Search Results (172)

The prevalence of food allergies has been steadily increasing in recent years. β-lactoglobulin (β-LG), the main allergenic protein of milk and dairy allergies, is more commonly observed in infants and children. In this study, a β-LG-specific aptamer was selected using the combinatorial chemistry process known as systematic evolution of ligands by exponential enrichment (SELEX), and a quartz crystal microbalance with dissipation monitoring (QCM-D)-based aptasensor was developed using a novel surface functionalization technique, which mimics an artificial cell membrane on the QCM-D sensor surface, creating a physiologically relevant environment for the binding of the target to the sensor. Through SELEX combined with next-generation sequencing (NGS), the aptamer Apt 356 was identified. Its binding to β-LG was confirmed via dot blot analysis. The selected Apt 356 was then used for the development of a QCM-D-based sensor. To fabricate the sensor, the quartz surface was functionalized with a supported lipid bilayer (SLB). The β-LG-specific aptamer was immobilized onto this SLB. The results demonstrated that the QCM-D system allows real-time observation and evaluation of the binding of β-LG. While there have been some studies on aptasensors for the β-LG protein, to the best of our knowledge, this is the first QCM-D-based aptasensor developed specifically for β-LG protein detection. Full article

Cell-free biosensors harness the selectivity of cellular machinery without living cells’ constraints, offering advantages in environmental monitoring, medical diagnostics, and biotechnological applications. This review examines recent advances in cell-free biosensor development, highlighting their ability to detect diverse analytes including heavy metals, organic pollutants, pathogens, and clinical biomarkers with high sensitivity and specificity. We analyze technological innovations in cell-free protein synthesis optimization, preservation strategies, and field deployment methods that have enhanced sensitivity, and practical applicability. The integration of synthetic biology approaches has enabled complex signal processing, multiplexed detection, and novel sensor designs including riboswitches, split reporter systems, and metabolic sensing modules. Emerging materials such as supported lipid bilayers, hydrogels, and artificial cells are expanding biosensor capabilities through microcompartmentalization and electronic integration. Despite significant progress, challenges remain in standardization, sample interference mitigation, and cost reduction. Future opportunities include smartphone integration, enhanced preservation methods, and hybrid sensing platforms. Cell-free biosensors hold particular promise for point-of-care diagnostics in resource-limited settings, environmental monitoring applications, and food safety testing, representing essential tools for addressing global challenges in healthcare, environmental protection, and biosecurity. Full article

This study developed phospholipid-based liposomes loaded with extract from wild thyme (Thymus serpyllum L.) tea processing residues to enhance polyphenol stability and delivery. Liposomes were prepared with phospholipids alone or combined with 10–30 mol% cholesterol or β-sitosterol. The effect of different lipid compositions on encapsulation efficiency (EE), particle size, polydispersity index (PDI), zeta potential, stability, thermal properties, diffusion coefficient, and diffusion resistance of the liposomes was investigated. Liposomes with 10 mol% sterols (either cholesterol or β-sitosterol) exhibited the highest EE of polyphenols, while increasing sterol content to 30 mol% resulted in decreased EE. Particle size and PDI increased with sterol content, while liposomes prepared without sterols showed the smallest vesicle size. Encapsulation of the extract led to smaller liposomal diameters and slight increases in PDI values. Zeta potential measurements revealed that sterol incorporation enhanced the surface charge and stability of liposomes, with β-sitosterol showing the most pronounced effect. Stability testing demonstrated minimal changes in size, PDI, and zeta potential during storage. UV irradiation and lyophilization processes did not cause significant polyphenol leakage, although lyophilization slightly increased particle size and PDI. Differential scanning calorimetry revealed that polyphenols and sterols modified the lipid membrane transitions, indicating interactions between extract components and the liposomal bilayer. FT-IR spectra confirmed successful integration of the extract into the liposomes, while UV exposure did not significantly alter the spectral features. Thiobarbituric acid reactive substances (TBARS) assay demonstrated the extract’s efficacy in mitigating lipid peroxidation under UV-induced oxidative stress. In contrast, liposomes enriched with sterols showed enhanced peroxidation. Polyphenol diffusion studies showed that encapsulation significantly delayed release, particularly in sterol-containing liposomes. Release assays in simulated gastric and intestinal fluids confirmed controlled, pH-dependent polyphenol delivery, with slightly better retention in β-sitosterol-enriched systems. These findings support the use of β-sitosterol- and cholesterol-enriched liposomes as stable carriers for polyphenolic compounds from wild thyme extract, as bioactive antioxidants, for food and nutraceutical applications. Full article

The multi-domain muscle protein titin provides elasticity and mechanosensing functions to the sarcomere. Titin’s PEVK domain is intrinsically disordered due to the presence of a large number of prolines and highly charged residues. Although PEVK does not have canonical actin-binding motifs, it has been shown to bind F-actin. Here, we explored whether the PEVK domain may also affect actin assembly. We cloned the middle, 733-residue-long segment (called PEVKII) of the full-length PEVK domain, expressed in E. coli and purified by using His- and Avi-tags engineered to the N- and C-termini, respectively. Actin assembly was monitored by the pyrene assay in the presence of varying PEVKII concentrations. The structural features of PEVKII-associated F-actin were studied with atomic force microscopy. The added PEVKII enhanced the initial and log-phase rates of actin assembly and the peak F-actin quantity in a concentration-dependent way. However, the critical concentration of actin polymerization was unaltered. Thus, PEVK accelerates actin polymerization by facilitating its nucleation. This effect was highlighted in the AFM images of F-actin–PEVKII adsorbed to the supported lipid bilayer. The sample was dominated by radially symmetric complexes of short actin filaments. PEVK’s actin polymerization-modulating effect may, in principle, have a function in regulating sarcomeric actin length and turnover. Altogether, titin’s PEVK domain is not only a non-canonical actin-binding protein that regulates sarcomeric shortening, but one that may modulate actin polymerization as well. Full article

Supported lipid bilayers (SLBs) that model neuronal membranes are needed to explore the role of membrane lipids in the misfolding and aggregation of amyloid proteins associated with neurodegenerative diseases, including Parkinson’s and Alzheimer’s disease. The neuronal membranes include not only phospholipids, but also significant amounts of cholesterol, sphingomyelin, and gangliosides, which are critical to its biological function. In this study, we explored the conditions for the formation of an SLB, for the five-component lipid mixture composed of zwitterionic 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), anionic 1,2-dioleoyl- sn-glycero-3-phospho-L-serine (DOPS), nonionic cholesterol (Chol), zwitterionic sphingomyelin (SM), and anionic ganglioside (GM), using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, by varying experimental parameters such as pH, buffer type, temperature, vesicle size, and osmotic stress. SLB formation from this multicomponent lipid system was found challenging because the vesicles adsorbed intact on the quartz crystal and failed to rupture. For most of the variables tested, other than osmotic stress, we found no or only partial vesicle rupture leading to either a supported layer of vesicles or a partial SLB that included unruptured vesicles. When osmotic stress was applied to the vesicles already adsorbed on the surface, by having a different salt concentration in the rinse buffer that follows vesicle flow compared to that of the dilution buffer during vesicle flow and adsorption, vesicle rupture increased, but it remained incomplete. In contrast, when osmotic stress was applied during vesicle flow and adsorption on the surface, by having different salt concentrations in the dilution buffer in which vesicles flowed compared to the hydration buffer in which vesicles were prepared, complete vesicle rupture and successful formation of a rigid SLB was demonstrated. The robustness of this approach to form SLBs by applying osmotic stress during vesicle adsorption was found to be independent of the number of lipid components, as shown by SLB formation from the 1-, 2-, 3-, 4-, and 5-component lipid systems. Full article

Various pathogens use receptors on the host’s plasma membrane for their cellular uptake. For the bacterium Pseudomonas aeruginosa, interactions between its lectin LecA and the host cell glycosphingolipid globotriaosylceramide (also known as Gb3) are crucial for its internalization via the so-called lipid zipper mechanism. In this study, we investigated the interactions of the P. aeruginosa strain PAO1 with phase-separated lipid bilayers containing Gb3. Surprisingly, bacteria are mostly bound to the interphase of liquid-ordered (Lo) and liquid-disordered (Ld) membrane domains. Simultaneously with the formation of bacterial aggregates and the accumulation of membrane lipids, the lipid bilayers were drastically reorganized and Lo domains were dissolved. Surprisingly, Gb3 was found to play a role in the localization of the bacterium at the interface, less so LecA. When microspheres were used as a minimal mimic of the bacterium, these beads also localized preferentially at the Lo–Ld phase boundaries, but in contrast to living bacteria, beads were unable to cause membrane reorganization and dissolution of the Lo domain, even when coated with LecA. Targeting phase boundaries as “weak points” in membranes and thereby reorganizing and destabilizing the host cell plasma membrane could be an attractive entry strategy for P. aeruginosa and many other bacteria and viruses. Full article

Water in membrane interphases is vital for cellular biological functions, but despite its importance, the structure and function of biological water remain elusive. Here, by studying the OH stretching mode in partially hydrated lipid multilayers by FTIR measurements, relevant information on the water structure near the surface with lipid membranes has been gathered. The water hydrogen bond network is highly perturbed in the first layers that are in contact with the lipid membrane, exhibiting strong deviations from tetrahedral symmetry and a significant number of defects, such as isolated water molecules and a large number of hydrogen-bonded water dimers in the interphase region. These findings support the hypothesis that water chains form in phospholipid membranes, and are involved in the proton transfer across lipid bilayers by phosphate groups of opposing lipids. Furthermore, we have determined that even at very low hydration levels, a small amount of water is embedded within the confined spaces of the hydrocarbon region of phospholipid bilayers, which could potentially contribute to the structural stability of the lipid membrane. Full article

Palladium(II) complexes with tris(2-carboxyethyl)phosphine (PdTCEP) show promise for biomedical applications due to their distinct chemical characteristics. This study explored the toxicity of PdTCEP towards normal human cells and examined its interactions with model cell membranes. Two cell types were used to evaluate cytotoxicity: human microvascular endothelial cells (HMEC-1) and red blood cells (RBCs). In HMEC-1 cells, PdTCEP reduced survival to about 80% at 15 µM, with the most significant drop—down to 40%—occurring at 40 µM. The production of reactive oxygen species (ROS) increased in a manner dependent on both dose and time, especially after 72 h of incubation. Despite these effects, PdTCEP caused only minor hemolysis in RBCs, with hemolysis levels staying below 10% even at higher concentrations. Fluorescence anisotropy measurements showed that PdTCEP minimally affects the hydrophobic core of the lipid bilayer, with slight changes observed at concentrations above 40 µM. Generalized polarization (GP) analysis indicated a slight decrease in lipid polar head packing with increasing PdTCEP concentration. Complementary FTIR analysis supported these findings by providing detailed insights into PdTCEP-membrane interactions. This research underscores PdTCEP’s selective cytotoxicity and structural effects on membranes, suggesting its promise for more in-depth biological and pharmacological studies. Full article

Functional amyloids (protein nanofibrils, PNF) synthesized from plant sources exhibit unique physicochemical and nanomechanical properties that could improve food texture. While environmental factors affecting PNFs are well-known, scientific evidence on how cells (focus on the oral cavity) respond to them under physiological conditions is lacking. Self-assembled PNFs synthesized from fava bean whole protein isolate show a strong pH- and solvent-dependent morphology and elasticity modification measured by atomic force microscopy (AFM). After incubation of PNFs with an oral mechanosensitive model cell line at pH 7.3, difference in cell-surface roughness without significant changes in the overall cell elasticity were measured. The role of cell membrane composition on supported lipid bilayers was also tested, showing an increase in membrane elasticity with increasing fibril concentration and the possible impact of annular phospholipids in binding. Genetic responses of membrane proteins involved in texture and fat perception were detected at the mRNA level by RT-qPCR assay and both mechano- and chemosensing proteins displayed responses highlighting an interface dependent interaction. The outcomes of this study provide a basis for understanding the changing physicochemical properties of PNFs and their effect on flavor perception by altering mouthfeel and fat properties. This knowledge is important in the development of plant-based texture enhancers for sensory-appealing foods that require consumer acceptance and further promote healthy diets. Full article

Non-thermal plasma (NTP) synergistic anticancer strategies are a current hotspot of interest at the intersection of plasma biomedicine. Melittin (MEL) has been shown to inhibit cancer in many malignant tumors; however, its clinical application is controversial. Therefore, the transmembrane process and mechanism of MEL activity in different cell systems were studied and the combination of MEL and NTP was proposed in this paper. The results showed that the electrostatic attraction between MEL and the lipid bilayer contributes to the stable orientation of MEL on the membrane surface. In addition, sialic acid overexpression affects the degree to which MEL binds the membrane system and the stability of the membrane structure. The use of NTP to reduce the dosage of MEL and its related nonspecific cytolysis activity has certain clinical application value. The results of this study provide theoretical support for improving the clinical applicability of MEL and contribute to the further development of plasma biomedicine. Full article

