Surface-enhanced Raman sensor with molecularly imprinted nanoparticles as highly sensitive recognition material for cancer marker amino acids.

Surface-enhanced Raman scattering (SERS) is a powerful technique primarily due to its high sensitivity and signal-enhancing properties, which enable the identification of unique vibrational fingerprints. These fingerprints can be used for the diagnosis and monitoring of diseases such as cancer. It is crucial to selectively identify cancer biomarkers for early diagnosis. A correlation has been established between the reduction in the concentration of specific amino acids and the stage of the disease, particularly tryptophan (TPP) and tyrosine (TRS) in individuals diagnosed with prostate cancer. In this work, we present a strategy to analyze TPP and TRS amino acids using molecularly imprinted polymer nanoparticles (nanoMIPs), which selectively detect target molecules in a SERS sensor. NanoMIPs are synthesized using the solid-phase molecular imprinting method with TPP and TRS as templates. These are then immobilized on a SERS substrate with gold nanoparticles to measure samples prepared from tryptophan and tyrosine in phosphate-buffered saline. The detection and quantification limits of the designed sensor are 7.13 µM and 23.75 µM for TPP, and 22.11 µM and 73.72 µM for TRS, respectively. Our study lays the groundwork for future investigations utilizing nanoMIPs in SERS assessments of TPP and TRS as potential biomarkers for prostate cancer detection.

Differential Proteoglycan Expression in Atherosclerosis Alters Platelet Adhesion and Activation.

Proteoglycans are differentially expressed in different atherosclerotic plaque phenotypes, with biglycan and decorin characteristic of ruptured plaques and versican and hyaluronan more prominent in eroded plaques. Following plaque disruption, the exposure of extracellular matrix (ECM) proteins triggers platelet adhesion and thrombus formation. In this study, the impact of differential plaque composition on platelet function and thrombus formation was investigated. Platelet adhesion, activation and thrombus formation under different shear stress conditions were assessed in response to individual proteoglycans and composites representing different plaque phenotypes. The results demonstrated that all the proteoglycans tested mediated platelet adhesion but not platelet activation, and the extent of adhesion observed was significantly lower than that observed with type I and type III collagens. Thrombus formation upon the rupture and erosion ECM composites was significantly reduced (p < 0.05) compared to relevant collagen alone, indicating that proteoglycans negatively regulate platelet collagen responses. This was supported by results demonstrating that the addition of soluble biglycan or decorin to whole blood markedly reduced thrombus formation on type I collagen (p < 0.05). Interestingly, thrombus formation upon the erosion composite displayed aspirin sensitivity, whereas the rupture composite was intensive to aspirin, having implications for current antiplatelet therapy regimes. In conclusion, differential platelet responses and antiplatelet efficacy are observed on ECM composites phenotypic of plaque rupture and erosion. Proteoglycans inhibit thrombus formation and may offer a novel plaque-specific approach to limit arterial thrombosis.

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.

Nanozyme-Based Lateral Flow Immunoassay (LFIA) for Extracellular Vesicle Detection.

Extracellular vesicles (EVs) are biological nanoparticles of great interest as novel sources of biomarkers and as drug delivery systems for personalized therapies. The research in the field and clinical applications require rapid quantification. In this study, we have developed a novel lateral flow immunoassay (LFIA) system based on Fe3O4 nanozymes for extracellular vesicle (EV) detection. Iron oxide superparamagnetic nanoparticles (Fe3O4 MNPs) have been reported as peroxidase-like mimetic systems and competent colorimetric labels. The peroxidase-like capabilities of MNPs coated with fatty acids of different chain lengths (oleic acid, myristic acid, and lauric acid) were evaluated in solution with H2O2 and 3,3,5,5-tetramethylbenzidine (TMB) as well as on strips by biotin-neutravidin affinity assay. As a result, MNPs coated with oleic acid were applied as colorimetric labels and applied to detect plasma-derived EVs in LFIAs via their nanozyme effects. The visual signals of test lines were significantly enhanced, and the limit of detection (LOD) was reduced from 5.73 × 107 EVs/µL to 2.49 × 107 EVs/µL. Our work demonstrated the potential of these MNPs as reporter labels and as nanozyme probes for the development of a simple tool to detect EVs, which have proven to be useful biomarkers in a wide variety of diseases.

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.

Magnetic Lateral Flow Immunoassay for Small Extracellular Vesicles Quantification: Application to Colorectal Cancer Biomarker Detection.

Colorectal cancer (CRC) is the third leading cause of cancer death and the fourth most common cancer in the world. Colonoscopy is the most sensitive test used for detection of CRC; however, their procedure is invasive and expensive for population mass screening. Currently, the fecal occult blood test has been widely used as a screening tool for CRC but displays low specificity. The lack of rapid and simple methods for mass screening makes the early diagnosis and therapy monitoring difficult. Extracellular vesicles (EVs) have emerged as a novel source of biomarkers due to their contents in proteins and miRNAs. Their detection would not require invasive techniques and could be considered as a liquid biopsy. Specifically, it has been demonstrated that the amount of CD147 expressed in circulating EVs is significant higher for CRC cell lines than for normal colon fibroblast cell lines. Moreover, CD147-containing EVs have been used as a biomarker to monitor response to therapy in patients with CRC. Therefore, this antigen could be used as a non-invasive biomarker for the detection and monitoring of CRC in combination with a Point-of-Care platform as, for example, Lateral Flow Immunoassays (LFIAs). Here, we propose the development of a quantitative lateral flow immunoassay test based on the use of magnetic nanoparticles as labels coupled to inductive sensor for the non-invasive detection of CRC by CD147-positive EVs. The results obtained for quantification of CD147 antigen embedded in EVs isolated from plasma sample have demonstrated that this device could be used as a Point-of-Care tool for CRC screening or therapy monitoring thanks to its rapid response and easy operation.

