| 1. |
- Mapar, M., et al.
(författare)
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A comparative study of the mini-punch grafting and hair follicle transplantation in the treatment of refractory and stable vitiligo
- 2014
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Ingår i: Journal of the American Academy of Dermatology. - : Elsevier BV. - 1097-6787 .- 0190-9622. ; 70:4, s. 743-747
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Tidskriftsartikel (refereegranskat)abstract
- Background: Some vitiligo lesions are resistant to all medical treatments. Objective: We sought to compare the efficacy of hair follicle transplantation and mini-punch grafting for the treatment of refractory vitiligo lesions. Methods: A total of 25 patients with stable and resistant vitiligo participated in the study. In each patient, a resistant vitiligo patch was divided into 2 equal parts. One part was treated with hair follicle transplantation and the other part with mini-punch grafting. Postsurgically, the recipient areas were exposed to narrowband ultraviolet B twice a week for 6 months. The diameter of the repigmentation around each graft was measured monthly. Results: At the end of the sixth month, 68% of follicle grafts, and 72% of mini-punch grafts, had repigmentation. The mean diameter of repigmentation around follicle grafts was 5 +/- 1.7 mm and around punch grafts was 5.3 +/- 1.6 mm. There was no significant difference between the 2 groups statistically (P = .18). Limitations: Small sample size and short time of follow-up are limitations. Conclusions: Because the results of the 2 methods are not statistically different and mini-punch grafting is much easier to do than follicular transplantation, we recommend mini-punch grafting to treat drug-resistant vitiligo.
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| 2. |
- Agnarsson, Björn, 1977, et al.
(författare)
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Evanescent Light-Scattering Microscopy for Label-Free Interfacial Imaging: From Single Sub-100 nm Vesicles to Live Cells
- 2015
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 9:12, s. 11849-11862
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Tidskriftsartikel (refereegranskat)abstract
- Advancement in the understanding of biomolecular interactions has benefited greatly from the development of surface-sensitive bioanalytical sensors. To further increase their broad impact, significant efforts are presently being made to enable label-free and specific biomolecule detection with high sensitivity, allowing for quantitative interpretation and general applicability at low cost. In this work, we have addressed this challenge by developing a waveguide chip consisting of a flat silica core embedded in a symmetric organic cladding with a refractive index matching that of water. This is shown to reduce stray light (background) scattering and thereby allow for label-free detection of faint objects, such as individual sub-20 rim gold nanoparticles as well as sub-100 nm lipid vesicles. Measurements and theoretical analysis revealed that light-scattering signals originating from single surface-bound lipid vesicles enable characterization of their sizes without employing fluorescent lipids as labels. The concept is also demonstrated for label-free measurements of protein binding to and enzymatic (phospholipase A2) digestion of individual lipid vesicles, enabling an analysis of the influence on the measured kinetics of the dye-labeling of lipids required in previous assays. Further, diffraction-limited imaging of cells (platelets) binding to a silica surface showed that distinct subcellular features could be visualized and temporally resolved during attachment, activation, and spreading. Taken together, these results underscore the versatility and general applicability of the method, which due to its simplicity and compatibility with conventional microscopy setups may reach a widespread in life science and beyond.
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| 3. |
- Agnarsson, Björn, 1977, et al.
(författare)
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Low-temperature fabrication and characterization of a symmetric hybrid organic–inorganic slab waveguide for evanescent light microscopy
- 2018
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Ingår i: Nano Futures. - : IOP Publishing. - 2399-1984. ; 2:2
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Tidskriftsartikel (refereegranskat)abstract
- Organic and inorganic solid materials form the building blocks for most of today's high-technological instruments and devices. However, challenges related to dissimilar material properties have hampered the synthesis of thin-film devices comprised of both organic and inorganic films. We here give a detailed description of a carefully optimized processing protocol used for the construction of a three-layered hybrid organic–inorganic waveguide-chip intended for combined scattering and fluorescence evanescent-wave microscopy in aqueous environments using conventional upright microscopes. An inorganic core layer (SiO2 or Si3N4), embedded symmetrically in an organic cladding layer (CYTOP), aids simple, yet efficient in-coupling of light, and since the organic cladding layer is refractive index matched to water, low stray-light (background) scattering of the propagating light is ensured. Another major advantage is that the inorganic core layer makes the chip compatible with multiple well-established surface functionalization schemes that allows for a broad range of applications, including detection of single lipid vesicles, metallic nanoparticles or cells in complex environments, either label-free—by direct detection of scattered light—or by use of fluorescence excitation and emission. Herein, focus is put on a detailed description of the fabrication of the waveguide-chip, together with a fundamental characterization of its optical properties and performance, particularly in comparison with conventional epi illumination. Quantitative analysis of images obtained from both fluorescence and scattering intensities from surface-immobilized polystyrene nanoparticles in suspensions of different concentrations, revealed enhanced signal-to-noise and signal-to-background ratios for the waveguide illumination compared to the epi-illumination.
