A microfluidics-integrated impedance/surface acoustic resonance tandem sensor

We demonstrate a dual sensor concept for lab-on-a-chip in-liquid sensing through integration of surface acoustic wave resonance (SAR) sensing with electrochemical impedance spectroscopy (EIS) in a single device. In this concept, the EIS is integrated within the building blocks of the SAR sensor, but features a separate electrical port. The two-port sensor was designed, fabricated, and embedded in a soft polymer microfluidic delivery system, and subsequently characterized. The SAR-EIS tandem sensor features low cross-talk between SAR and EIS ports, thus promoting non-interfering gravimetric and impedimetric measurements. The EIS was characterized by means of the modified Randle\u27s cell lumped element model. Four sensitive parameters could be established from the tandem sensor readout, and subsequently employed in a proof of principle study of liposome layers and their interaction with Ca2+ ions, leading to transformation into molecular film structures. The associated shift of the sensing quantities is analysed and discussed. The combination of impedimetric and gravimetric sensing quantities provides a unique and detailed description of physicochemical surface phenomena as compared to a single mode sensing routine

Generation of interconnected vesicles in a liposomal cell model

We introduce an experimental method based upon a glass micropipette microinjection technique for generating a multitude of interconnected vesicles (IVs) in the interior of a single giant unilamellar phospholipid vesicle (GUV) serving as a cell model system. The GUV membrane, consisting of a mixture of soybean polar lipid extract and anionic phosphatidylserine, is adhered to a multilamellar lipid vesicle that functions as a lipid reservoir. Continuous IV formation was achieved by bringing a micropipette in direct contact with the outer GUV surface and subjecting it to a localized stream of a Ca2+ solution from the micropipette tip. IVs are rapidly and sequentially generated and inserted into the GUV interior and encapsulate portions of the micropipette fluid content. The IVs remain connected to the GUV membrane and are interlinked by short lipid nanotubes and resemble beads on a string. The vesicle chain-growth from the GUV membrane is maintained for as long as there is the supply of membrane material and Ca2+ solution, and the size of the individual IVs is controlled by the diameter of the micropipette tip. We also demonstrate that the IVs can be co-loaded with high concentrations of neurotransmitter and protein molecules and displaying a steep calcium ion concentration gradient across the membrane. These characteristics are analogous to native secretory vesicles and could, therefore, serve as a model system for studying secretory mechanisms in biological systems

A high-performance lab-on-a-chip liquid sensor employing surface acoustic wave resonance: part II

We recently introduced an in-liquid sensing concept based on surface acoustic resonance (SAR) in a lab-on-a-chip resonant device with one electrical port. The 185 MHz one-port SAR sensor has a sensitivity comparable to other surface acoustic wave (SAW) in-liquid sensors, while offering a high quality factor (Q) in water, low impedance, and fairly low susceptibility to viscous damping. In this work, we present significant design and performance enhancements of the original sensor presented in part I. A novel \u27lateral energy confinement\u27 (LEC) design is introduced, where the spatially varying reflectivity of the SAW reflectors enables strong SAW localization inside the sensing domain at resonance. An improvement in mass-sensitivity greater than 100% at resonance is achieved, while the measurement noise stays below 0.5 ppm. Sensing performance was evaluated through real-time measurements of the binding of 40 nm neutravidin-coated SiO2nanoparticles to a biotin-labeled lipid bilayer. Two complementary sensing parameters are studied, the shift of resonance frequency and the shift of conductance magnitude at resonance

Interaction of Calcium Ions with Lipid Membranes

Bilayer membranes enclose and shield the biological cell and its inner compartments, as well as the tubular networks that exist within and between the cells. Due to their fluidic nature, the membranes are incredibly dynamic and flexible, which allows them to bend, reshape and fuse in response to mechanical and chemical stimuli within their natural microenvironments. Variations in calcium ion concentration are of particular importance for chemical stimulation, as calcium ions play a major role in many cellular processes, including signaling, proliferation, cell division, migration, and exocytosis. Previous studies with lipid membranes showed that binding of calcium ions to the membrane results in dehydration of the lipid head groups, ordering of the hydrocarbon tails, and in the increase of membrane rigidity and tension. At the same time, direct membrane remodeling upon stimulation with calcium ions remains largely unknown. In this thesis, giant unilamellar vesicles (GUVs) and nanotubes were used as model systems of cellular membranes. These lipid membranes were subjected to variations in local calcium ion concentration close to the membrane surface using the microinjection technique and imaged with fluorescence microscopy.\ua0\ua0\ua0\ua0\ua0\ua0\ua0\ua0\ua0\ua0\ua0 The results of our studies on the membrane model systems provide evidence for formation of highly curved membrane structures such as membrane tubular protrusions in GUVs (Paper I, II), and lipid aggregates (bulges) in lipid nanotubes (Paper III), as consequences of controlled calcium ion exposure, which cannot be obtained under bulk conditions. It is also possible to move these highly curved membrane structures by repositioning the source of the calcium ion gradients along the GUV surfaces. Our findings demonstrate how elevated calcium ion concentration close to the GUV surface can trigger membrane remodeling, and how calcium gradients can be used to manipulate and guide lipid-based systems. It is further shown that calcium ion gradients can trigger directed movement and reorientation of phase-separated free-floating GUVs towards the calcium ion source (Paper IV), suggesting possible aspects of protocell migration due to changes in the chemical microenvironment. Lastly, in Paper V, we developed a technique to produce artificial intracellular vesicles (AIVs) inside a GUV. The formation of AIVs occurred upon localized microinjection of calcium solution using a glass micropipette, which was placed in direct contact with the outer surface of the GUV membrane. The AIVs can be used to study mechanisms of exo- and endocytosis, and due to the capability to entrap high concentrations of proteins within the AIVs, the findings bear potential for drug delivery applications

Ca2+ Gradient Induces Membrane Bending and Formation of Nanotubes in Giant Lipid Vesicles

Reshaping and bending of the cell membrane is imperative in many processes such as cell division, filopodia formation, and endocytosis. Understanding these shape transitions, will help to elucidate the underlying mechanisms of these essential cellular processes. In our work, we investigate an interplay between cell membrane morphology and chemical stimulation by constructing a biomimetic model system. More specifically, giant lipid vesicles were exposed to a chemical gradient of Ca2+, which was established over the membrane surface

Contactless Stimulation and Control of Biomimetic Nanotubes by Calcium Ion Gradients

Membrane tubular structures are important communication pathways between cells and cellular compartments. Studying these structures in their native environment is challenging, due to the complexity of membranes and varying chemical conditions within and outside of the cells. This work demonstrates that a calcium ion gradient, applied to a synthetic lipid nanotube, triggers lipid flow directed toward the application site, resulting in the formation of a bulge aggregate. This bulge can be translated in a contactless manner by moving a calcium ion source along the lipid nanotube. Furthermore, entrapment of polystyrene nanobeads within the bulge does not tamper the bulge movement and allows transporting of the nanoparticle cargo along the lipid nanotube. In addition to the synthetic lipid nanotubes, the response of cell plasma membrane tethers to local calcium ion stimulation is investigated. The directed membrane transport in these tethers is observed, but with slower kinetics in comparison to the synthetic lipid nanotubes. The findings of this work demonstrate a novel and contactless mode of transport in lipid nanotubes, guided by local exposure to calcium ions. The observed lipid nanotube behavior can advance the current understanding of the cell membrane tubular structures, which are constantly reshaped during dynamic cellular processes

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping