magnetic nanoparticles were synthesized and used in an electrochemical sandwich-type magnetic immunoassay. Screen-printed carbon electrodes (SPCE) were used as transducers, and electrochemical detection was performed by chronoamperometry using a secondary horseradish peroxidase-labeled antibody and 3,3',5,5'-tetramethylbenzidine. This magnetic assay was used to determine the E. coli O157:H7 strain in the linear range from 20 to 2 × 106 CFU/mL, with a limit of detection of 20 CFU/mL. The selectivity of the assay was tested using Listeria monocytogenes p60 protein, and the applicability of the assay was assessed by analyzing a commercial milk sample, demonstrating the usefulness of the synthesized nanoparticles in the developed magnetic immunoassay.

Stability of Non-Ionic Surfactant Vesicles Loaded with Rifamycin S.

These days, the eradication of bacterial infections is more difficult due to the mechanism of resistance that bacteria have developed towards traditional antibiotics. One of the medical strategies used against bacteria is the therapy with drug delivery systems. Non-ionic vesicles are nanomaterials with good characteristics for encapsulating drugs, due to their bioavailability and biodegradability, which allow the drugs to reach the specific target and reduce their side effects. In this work, the antibiotic Rifamycin S was encapsulated. The rifamycin antibiotics family has been widely used against Mycobacterium tuberculosis, but recent studies have also shown that rifamycin S and rifampicin derivatives have bactericidal activity against Staphylococcus epidermidis and Staphylococcus aureus. In this work, a strain of S. aureus was selected to study the antimicrobial activity through Minimum Inhibitory Concentration (MIC) assay. Three formulations of niosomes were prepared using the thin film hydration method by varying the composition of the aqueous phase, which included MilliQ water, glycerol solution, or PEG400 solution. Niosomes with a rifamycin S concentration of 0.13 µg/g were satisfactorily prepared. Nanovesicles with larger size and higher encapsulation efficiency (EE) were obtained when using glycerol and PEG400 in the aqueous media. Our results showed that niosomes consisting of an aqueous glycerol solution have higher stability and EE across a diversity of temperatures and pHs, and a lower MIC of rifamycin S against S. aureus.

Lipid-Polymer Hybrids Encapsulating Iron-Oxide Nanoparticles as a Label for Lateral Flow Immunoassays.

The feasibility of using Superparamagnetic Iron Oxide Nanoparticles (SPIONs) encapsulated by lipid-polymer nanoparticles as labels in lateral flow immunoassays (LFIA) was studied. First, nanoparticles were synthesized with average diameters between 4 and 7 (nm) through precipitation in W/O microemulsion and further encapsulated using lipid-polymer nanoparticles. Systems formulated were characterized in terms of size and shape by DLS (Nanozetasizer from Malvern) and TEM. After encapsulation, the average size was around (≈20 and 50 nm). These controlled size agglomerates were tested as labels with a model system based on the biotin-neutravidin interaction. For this purpose, the encapsulated nanoparticles were conjugated to neutravidin using the carbodiimide chemistry, and the LFIA was carried out with a biotin test line. The encapsulated SPIONs showed that they could be promising candidates as labels in LFIA test. They would be useful for immunomagnetic separations, that could improve the limits of detection by means of preconcentration.

Microemulsion Synthesis of Superparamagnetic Nanoparticles for Bioapplications.