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| 4. |
- Aliakbarinodehi, Nima, 1986, et al.
(författare)
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Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion
- 2022
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 16:12, s. 20163-20173
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Tidskriftsartikel (refereegranskat)abstract
- Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.
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| 5. |
- Dahlin, Andreas, 1980, et al.
(författare)
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Plasmonic Nanopores in Metal-Insulator-Metal Films
- 2014
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Ingår i: Advanced Optical Materials. - : Wiley. - 2195-1071. ; 2:6, s. 556-564
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Tidskriftsartikel (refereegranskat)abstract
- A novel type of plasmonic nanopore array in a metal-insulator-metal thin film is presented. The optical properties of this structure are described using a generic theoretical framework for surface waves in a coupled multilayer system. The characteristic spacing (short-range order) of the pores enables grating-type coupling to hybridized surface plasmons, with stronger coupling to some modes than others. The nature of the optical resonances and their excitation mechanisms can be conceptually understood from a charge distribution argument. The experimental results are further verified by numerical simulations, which also enable visualization of the near field. This study illustrates the surface plasmon characteristics (sensitive to periodicity) of the extinction maximum in the asymmetric spectral resonance induced by aperture arrays, while the transmission maximum corresponds to a resonance of localized character (sensitive to pore shape). Finally, the use of these nanopores for sensing applications through changes in the refractive index is evaluated.
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| 6. |
- Jõemetsa, Silver, 1990, et al.
(författare)
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Independent Size and Fluorescence Emission Determination of Individual Biological Nanoparticles Reveals that Lipophilic Dye Incorporation Does Not Scale with Particle Size
- 2020
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 36:33, s. 9693-9700
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Tidskriftsartikel (refereegranskat)abstract
- Advancements in nanoparticle characterization techniques are critical for improving the understanding of how biological nanoparticles (BNPs) contribute to different cellular processes, such as cellular communication, viral infection, as well as various drug-delivery applications. Since BNPs are intrinsically heterogeneous, there is a need for characterization methods that are capable of providing information about multiple parameters simultaneously, preferably at the single-nanoparticle level. In this work, fluorescence microscopy was combined with surface-based two-dimensional flow nanometry, allowing for simultaneous and independent determination of size and fluorescence emission of individual BNPs. In this way, the dependence of the fluorescence emission of the commonly used self-inserting lipophilic dye 3,3′-dioctadecyl-5,5′-di(4-sulfophenyl)oxacarbocyanine (SP-DiO) could successfully be correlated with nanoparticle size for different types of BNPs, including synthetic lipid vesicles, lipid vesicles derived from cellular membrane extracts, and extracellular vesicles derived from human SH-SY5Y cell cultures; all vesicles had a radius, r, of ∼50 nm and similar size distributions. The results demonstrate that the dependence of fluorescence emission of SP-DiO on nanoparticle size varies significantly between the different types of BNPs, with the expected dependence on membrane area, r2, being observed for synthetic lipid vesicles, while a significant weaker dependence on size was observed for BNPs with more complex composition. The latter observation is attributed to a size-dependent difference in membrane composition, which may influence either the optical properties of the dye and/or the insertion efficiency, indicating that the fluorescence emission of this type of self-inserting dye may not be reliable for determining size or size distribution of BNPs with complex lipid compositions.
