QSense
User Days
2-3 November 2021

Cellular membranes are complex structures and simplified analogues in the form of model membranes or biomembranes are used as platforms to understand fundamental properties of the membrane itself as well as interactions with various biomolecules such as drugs, peptides and proteins. Model membranes at solid-liquid interfaces can be studied using a range of complementary surface-sensitive techniques to give a detailed picture of both the structure and physicochemical properties of the membrane and its resulting interactions.

In this talk, I will present the state of the art on the formation of solid supported lipid bilayers on flat and curved surfaces by vesicle fusion. I will then briefly present the principles as well as the main type of information on molecular interactions at model membranes accessible using quartz crystal microbalance with dissipation monitoring, ellipsometry, atomic force microscopy, Infrared spectroscopy, and neutron and X-ray reflectometry. Consistent examples for following bio- molecular interactions at model membranes will be used across many of the techniques including the well-studied antimicrobial peptide Melittin. The overall objective is to establish an understanding of the information accessible from each technique, their respective advantages and limitations, and their complementarity.

Self-assembled monolayers (SAM) represent a common technique of surface modification, applied in different fields of research, including development of chemical sensors and studies on biological interactions. In this set-up, smaller molecular units spontaneously arrange on a solid surface, forming a molecular film of a well-defined thickness and layered architecture. In the set-up of conventional SAMs, active surface molecules are coupled to the alkyl tether covalently attached to the gold surface1. This model however, can in many cases not fully reflect the fluid biological conditions, where membrane glycans are free to migrate within lipid bilayer and bind multivalent targets. Polyvalent interactions play a major role in biochemical processes like ligand - receptor mediated signaling and cell - cell recognition. Lacking of this dynamic feature and ability for flexible spatial organization leads to much weaker binding for covalent SAMs as compared to real biological conditions.

Introduced in our group concept of reversible self-assembled monolayers (rSAM) shares the simplicity of conventional SAMs, adding the feature of liquid-like nature. In this type of arrangement, primary covalent layer on the gold surface is expanded with an additional layer of benzamidine amphiphiles4. Presented in this communication studies describe interactions between galactose – presenting surfaces and LecA (PA-IL)/LecB (PA-IIL) lectins from Pseudomonas aeruginosa. Binding kinetics for separate bacterial lectins were compared for both rSAM and conventional SAM surfaces. Mass uptake and changes in surface thickness were estimated using simplified Sauerbrey equation and viscoelastic Voigt’s modelling method. Calculated increase in surface thickness was additionally compared with our previous studies using null ellipsometry.

This talk will introduce the class of antimicrobial lipids, including medium-chain fatty acids and monoglycerides, that are being developed for a wide range of human biomedical and livestock agricultural applications. Particular focus will be placed on how the QCM-D technique is well suited to characterize the interactions of  antimicrobial lipids with supported lipid bilayers, enabling detailed evaluation of the corresponding membrane-disruptive mechanisms of action and potency levels. 

We have developed biocompatible polymeric materials which have a repellent property of protein and cell and applying for medical devices such as biosensors, artificial organs, and cell culture devices. For the development of biomaterials in medical devices, study on biointerface between biomolecules such as protein and cell, and biomaterials such as metal, ceramic, and polymer is significant. Because the protein adsorption on material surfaces is the first event when medical devices are in contact with blood. How to evaluate the biointerface?  

In this seminar, firstly a mechanism of protein adsorption and cell adhesion on material surfaces is reviewed considering blood coagulation and thrombus formation. And then surface analytical methods of properties of the material surface, adsorption of proteins, and adhesion of cells are explained.  

Our experimental results about ΔD-ΔF plots of proteins and cells on the surface with different chemical characteristics (-CH3, -COOH, -NH2, -OH) by QCM-D showed the interaction forces of the biointerface. The interaction analysis of QCM-D was a better understanding with the use of imaging analysis of microscopy.   

From these results, we believe that QCM-D is a powerful tool not only for analyzing the amount of protein adsorption on materials, but also for evaluation of an interaction between biomolecules and material surfaces. 

Through the years, Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) system widely used to study the soft layers has extended its use to the biomedical research. In the present work, we optimized the QCM-D system to study platelet adhesion and aggregation and studied the role of the prostacyclin (PGI2)-, nitric oxide (NO)-, and carbon monoxide (CO)-dependent pathways in the early stages of αIIbβ3 receptor-mediated platelet signaling mechanisms. Studies were carried out with washed human platelets using the fibrinogenmodified gold sensors with the polystyrene layer.
The negative frequency (Δfn) and positive dissipation (ΔDn) shifts registered during the convulxin-stimulated platelets analysis showed better sensitivity of QCM-D to the concentration of platelet suspensions (10 000 plt/µl, 100 000 plt/µl, 200 000 plt/µl) and convulxin concentration (1, ng/ml, 10 ng/ml, 30 ng/ml) in contrast to 200 000 plt/µl and EC50=30.77 ng/ml of the agonist required for Light Transmission Aggregometry (LTA) analysis.
The late αIIbβ3-fibrinogen interactions in QCM-D were reduced by eptifibatide at 5 µg/ml in comparison to IC50=0.05 µg/ml established by LTA. However, the high concentration of eptifibatide was not sufficient to decrease ΔDn values below the critical dissipation shifts values (ΔDn > 2 ‧10-6) that confirmed the viscoelastic properties of the layers formed by adhered platelets. The fitting of the linear regression to Δfn values indicated the heterogeneous properties of the platelets layers formed on the sensors surfaces.
The paradoxical effects of the positive frequency shifts were registered collaterally to the positive dissipation shifts during the first phase of stimulated platelet-sensor interactions and the non-stimulated platelet suspensions analysis. The positive frequency shifts were not related to the buffer interferences or the platelet interactions with the non-modified polystyrene gold sensors. The preincubation of convulxin-stimulated platelets with eptifibatide (5 µg/ml)
suppressed the positive frequency shifts. The treatment of the non-stimulated platelets with carbon monoxide-releasing molecule (CORM-A1, 300 µM) totally suppressed the positive frequency and dissipation shifts while nitric oxide donor (PAPA-NO, 10 µM) or carbaprostacyclin (cPGI2, 1000 ng/ml) had slight effects.
In conclusion, QCM-D system is suitable for studying αIIbβ3 receptor-dependent platelet adhesion giving the unique insight into the early phase of platelets activation. The positive frequency shift phenomenon is related to the early stages of platelet-fibrinogen contact, and could be approximated by the sphere-surface contact approach. This early platelet response is followed by the frequency shifts collapses related to the full platelet activation, adhesion and aggregation. The full suppression of positive frequency shifts by CORM-A1, an effective inhibitor of platelets metabolism (P. Kaczara et al. Arterioscler Thromb Vasc Biol. 2020 Oct;40(10):2376-2390; doi: 10.1161/ATVBAHA.120.314284), with minor effects of PAPA-NO or cPGI2 (cGMP, cAMP activators, respectively) suggests that the active modulation of the early stages of αIIbβ3 receptor-mediated platelet signaling is independent on cGMP-mediated or cAMP-mediated pathways but rather dependent on early activation of platelet energy metabolism.

During biomanufacturing,  common unit operations can exert considerable physical and interfacial stress on therapeutic protein molecules. In certain cases, such stress factors have been associated with unwanted adsorption to process surfaces and an increased risk of aggregation. At Pfizer, we aim to understand and control such events to deliver the highest quality therapeutic proteins to our patients. Recently, we’ve applied QCM-D technology to simulate protein behavior at the solid-liquid interface, analogous to bioprocessing conditions. The QCM-D response shows promise as a predictive marker to inform risk assessment, well in-advance of manufacturing.

Interactions between molecules and substrates are one of the major determinants of biological activity. We have been using Quartz Crystal Microbalance with Dissipation (QCMD) to understand some aspect of these interactions to complement our studies using X-ray scattering and atomic force microscopy. This talk will highlight two of these studies. In one, structural transformations that occur in polymeric nanospheres as they are adsorbed onto surfaces, and which affect the release of encapsulate drugs, was investigated with three different types of nanospheres and five different types of substrates. Poly(ethylene glycol), PEG, was used to modulate the surface chemistries. Our results showed that while nanospheres assemble into a close-packed structure on hydrophobic surfaces, on other surfaces, especially PEGylated ones, the nanospheres disassemble and form continuous film. This transformation was followed in real time by monitoring the Δf-ΔD plots. Implications of such conformational changes on drug delivery, and on designing nanospheres will be discussed. In the second study, the interaction between proteins were followed by carrying sequential deposition of the proteins on the QCMD sensors. The results show that the widely used Vrooman effect to interpret such data is not universally observed. Examples will be presented that explore the factors that influence these interactions. The utility of these studies in blood clot formation and for enzyme stabilization will be discussed.

In this webinar, we will discuss a couple of food applications using the QCM-D characterization technique as a complementary method to shed light on (i) the interaction of folic acid with ovalbumin to improve the bioavailability and shelflife stability of folic acid and (ii) mechanistic understanding of whey protein interaction with oral cavity mucin to decode astringency. 

In food manufacturing, fouling of surfaces has deleterious effects on the efficiency of operations. Fouling is a complex phenomenon that is significantly affected by product formulation and processing conditions (i.e., temperature, residence time, flow regime). The adsorption and fouling of complex, dairy-based beverages was investigated using QCM-D at temperatures ranging from 25 °C to 135 °C. For higher temperatures (> 100 °C), a novel high-pressure high-temperature QCM-D was employed. Results indicate QCM-D technology is a small-scale tool which provides better understanding of the mechanisms of fouling and the potential to predict fouling on larger scales.