Search Results (26)

Diffusion and immobilization of molecules in biomembranes are essential for life. Understanding it is crucial for biomimetic approaches where well-defined substrates are created for live cell assays or biomaterial development. Here, we present biomimetic model systems consisting of a supported lipid bilayer and membrane coupled proteins to study the influence of lipid–lipid and lipid–protein interactions on membrane mobility. To characterize the diffusion of lipids or proteins, the continuous photobleaching technique is used. Either Neutravidin coupled to DOPE-cap-Biotin lipids or GFP coupled to DOGS-NTA lipids is studied at 0.005–0.5 mol% concentration of the linker lipid. Neutravidin creates mobile obstacles in the membrane, while GFP coupling results in immobile obstacles. By actin filament coupling to Neutravidin-lipid complexes, obstacles are crosslinked, resulting in lipid mobility reduction along with the appearance of a membrane texture. Theoretical considerations accurately describe lipid diffusion changes at high obstacle concentration as a function of obstacle size and viscous effects. The mobility of membrane lipids depends on the concentration of protein-binding lipids and on the concentration and charge of the coupled protein. Next to diffusion and friction coefficients, we determine the effective obstacle size as well as a charge-dependent effect that dominates the decrease in lipid mobility. Full article

Inflammation is a physiological response to a damaging stimulus but sometimes can be the cause of the onset of neurodegenerative diseases, atherosclerosis, and cancer. These pathologies are characterized by the overexpression of inflammatory markers like endothelial adhesion molecules, such as Vascular Cell Adhesion Molecule-1 (VCAM-1). In the present work, the development of liposomes for therapeutic targeted delivery to inflamed endothelia is described. The idea is to exploit a three-step pretargeting system based on the biotin–avidin high-affinity interaction: the first step involves a previously described biotin derivative bearing a VCAM-1 binding peptide; in the second step, the avidin derivative NeutrAvidin^TM, which strongly binds to the biotin moiety, is injected; the final step is the administration of biotinylated liposomes that would bind to Neutravidin^TM immobilized onto VCAM-1 overexpressing endothelium. Stealth biotinylated liposomes, prepared via the thin film hydration method followed by extrusion and purification via size exclusion chromatography, have been thoroughly characterized for their chemico-physical and morphological features and loaded with metformin hydrochloride, a potential anti-inflammatory agent. The three-step system, tested in vitro on different cell lines via confocal microscopy, FACS analysis and metformin uptake, has proved its suitability for therapeutic applications. Full article

G protein-coupled receptors (GPCRs) are of outstanding pharmacological interest as they are abundant in cell membranes where they perform diverse functions that are closely related to the vitality of cells. The analysis of GPCRs in natural membranes is laborious, as established methods are almost exclusively cell culture-based and only a few methods for immobilization in a natural membrane outside the cell are known. Within this study, we present a one-step, fast and robust immobilization strategy of the GPCR glucagon-like peptide 1 receptor (GLP-1R). GLP-1R was synthesized in eukaryotic lysates harboring endogenous endoplasmic reticulum-derived microsomes enabling the embedment of GLP-1R in a natural membrane. Interestingly, we found that these microsomes spontaneously adsorbed to magnetic Neutravidin beads thus providing immobilized membrane protein preparations which required no additional manipulation of the target receptor or its supporting membrane. The accessibility of the extracellular domain of membrane-embedded and bead-immobilized GLP-1R was demonstrated by bead-based enzyme-linked immunosorbent assay (ELISA) using GLP-1R-specific monoclonal antibodies. In addition, ligand binding of immobilized GLP-1R was verified in a radioligand binding assay. In summary, we present an easy and straightforward synthesis and immobilization methodology of an active GPCR which can be beneficial for studying membrane proteins in general. Full article

Serum level of CCL5 chemokine is considered an emerging biomarker for multiple sclerosis (MS). Due to the lack of specific assays for this disease, the development of a point-of-care test for rapid detection of MS could lead to avoiding diagnostics delays. In this paper, we report the first electrochemical immunoplatform for quantification of the CCL5 biomarker at the clinically required levels, able to discriminate between patients diagnosed with MS and healthy individuals. The immunosensing device involves protein capture from biological samples by complexation with biotinylated specific antibodies immobilized onto neutravidin-functionalized microparticles and sandwich assay with anti-CCL5 antibody and IgG labelled with horseradish peroxidase (HRP) for the enzyme-catalyzed amperometric detection of H₂O₂ using hydroquinone (HQ) as the redox mediator. The method shows excellent analytical performance for clinical application with a wide linear range of concentrations (0.1–300 ng·mL⁻¹ CCL5, R² = 0.998) and a low detection limit (40 pg·mL⁻¹ CCL5). The biosensing platform was applied to the determination of the CCL5 endogenous content in 100-fold diluted sera both from healthy individuals and patients diagnosed with MS, with no further sample treatment in just two hours. The results were successfully compared with those obtained by the ELISA methodology. Full article

The conventional signal amplification strategies for surface plasmon resonance (SPR) biosensors involve the immobilization of receptors, the capture of target analytes and their recognition by signal reporters. Such strategies work at the expense of simplicity, rapidity and real-time measurement of SPR biosensors. Herein, we proposed a one-step, real-time method for the design of SPR biosensors by integrating magnetic preconcentration and separation. The target analytes were captured by the receptor-modified magnetic nanoparticles (MNPs), and then the biotinylated recognition elements were attached to the analyte-bound MNPs to form a sandwich structure. The sandwich hybrids were directly delivered to the neutravidin-modified SPR fluidic channel. The MNPs hybrids were captured by the chip through the neutravidin–biotin interaction, resulting in an enhanced SPR signal. Two SPR biosensors have been constructed for the detection of target DNA and beta-amyloid peptides with high sensitivity and selectivity. This work, integrating the advantages of one-step, real-time detection, multiple signal amplification and magnetic preconcentration, should be valuable for the detection of small molecules and ultra-low concentrations of analytes. Full article

The exceptional strength and stability of noncovalent avidin-biotin binding is widely utilized as an effective bioconjugation strategy in various biosensing applications, and neutravidin and streptavidin proteins are two commonly used avidin analogues. It is often regarded that the biotin-binding abilities of neutravidin and streptavidin are similar, and hence their use is interchangeable; however, a deeper examination of how these two proteins attach to sensor surfaces is needed to develop reliable surface functionalization options. Herein, we conducted quartz crystal microbalance-dissipation (QCM-D) biosensing experiments to investigate neutravidin and streptavidin binding to biotinylated supported lipid bilayers (SLBs) in different pH conditions. While streptavidin binding to biotinylated lipid receptors was stable and robust across the tested pH conditions, neutravidin binding strongly depended on the solution pH and was greater with increasingly acidic pH conditions. These findings led us to propose a two-step mechanistic model, whereby streptavidin and neutravidin binding to biotinylated sensing interfaces first involves nonspecific protein adsorption that is mainly influenced by electrostatic interactions, followed by structural rearrangement of adsorbed proteins to specifically bind to biotin functional groups. Practically, our findings demonstrate that streptavidin is preferable to neutravidin for constructing SLB-based sensing platforms and can improve sensing performance for detecting antibody–antigen interactions. Full article

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. Full article

Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise β-cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116^{p53−/−/PSCA} cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4^PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities. Full article

This review is focused on the biosensing assay based on AuNPs (AuNPs) modified by proteins, peptides and nucleic acid aptamers. The unique physical properties of AuNPs allow their modification by proteins, peptides or nucleic acid aptamers by chemisorption as well as other methods including physical adsorption and covalent immobilization using carbodiimide chemistry or based on strong binding of biotinylated receptors on neutravidin, streptavidin or avidin. The methods of AuNPs preparation, their chemical modification and application in several biosensing assays are presented with focus on application of nucleic acid aptamers for colorimetry assay for determination of antibiotics and bacteria in food samples. Full article

The heterogeneous assays of proteases usually require the immobilization of peptide substrates on the solid surface for enzymatic hydrolysis reactions. However, immobilization of peptides on the solid surface may cause a steric hindrance to prevent the interaction between the substrate and the active center of protease, thus limiting the enzymatic cleavage of the peptide. In this work, we reported a heterogeneous surface plasmon resonance (SPR) method for protease detection by integration of homogeneous reaction. The sensitivity was enhanced by the signal amplification of streptavidin (SA)-conjugated immunoglobulin G (SA-IgG). Caspase-3 (Cas-3) was determined as the model. A peptide labeled with two biotin tags at the N- and C-terminals (bio-GDEVDGK-bio) was used as the substrate. In the absence of Cas-3, the substrate peptide was captured by neutravidin (NA)-covered SPR chip to facilitate the attachment of SA-IgG by the avidin-biotin interaction. However, once the peptide substrate was digested by Cas-3 in the aqueous phase, the products of bio-GDEVD and GK-bio would compete with the substrate to bond NA on the chip surface, thus limiting the attachment of SA-IgG. The method integrated the advantages of both heterogeneous and homogeneous assays and has been used to determine Cas-3 inhibitor and evaluate cell apoptosis with satisfactory results. Full article