Superparamagnetic nanoparticles have seen increased potential in medical and environmental applications. Their preparation is traditionally made by the coprecipitation method, with limited control over the particle size distribution. Microemulsion methods could be advantageous due to the efficient control of the size, shape, and composition of the nanoparticles obtained. Water-in-oil (W/O) microemulsions consist of aqueous microdomains dispersed in a continuous oil phase, stabilized by surfactant molecules. These work as nanoreactors where the synthesis of the desired nanoparticles takes place through a co-precipitation chemical reaction. In this work, superparamagnetic magnetite nanoparticles with average diameters between 5.4 and 7.2 nm and large monodispersity have been synthesized through precipitation in a W/O microemulsion, with Cetyl Trimethyl Ammonium Bromide (CTAB) as a main surfactant, 1-butanol as a cosurfactant, and with 1-hexanol as the continuous oily phase. The optimization of the corresponding washing protocol has also been established since a strict control is required when using these materials for bioapplications. Their applicability in those has been proved by their encapsulation in liposomes, being tested as signal enhancers for lateral flow immunoassays by using the affinity neutravidin-biotin model system. Due to their magnetic behaviour, they were also tested for magnetic separation. These novel materials have been found to be useful for analytical applications requiring high sensitivity and the removal of interferences.

Extracellular Vesicles: Current Analytical Techniques for Detection and Quantification.

Since their first observation, understanding the biology of extracellular vesicles (EV) has been an important and challenging field of study. They play a key role in the intercellular communication and are involved in important physiological and pathological functions. Therefore, EV are considered as potential biomarkers for diagnosis, prognosis, and monitoring the response to treatment in some diseases. In addition, due to their properties, EV may be used for therapeutic purposes. In the study of EV, three major points have to be addressed: 1. How to isolate EV from cell culture supernatant/biological fluids, 2. how to detect them, and 3. how to characterize and quantify. In this review, we focus on the last two questions and provide the main analytical techniques up-to-date for detection and profiling of EV. We critically analyze the advantages and disadvantages of each one, aimed to be of relevance for all researchers working on EV biology and their potential applications.

Paper-Based Working Electrodes Coated with Mercury or Bismuth Films for Heavy Metals Determination.

Paper-based carbon working electrodes were modified with mercury or bismuth films for the determination of trace metals in aqueous solutions. Both modification procedures were optimized in terms of selectivity and sensitivity for the determination of different heavy metals, aiming their simultaneous determination. Cd (II), Pb (II) and In (III) could be quantified with both films. However, Cu (II) could not be determined with bismuth films. The modification with mercury films led to the most sensitive method, with linear ranges between 0.1 and 10 µg/mL and limits of detection of 0.4, 0.1, 0.04 and 0.2 µg/mL for Cd (II), Pb (II), In (III) and Cu (II), respectively. Nevertheless, the bismuth film was a more sustainable alternative to mercury. Tap-water samples were analyzed for the determination of metals by standard addition methodology with good accuracy, by using a low-cost and easily disposable paper-based electrochemical platform. This system demonstrated its usefulness for monitoring heavy metals in water.

Selected Tetraspanins Functionalized Niosomes as Potential Standards for Exosome Immunoassays.

Quantitative detection of exosomes in bio-fluids is a challenging task in a dynamic research field. The absence of a well-established reference material (RM) for method development and inter-comparison studies could be potentially overcome with artificial exosomes: lab-produced biomimetic particles with morphological and functional properties close to natural exosomes. This work presents the design, development and functional characteristics of fully artificial exosomes based on tetraspanin extracellular loops-coated niosomes, produced by bio-nanotechnology methods based on supra-molecular chemistry and recombinant protein technology. Mono- and double-functionalized particles with CD9/CD63 tetraspanins have been developed and characterized from a morphological and functional point of view. Produced bio-particles showed close similarities with natural entities in terms of physical properties. Their utility for bioanalysis is demonstrated by their detection and molecular-type discrimination by enzyme-linked immunosorbent assays (ELISAs), one of the most frequent bio-analytical method found in routine and research labs. The basic material based on streptavidin-coated niosomes allows the surface functionalization with any biotinylated protein or peptide, introducing versatility. Although promising results have been reported, further optimizations and deeper characterization will help this innovative biomaterial become a robust RM for validation and development of diagnostic tools for exosomes determination.

Effect of drug molecular weight on niosomes size and encapsulation efficiency.

