Lipid-Based Surface Modifications: towards the Development of Membrane-Protein Arrays

Mimics of the natural cell membrane, such as lipid vesicles and supported lipid membranes, have gained significant attention in recent years. This is primarily due to their ability to provide scientists with a model system allowing a large arsenal of scientific tools to be used in studies of biological reactions that are naturally controlled by the cell membrane and its components. An in depth understanding of these processes, which are essential for the integrity and function of cells in all living organisms, is not only of high scientific interest, but also a key component in the development of therapeutic drugs and in disease diagnostics. In the latter contexts, various surface-based biosensor platforms have recently gained increased attention. This stems from the potential of such platforms to provide real-time, label-free and array-based analysis of cell-membrane mediated processes. However, progress in this direction is not limited to new sensor concepts alone, but also includes sophisticated surface modification schemes, which are compatible with array-based sensor platforms. This thesis work has been focused on the two latter issues, and contributes in essence with (i) a new means of distributing lipid vesicles on surface based array formats, using site-selective and sequence-specific sorting of DNA-modified vesicles on DNA arrays (Paper I to III; this is the major part of the thesis) and (ii) a new platform for electrochemical impedance spectroscopy (EIS) studies of supported membranes that span nanoscale holes in thin (~50 nm) transmission electron microscopy (TEM) windows, designed to be compatible with studies of single ion-channel processes (Paper V and Progress Report in Chapter 6). The concept used to modify lipid vesicles with DNA was based on self incorporating cholesterol-modified DNA. Significant efforts were put on increasing the strength of this coupling (Paper III), and a new method to quantify the DNA density on vesicles has been developed (Paper IV). In addition, as a precursor step towards nano-aperture spanning lipid membranes, an investigation was made of the influence on lateral lipid diffusivity in supported lipid membranes formed on substrates with nanoscale pits (Paper V). The lipid-based surface-modification protocols developed within this work have in common that they are compatible with studies of different types of membrane residing proteins on a single chip, making it likely that the concepts developed will contribute significantly to the field of array-based detection of membrane protein function. The work on nano-holes and hole-spanning lipid membranes has created a valuable platform towards the goal of studying single trans-membrane proteins, specifically ion channels

Lipid-Based Surface Modifications: towards the Development of Membrane-Protein Arrays

Mimics of the natural cell membrane, such as lipid vesicles and supported lipid membranes, have gained significant attention in recent years. This is primarily due to their ability to provide scientists with a model system allowing a large arsenal of scientific tools to be used in studies of biological reactions that are naturally controlled by the cell membrane and its components. An in depth understanding of these processes, which are essential for the integrity and function of cells in all living organisms, is not only of high scientific interest, but also a key component in the development of therapeutic drugs and in disease diagnostics. In the latter contexts, various surface-based biosensor platforms have recently gained increased attention. This stems from the potential of such platforms to provide real-time, label-free and array-based analysis of cell-membrane mediated processes. However, progress in this direction is not limited to new sensor concepts alone, but also includes sophisticated surface modification schemes, which are compatible with array-based sensor platforms. This thesis work has been focused on the two latter issues, and contributes in essence with (i) a new means of distributing lipid vesicles on surface based array formats, using site-selective and sequence-specific sorting of DNA-modified vesicles on DNA arrays (Paper I to III; this is the major part of the thesis) and (ii) a new platform for electrochemical impedance spectroscopy (EIS) studies of supported membranes that span nanoscale holes in thin (~50 nm) transmission electron microscopy (TEM) windows, designed to be compatible with studies of single ion-channel processes (Paper V and Progress Report in Chapter 6). The concept used to modify lipid vesicles with DNA was based on self incorporating cholesterol-modified DNA. Significant efforts were put on increasing the strength of this coupling (Paper III), and a new method to quantify the DNA density on vesicles has been developed (Paper IV). In addition, as a precursor step towards nano-aperture spanning lipid membranes, an investigation was made of the influence on lateral lipid diffusivity in supported lipid membranes formed on substrates with nanoscale pits (Paper V). The lipid-based surface-modification protocols developed within this work have in common that they are compatible with studies of different types of membrane residing proteins on a single chip, making it likely that the concepts developed will contribute significantly to the field of array-based detection of membrane protein function. The work on nano-holes and hole-spanning lipid membranes has created a valuable platform towards the goal of studying single trans-membrane proteins, specifically ion channels

Formation of pit-spanning phospholipid bilayers on nanostructured silicon dioxide surfaces for studying biological membrane events

Zwitterionic phospholipid vesicles are known to adsorb and ultimately rupture on flat silicon dioxide (SiO 2 ) surfaces to form supported lipid bilayers. Surface topography, however, alters the kinetics and mechanistic details of vesicles adsorption, which under certain conditions may be exploited to form a suspended bilayer. Here we describe the use of nanostructured SiO 2 surfaces prepared by the colloidal lithography technique to scrutinize the formation of suspended 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) lipid bilayers from a solution of small unilamellar lipid vesicles (SUV s ). Atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) were employed to characterize nanostructure fabrication and lipid bilayer assembly on the surface. \ua9 2013 Springer Science+Business Media New York

Bivalent Cholesterol-based coupling of oligonucleotides to lipid membrane assemblies

By mimicking Nature\u27s way of utilizing multivalent interactions, we introduce in the present work a novel method to improve the strength of cholesterol-based DNA coupling to lipid membranes. The bivalent coupling of DNA was accomplished by hybridization between a 15-mer DNA and a 30-mer DNA, being modified with cholesterol in the 3′ and 5′ end, respectively. Compared with DNA modified with one cholesterol moiety only, the binding strength to lipid membranes appears to be significantly stronger and even irreversible over the time scale investigated (∼1 hr). First, this means that the bivalent coupling can be used to precisely control the number of DNA per lipid-membrane area. Second, the strong coupling is demonstrated to facilitate DNA-hybridization kinetics studies. Third, exchange of DNA between differently DNA-modified vesicles was demonstrated to be significantly reduced. The latter condition was verified via site-selective and sequence-specific sorting of differently DNA-modified lipid vesicles on a low-density cDNA array. This means of spatially control the location of different types of lipid vesicles is likely to find important applications in relation to the rapid progress currently made in the protein chip technology and the emerging need for efficient ways to develop membrane protein arrays

Bivalent Cholesterol-based coupling of oligonucleotides to lipid membrane assemblies

By mimicking Nature\u27s way of utilizing multivalent interactions, we introduce in the present work a novel method to improve the strength of cholesterol-based DNA coupling to lipid membranes. The bivalent coupling of DNA was accomplished by hybridization between a 15-mer DNA and a 30-mer DNA, being modified with cholesterol in the 3′ and 5′ end, respectively. Compared with DNA modified with one cholesterol moiety only, the binding strength to lipid membranes appears to be significantly stronger and even irreversible over the time scale investigated (∼1 hr). First, this means that the bivalent coupling can be used to precisely control the number of DNA per lipid-membrane area. Second, the strong coupling is demonstrated to facilitate DNA-hybridization kinetics studies. Third, exchange of DNA between differently DNA-modified vesicles was demonstrated to be significantly reduced. The latter condition was verified via site-selective and sequence-specific sorting of differently DNA-modified lipid vesicles on a low-density cDNA array. This means of spatially control the location of different types of lipid vesicles is likely to find important applications in relation to the rapid progress currently made in the protein chip technology and the emerging need for efficient ways to develop membrane protein arrays