A multiharmonic quartz crystal microbalance (QCM) has been applied to study the viscoelastic properties of the aptamer-based sensing layers at the surface of a QCM transducer covered by neutravidin following interaction with bacteria Listeria innocua. Addition of bacteria in the concentration range 5 × 10³–10⁶ CFU/mL resulted in a decrease of resonant frequency and in an increase of dissipation. The frequency decrease has been lower than one would expect considering the dimension of the bacteria. This can be caused by lower penetration depth of the acoustics wave (approximately 120 nm) in comparison with the thickness of the bacterial layer (approximately 500 nm). Addition of E. coli at the surface of neutravidin as well as aptamer layers did not result in significant changes in frequency and dissipation. Using the Kelvin–Voight model the analysis of the viscoelastic properties of the sensing layers was performed and several parameters such as penetration depth, Γ, viscosity coefficient, η, and shear modulus, μ, were determined following various modifications of QCM transducer. The penetration depth decreased following adsorption of the neutravidin layer, which is evidence of the formation of a rigid protein structure. This value did not change significantly following adsorption of aptamers and Listeria innocua. Viscosity coefficient was higher for the neutravidin layer in comparison with the naked QCM transducer in a buffer. However, a further increase of viscosity coefficient took place following attachment of aptamers suggesting their softer structure. The interaction of Listeria innocua with the aptamer layer resulted in slight decrease of viscosity coefficient. The shearing modulus increased for the neutravidin layer and decreased following aptamer adsorption, while a slight increase of µ was observed after the addition of Listeria innocua. Full article

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. Full article

Monolithic quartz crystal microbalance (MQCM) has recently emerged as a very promising technology suitable for biosensing applications. These devices consist of an array of miniaturized QCM sensors integrated within the same quartz substrate capable of detecting multiple target analytes simultaneously. Their relevant benefits include high throughput, low cost per sensor unit, low sample/reagent consumption and fast sensing response. Despite the great potential of MQCM, unwanted environmental factors (e.g., temperature, humidity, vibrations, or pressure) and perturbations intrinsic to the sensor setup (e.g., mechanical stress exerted by the measurement cell or electronic noise of the characterization system) can affect sensor stability, masking the signal of interest and degrading the limit of detection (LoD). Here, we present a method based on the discrete wavelet transform (DWT) to improve the stability of the resonance frequency and dissipation signals in real time. The method takes advantage of the similarity among the noise patterns of the resonators integrated in an MQCM device to mitigate disturbing factors that impact on sensor response. Performance of the method is validated by studying the adsorption of proteins (neutravidin and biotinylated albumin) under external controlled factors (temperature and pressure/flow rate) that simulate unwanted disturbances. Full article

COVID-19 is a pandemic respiratory disease that is caused by the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Anti-SARS-CoV-2 antibodies are essential weapons that a patient with COVID-19 has to combat the disease. When now repurposing a drug, namely an aptamer that interacts with SARS-CoV-2 proteins for COVID-19 treatment (BC 007), which is, however, a neutralizer of pathogenic autoantibodies in its original indication, the possibility of also binding and neutralizing anti-SARS-CoV-2 antibodies must be considered. Here, the highly specific virus-neutralizing antibodies have to be distinguished from the ones that also show cross-reactivity to tissues. The last-mentioned could be the origin of the widely reported SARS-CoV-2-induced autoimmunity, which should also become a target of therapy. We, therefore, used enzyme-linked immunosorbent assay (ELISA) technology to assess the binding of well-characterized publicly accessible anti-SARS-CoV-2 antibodies (CV07-209 and CV07-270) with BC 007. Nuclear magnetic resonance spectroscopy, isothermal calorimetric titration, and circular dichroism spectroscopy were additionally used to test the binding of BC 007 to DNA-binding sequence segments of these antibodies. BC 007 did not bind to the highly specific neutralizing anti-SARS-CoV-2 antibody but did bind to the less specific one. This, however, was a lot less compared to an autoantibody of its original indication (14.2%, range 11.0–21.5%). It was also interesting to see that the less-specific anti-SARS-CoV-2 antibody also showed a high background signal in the ELISA (binding on NeutrAvidin-coated or activated but noncoated plastic plate). These initial experiments suggest that the risk of binding and neutralizing highly specific anti-SARS CoV-2 antibodies by BC 007 should be low. Full article

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. Full article