Encapsulation into nanocarriers, such as niosomes, is a promising way to protect them from degradation, and allow controll and target delivery of bioactive compounds. For biotechnological applications, a tight control of particle size with acceptable encapsulation efficiencies (EE) is a technological challenge, especially for hydrophilic compounds due to its capability to diffuse across biological barriers. Niosomes formulated with mixture of surfactants represent promising nanocarriers due to the advantages of non-ionic surfactants, such as low cost, versatility and enhanced physico-chemical properties. In this work, the effect of both, composition of the hydrating solution and molecular weight of the loaded compound, on the particle size and EE of niosomes prepared by using the thin film hydration method was studied. Particularly, mili-Q water, glycerol solution and PEG-400 solution were tested for niosomes formulated with Span®80-Tween®80 with/without dodecanol as membrane stabilizer. It was found that particle size highly depends on hydration media composition and an interaction with compound MW could exist. Larger vesicles results in an increase in EE, which could be purely related with physical aspects such as vesicle loading volume capacity. The effect of hydration solution composition could be related with their ability to change the bilayer packing and physical properties, as observed by differential scanning calorimetry. Finally, it was possible to compare the suitability of dialysis and gel filtration as purification methods, demonstrating that gel filtration is not an adequate purification method when viscous solutions are used, since they could affect the particle vesicles retention and hence EE measurements would be misrepresentative.

Micropipette Tip-Based Immunoassay with Electrochemical Detection of Antitissue Transglutaminase to Diagnose Celiac Disease Using Staples and a Paper-Based Platform.

In this work, 1-200 µL polypropylene micropipette tips were used as platforms for performing immunoassays after converting their inner surfaces on a capture zone for the analyte of interest. We have used a micropipette-tip immunoelectroanalytical platform for the detection of antitissue transglutaminase (IgA), the main biomarker for celiac disease. Modification of the tip wall with poly-l-lysine allowed adsorption of tissue transglutaminase (tTG), which will capture later anti-tTG (IgA) antibodies developed in celiac-affected people. A sandwich-type format was followed, incubating simultaneously the analyte and the detection antibody, labeled with horseradish peroxidase. With this new application for an extremely common lab material, we can perform quantitative analysis by dispensing the liquid into a low-cost and miniaturized staple-based paper electrochemical platform. The analytical signal was the reduction of the enzymatically oxidized substrate, recorded chronoamperometrically (i-t curve). The intensity of the current obtained at a fixed time after the application of the cathodic potential followed a linear relationship with anti-tTG (IgA) concentration. The relative standard deviation obtained for immunoassays performed in different tips indicates the adequate precision of this new methodology, which is very promising for decentralized analysis. Negative and positive controls produced results that were in accordance with those obtained with spectrophotometric enzyme linked-immunosorbent assays.

Extracellular Vesicles: From Biology to Biomedical Applications.

Legend says that Philippides ran to Athens to announce the victory against the Persians in the Battle of Marathon [...].

Continuous flow production of size-controllable niosomes using a thermostatic microreactor.

The new roles of vesicular systems in advanced biomedical, analytical and food science applications demand novel preparation processes designed to reach the new standards. Particle size and monodispersity have become essential properties to control. In this work, key parameters, involved in a microfluidic reactor with hydrodynamic flow focusing, were investigated in order to quantify their effects on niosomes morphology. Particular attention was given to temperature, which is both a requirement to handle non-ionic surfactants with phase transition temperature above RT, and a tailoring variable for size and monodispersity control. With this aim, niosomes with two different sorbitan esters and cholesterol as stabilizer were formulated. High resolution and conventional 3D-printing technologies were employed for the fabrication of microfluidic reactor and thermostatic systems, since this additive technology has been essential for microfluidics development in terms of cost-effective and rapid prototyping. A customised device to control temperature and facilitate visualization of the process was developed, which can be easily coupled with commercial inverted microscopes. The results demonstrated the capability of microfluidic production of niosomes within the full range of non-ionic surfactants and membrane stabilizers.

