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| 2. |
- Pfeiffer, Indriati, 1974, et al.
(författare)
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Bivalent Cholesterol-based coupling of oligonucleotides to lipid membrane assemblies
- 2004
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 126:33, s. 10224-10225
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Tidskriftsartikel (refereegranskat)abstract
- By mimicking Nature's 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.
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| 3. |
- Pfeiffer, Indriati, 1974, et al.
(författare)
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Formation of pit-spanning phospholipid bilayers on nanostructured silicon dioxide surfaces for studying biological membrane events
- 2013
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029 .- 1064-3745. - 9781627033350 ; 991, s. 113-125
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- 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. © 2013 Springer Science+Business Media New York.
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| 5. |
- Pfeiffer, Indriati, 1974
(författare)
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Lipid-Based Surface Modifications: towards the Development of Membrane-Protein Arrays
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 6. |
- Pfeiffer, Indriati, 1974, et al.
(författare)
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Quantification of Oligonucleotide Modifications of Small Unilamellar Lipid Vesicles
- 2006
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 78:21, s. 7493-7498
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Tidskriftsartikel (refereegranskat)abstract
- We present a new method for quantification of the coupling efficiency between amphiphilic oligonucleotides and suspended small unilamellar lipid vesicles (SUVs). The method employs a supported (phospho) lipid bilayer (SLB)-modified sensor template, which upon exposure to a mixture of SUVs and amphiphilic DNA reacts neither with free SUVs nor with DNA-modified SUVs, but with free DNA only. Using calibration curves obtained by recording the concentration dependence of the initial binding rate of free amphiphilic DNA (in the absence of SUVs), it is demonstrated how concentration determinations of both free and bound DNA in the two-component mixture (amphiphilic DNA and lipid vesicles) can be obtained. The calibration curves and the binding analysis were obtained using a quartz crystal microbalance with dissipation (QCM-D) monitoring. The binding efficiency of DNA coupled to SUVs (theta similar to 50 nm) with two cholesterol moieties revealed that the bivalent coupling is essentially 100% in the range of similar to 1 to similar to 35 oligonucleotides per vesicle, whereas reversible coupling was confirmed in the case of monovalent coupling. Coupling of DNA via two cholesterol moieties was obtained by prehybridization of two single-stranded DNA strands modified with single cholesterol moieties in their 3' and 5' ends, respectively, and the monovalent coupling was obtained using single-stranded DNA. In the latter case, the analysis of the amount of free DNA at different DNA-SUV ratios also allowed for a determination of the maximum number of available binding sites on the SUVs, shown to be in good agreement with data obtained for DNA coupling on planar surfaces. With the only requirement that the SLB-modified sensor template react with one of the components in the two-component mixture only, as verified through fingerprint analysis of frequency, f, and energy dissipation, D, QCM-D measurements, it is emphasized that the method is generic and offers a fast and reliable method for evaluations of biomolecular modifications of any type of colloidal nanoparticles.
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| 7. |
- Pfeiffer, Indriati, 1974, et al.
(författare)
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Vesicle Adsorption and Phospholipid Bilayer Formation on Topographically and Chemically Nanostructured Surfaces
- 2010
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 114:13, s. 4623-4631
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the influence of combined nanoscale topography and surface chemistry on lipid vesicle adsorption and supported bilayer formation on well-controlled model surfaces. To this end, we utilized colloidal lithography to nanofabricate pitted Au-SiO2 surfaces, where the top surface and the walls of the pits consisted of silicon dioxide whereas the bottom of the pits was made of gold. The diameter and height of the pits were fixed at 107 and 25 nm, respectively. Using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique and atomic force microscopy (AFM), we monitored the processes occurring upon exposure of these nanostructured surfaces to a solution of extruded unilamellar 1-palmitolyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles with a nominal diameter of 100 nm. To scrutinize the influence of surface chemistry, we studied two cases: (1) the bare gold surface at the bottom of the pits and (2) the gold passivated by biotinamidocaproyl-labeled bovine serum albumin (BBSA) prior to vesicle exposure. As in our previous work on pitted silicon dioxide surfaces, we found that the pit edges promote bilayer formation on the SiO2 surface for the vesicle size used here in both cases. Whereas in the first case we observed a slow, continuous adsorption of intact vesicles onto the gold surface at the bottom of the pits, the presence of BBSA in the second case prevented the adsorption of intact vesicles into the pits. Instead, our experimental results, together with free energy calculations for various potential membrane configurations, indicate the formation of a continuous, supported lipid bilayer that spans across the pits. These results are significantly important for various biotechnology applications utilizing patterned lipid bilayers and highlight the power of the combined QCM-D/AFM approach to study the mechanism of lipid bilayer formation on nanostructured surfaces.
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| 8. |
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