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

Supported lipid bilayers (SLBs) are low-complexity biomimetic membranes, serving as popular experimental platforms to study membrane organization and lipid transfer, membrane uptake of nanoparticles and biomolecules, and many other processes. Quartz crystal microbalance with dissipation monitoring has been utilized to probe the influence of several parameters on the quality of SLBs formed on Au- and SiO₂-coated sensors. The influence of the aqueous medium (i.e., buffer type) and the adsorption temperature, above and below the lipid melting point, is neatly explored for SLBs of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine formed by a solvent exchange. Below the lipid melting temperature, quality variations are observed upon the formation on Au and SiO₂ surfaces, with the SLBs being more homogeneous for the latter. We further investigate how the buffer affects the detection of lipid melting in SLBs, a transition that necessitates high-sensitivity and time-consuming surface-sensitive techniques to be detected. Full article

The plasma membrane lipid distribution is asymmetric, with several anionic lipid species located in its inner leaflet. Among these, phosphatidylserine (PS) plays a crucial role in various important physiological functions. Over the last decade several methods have been developed that allow for the fabrication of large or giant unilamellar vesicles (GUVs) with an asymmetric lipid composition. Investigating the physicochemical properties of PS in such asymmetric lipid bilayers and studying its interactions with proteins necessitates the reliable fabrication of asymmetric GUVs (aGUVs) with a high degree of asymmetry that exhibit PS in the outer leaflet so that the interaction with peptides and proteins can be studied. Despite progress, achieving aGUVs with well-defined PS asymmetry remains challenging. Recently, a Ca²⁺-initiated hemifusion method has been introduced, utilizing the fusion of symmetric GUVs (sGUVs) with a supported lipid bilayer (SLB) for the fabrication of aGUVs. We extend this approach to create aGUVs with PS in the outer bilayer leaflet. Comparing the degree of asymmetry between aGUVs obtained via Ca²⁺ or Mg²⁺ initiated hemifusion of a phosphatidylcholine (PC) sGUVwith a PC/PS-supported lipid bilayer, we observe for both bivalent cations a significant number of aGUVs with near-complete asymmetry. The degree of asymmetry distribution is narrower for physiological salt conditions than at lower ionic strengths. While Ca²⁺ clusters PS in the SLB, macroscopic domain formation is absent in the presence of Mg²⁺. However, the clustering of PS upon the addition of Ca²⁺ is apparently too slow to have a negative effect on the quality of the obtained aGUVs. We introduce a data filtering method to select aGUVs that are best suited for further investigation. Full article

Doxorubicin (DOX) is a commonly used chemotherapeutic drug, from the anthracycline class, which is genotoxic to neoplastic cells via a DNA intercalation mechanism. It is effective and universal; however, it also causes numerous side effects. The most serious of them are cardiotoxicity and a decrease in the number of myeloid cells. For this reason, targeted DOX delivery systems are desirable, since they would allow lowering the drug dose and therefore limiting systemic side effects. Recently, synthetic dyes, in particular Congo red (CR), have been proposed as possible DOX carriers. CR is a planar molecule, built of a central biphenyl moiety and two substituted naphthalene rings, connected with diazo bonds. In water, it forms elongated ribbon-shaped supramolecular structures, which are able to selectively interact with immune complexes. In our previous studies, we have shown that CR aggregates can intercalate DOX molecules. In this way, they preclude DOX precipitation in water solutions and increase its uptake by MCF7 breast cancer cells. In the present work, we further explore the interactions between DOX, CR, and their aggregates (CR/DOX) with phospholipid membranes. In addition to neutral molecules, the protonated doxorubicin form, DXP, is also studied. Molecular dynamics simulations are employed to study the transfer of CR, DOX, DXP, and their aggregates through POPC bilayers. Interactions of CR, DOX, and CR/DOX with model monolayers are studied with Langmuir trough measurements. This study shows that CR may support the transfer of doxorubicin molecules into the bilayer. Both electrostatic and van der Waals interactions with lipids are important in this respect. The former promote the initial stages of the insertion process, the latter keep guest molecules inside the bilayer. Full article

Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers. Full article