Synthesis of Superparamagnetic Iron Oxide Nanoparticles: SWOT Analysis Towards Their Conjugation to Biomolecules for Molecular Recognition Applications.

Superparamagnetic Iron Oxide Nanoparticles (SPIONs) have attracted a great deal of research attention due to their enormous possibilities of utilisation in different bioapplications such as magnetic particle imaging, drug delivery, hyperthermia or magnetic bio-detection. The purpose of this review is to critically analyze the methods for synthesis of SPIONs reported at the literature taking into account their suitability in molecular recognition applications by means of their conjugation to biomolecules. In this work, we have summarized the main synthesis routes, and controlled agglomeration methods for enhancement of sensitivity at molecular recognition events. This includes conventional chemical precipitation methods, thermal decomposition, microemulsions, or microfluidic synthesis, and the main strategies for the preparation of nanocomposites or SPIONs nanoclusters, such as polymer or silica cross-linking reactions, entrapment in nanovesicles or flower-like structures through the appropriate use of different metals to get synergetic properties for the total nanoarquitecture. Since most of the actual applications in biomedicine require their conjugation to biomolecules, an analysis of the Strengths, Weaknesses, Opportunities and Threats of these methods was carried out for the first time, with a view to highlight the best routes for a given application at biomolecular recognition.

Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma.

BACKGROUND: Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis. METHODS: Using MICA as example of a tumour-derived antigen, endogenously expressed in metastatic melanoma and recruited to exosomes, we have developed two immunocapture-based assays for detection of different epitopes in nanovesicles. Although both techniques, enzyme-linked immunosorbent assay (ELISA) and Lateral flow immunoassays (LFIA) have the same theoretical basis, that is, using capture and detection antibodies for a colorimetric read-out, analysis of exosome-bound proteins poses methodological problems that do not occur when these techniques are used for detection of soluble molecules, due to the presence of multiple epitopes on the vesicle. RESULTS: Here we demonstrate that, in ELISA, the signal obtained was directly proportional to the amount of epitopes per exosome. In LFIA, the amount of detection antibody immobilized in Au-nanoparticles needs to be low for efficient detection, otherwise steric hindrance results in lower signal. We describe the conditions for detection of MICA in exosomes and prove, for the first time using both techniques, the co-existence in one vesicle of exosomal markers (the tetraspanins CD9, CD63 and CD81) and an endogenously expressed tumour-derived antigen. The study also reveals that scarce proteins can be used as targets for detection antibody in LFIA with a better result than very abundant proteins and that the conditions can be optimized for detection of the protein in plasma. CONCLUSIONS: These results open the possibility of analyzing biological samples for the presence of tumour-derived exosomes using high throughput techniques.

Therapeutic biomaterials based on extracellular vesicles: classification of bio-engineering and mimetic preparation routes.

Extracellular vesicles (EVs) are emerging as novel theranostic tools. Limitations related to clinical uses are leading to a new research area on design and manufacture of artificial EVs. Several strategies have been reported in order to produce artificial EVs, but there has not yet been a clear criterion by which to differentiate these novel biomaterials. In this paper, we suggest for the first time a systematic classification of the terms used to build up the artificial EV landscape, based on the preparation method. This could be useful to guide the derivation to clinical trial routes and to clarify the literature. According to our classification, we have reviewed the main strategies reported to date for their preparation, including key points such as: cargo loading, surface targeting strategies, purification steps, generation of membrane fragments for the construction of biomimetic materials, preparation of synthetic membranes inspired in EV composition and subsequent surface decoration.

Fully Artificial Exosomes: Towards New Theranostic Biomaterials.

Bionanotechnology routes have been recently developed to produce fully artificial exosomes: biomimetic particles designed to overcome certain limitations in extracellular vesicle (EV) biology and applications. These particles could soon become true therapeutic biomaterials. Here, we outline their current preparation techniques, their explored and future possibilities, and their present limits.

Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids.

Exosomes are cell-secreted nanovesicles (40-200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×10(5) exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.