Nano-Encapsulation of Mithramycin in Transfersomes and Polymeric Micelles for the Treatment of Sarcomas.

Sarcomas are aggressive tumors which often show a poor response to current treatments. As a promising therapeutic alternative, we focused on mithramycin (MTM), a natural antibiotic with a promising anti-tumor activity but also a relevant systemic toxicity. Therefore, the encapsulation of MTM in nano-delivery systems may represent a way to increase its therapeutic window. Here, we designed novel transfersomes and PLGA polymeric micelles by combining different membrane components (phosphatidylcholine, Span 60, Tween 20 and cholesterol) to optimize the nanoparticle size, polydispersity index (PDI) and encapsulation efficiency (EE). Using both thin film hydration and the ethanol injection methods we obtained MTM-loaded transferosomes displaying an optimal hydrodynamic diameter of 100-130 nm and EE values higher than 50%. Additionally, we used the emulsion/solvent evaporation method to synthesize polymeric micelles with a mean size of 228 nm and a narrow PDI, capable of encapsulating MTM with EE values up to 87%. These MTM nano-delivery systems mimicked the potent anti-tumor activity of free MTM, both in adherent and cancer stem cell-enriched tumorsphere cultures of myxoid liposarcoma and chondrosarcoma models. Similarly to free MTM, nanocarrier-delivered MTM efficiently inhibits the signaling mediated by the pro-oncogenic factor SP1. In summary, we provide new formulations for the efficient encapsulation of MTM which may constitute a safer delivering alternative to be explored in future clinical uses.

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.

Electrodecoration and Characterization of Superparamagnetic Iron Oxide Nanoparticles with Bioactive Synergistic Nanocopper: Magnetic Hyperthermia-Induced Ionic Release for Anti-Biofilm Action.

The urgency for the availability of new antibacterial/disinfectant agents has become a worldwide priority. At the same time, along with the extensive use of other metal nanoparticles (NPs), the investigation of magnetic NPs (MNPs) in antibacterial studies has turned out to be an increasingly attractive research field. In this context, we present the preparation and characterization of superparamagnetic iron oxide NPs, electrodecorated with antimicrobial copper NPs, able to modulate the release of bioactive species not only by the NP's stabilizer, but also through the application of a suitable magnetic field. Antimicrobial synergistic CuNPs stabilized by benzalkonium chloride have been used in the current study. We demonstrate the successful preparation of Cu@Fe3O4 MNPs composites through morphological and spectroscopic results. Additionally, an extensive magnetic characterization is reported, along with hyperthermia-induced copper ionic release. On the basis of our results, we propose a new generation of antimicrobial magnetic nanomaterials, whose bioactivity can be also tuned by the application of a magnetic field.

Vitamin D3 Loaded Niosomes and Transfersomes Produced by Ethanol Injection Method: Identification of the Critical Preparation Step for Size Control.

Vesicular nanocarriers have an important role in drug delivery and dietary supplements. Size control and optimization of encapsulation efficiency (EE) should be optimized for those applications. In this work, we report on the identification of the crucial step (injection, evaporation, or sonication) innanovesicles (transfersomes and niosomes) preparation by theethanol injection method (EI). The identification of each production step on the final vesicle size was analyzed in order to optimize further scale-up process. Results indicated that the final size of transfersomeswas clearly influenced by the sonication step while the final size of niosomes was mainly governed by the injection step. Measurements of final surface tension of the different vesicular systems prepared indicate a linear positive tendency with the vesicle size formed. This relation could help to better understand the process and design a vesicular size prediction model for EI. Vitamin D3 (VitD3) was encapsulated in the systems formulated with encapsulation efficiencies larger than 90%. Interaction between the encapsulated compound and the membrane layer components is crucial for vesicle stability. This work has an impact on the scaling-up production of vesicles for further food science applications.

Cu Nanoparticle-Loaded Nanovesicles with Antibiofilm Properties. Part I: Synthesis of New Hybrid Nanostructures.

Copper nanoparticles (CuNPs) stabilized by quaternary ammonium salts are well known as antimicrobial agents. The aim of this work was to study the feasibility of the inclusion of CuNPs in nanovesicular systems. Liposomes are nanovesicles (NVs) made with phospholipids and are traditionally used as delivery vehicles because phospholipids favor cellular uptake. Their capacity for hydrophilic/hydrophobic balance and carrier capacity could be advantageous to prepare novel hybrid nanostructures based on metal NPs (Me-NPs). In this work, NVs were loaded with CuNPs, which have been reported to have a biofilm inhibition effect. These hybrid materials could improve the effect of conventional antibacterial agents. CuNPs were electro-synthesized by the sacrificial anode electrolysis technique in organic media and characterized in terms of morphology through transmission electron microscopy (TEM). The NVs were prepared by the thin film hydration method in aqueous media, using phosphatidylcholine (PC) and cholesterol as a membrane stabilizer. The nanohybrid systems were purified to remove non-encapsulated NPs. The size distribution, morphology and stability of the NV systems were studied. Different quaternary ammonium salts in vesicular systems made of PC were tested as stabilizing surfactants for the synthesis and inclusion of CuNPs. The entrapment of charged metal NPs was demonstrated. NPs attached preferably to the membrane, probably due to the attraction of their hydrophobic shell to the phospholipid bilayers. The high affinity between benzyl-dimethyl-hexadecyl-ammonium chloride (BDHAC) and PC allowed us to obtain stable hybrid NVs c.a. 700 nm in diameter. The stability of liposomes increased with NP loading, suggesting a charge-stabilization effect in a novel antibiofilm nanohybrid material.

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.

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