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| 7. |
- Mapar, Mokhtar, 1983
(författare)
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Fabrication, development and characterization of a waveguide microscopy device for biological applications
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Our understanding of biological systems has advanced significantly thanks to the development of microscopy methods and suitable biological assays. Surface based methods have increasingly gained interest thanks to their superior sensitivity and ease of use. Still, most of them, such as quartz crystal microbalance (QCM) and surface plasmon resonance (SPR), provide information based on ensemble averaging of biomolecular interactions. In contrast, surface-sensitive microscopy methods provide the possibility to study biological processes on an individual biomolecular basis. Total internal reflection fluorescent (TIRF) microscopy is a surface-sensitive microscopy method that reaches sensitivities down to the level of single molecules, but it is limited by the need to fluorescently label the interaction partners. On the contrary, waveguide-based evanescent-light scattering microscopy provides a label-free surface-sensitive imaging technique that can also benefit from fluorescent labeling if desired. Here we present ongoing efforts to further develop a waveguide-based platform for evanescent light-scattering microscopy. By adopting the fabrication and processing steps to a transparent substrate and adding compatible microfluidics, we can now use high NA oil-immersion objectives thereby collecting more light and resolve details that could not be resolved when fabricated on an opaque substrate. Further, well-controlled microfluidic handling makes it possible to perform more complicated experiments and extract data on interaction dynamics. Moreover an image processing code has been developed that can be applied to the different experimental sequences, including both waveguide fluorescent and scattering modes, and thereby extract otherwise hidden information in the experimental data.
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| 8. |
- Mapar, Mokhtar, 1983, et al.
(författare)
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Label-free quantification of protein binding to lipid vesicles using transparent waveguide evanescent-field scattering microscopy with liquid control
- 2023
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Ingår i: Biomedical Optics Express. - 2156-7085. ; 14:8, s. 4003-4016
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Tidskriftsartikel (refereegranskat)abstract
- Recent innovations in microscopy techniques are paving the way for label-free studies of single nanoscopic biological entities such as viruses, lipid-nanoparticle drug carriers, and even proteins. One such technique is waveguide evanescent-field microscopy, which offers a relatively simple, yet sensitive, way of achieving label-free light scattering-based imaging of nanoparticles on surfaces. Herein, we extend the application of this technique by incorporating microfluidic liquid control and adapting the design for use with inverted microscopes by fabricating a waveguide on a transparent substrate. We furthermore formulate analytical models describing scattering and fluorescence intensities from single spherical and shell-like objects interacting with evanescent fields. The models are then applied to analyze scattering and fluorescence intensities from adsorbed polystyrene beads and to temporally resolve cholera-toxin B (CTB) binding to individual surface-immobilized glycosphingolipid GM1 containing vesicles. We also propose a self-consistent means to quantify the thickness of the CTB layer, revealing that protein-binding to individual vesicles can be characterized with sub-nm precision in a time-resolved manner.
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| 9. |
- Mapar, Mokhtar, 1983, et al.
(författare)
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Spatiotemporal Kinetics of Supported Lipid Bilayer Formation on Glass via Vesicle Adsorption and Rupture
- 2018
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Ingår i: Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 9:17, s. 5143-5149
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Tidskriftsartikel (refereegranskat)abstract
- Supported lipid bilayers (SLBs) represent one of the most popular mimics of the cell membrane. Herein, we have used total internal reflection fluorescence microscopy for in-depth characterization of the vesicle-mediated SLB formation mechanism on a common silica-rich substrate, borosilicate glass. Fluorescently labeling a subset of vesicles allowed us to monitor the adsorption of individual labeled vesicles, resolve the onset of SLB formation from small seeds of SLB patches, and track their growth via SLB-edge-induced autocatalytic rupture of adsorbed vesicles. This made it possible to perform the first quantitative measurement of the SLB front velocity, which is shown to increase up to 1 order of magnitude with time. This effect can be classified as dramatic because in many other physical, chemical, or biological kinetic processes the front velocity is either constant or decreasing with time. The observation was successfully described with a theoretical model and Monte Carlo simulations implying rapid local diffusion of lipids upon vesicle rupture.
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| 10. |
- Mapar, Mokhtar, 1983
(författare)
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Waveguide Evanescent-Field Microscopy for Label-Free Monitoring of Biological Nanoparticles: Fabrication, Characterization and Application
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The recent development of microscopy methods, biological assays and bioanalytical sensors has significantly advanced the understanding of biological systems. Surface-based bioanalytical sensors have in recent years gained increased interest thanks to improvements in sensitivity and simplicity to use. However, most of them, such as quartz crystal microbalance (QCM) and surface plasmon resonance (SPR), provide information based on ensemble averaging of biomolecular interactions. In contrast, with surface-sensitive microscopy methods, biological processes can be resolved down to the level of individual molecular interactions. Total internal reflection fluorescent microscopy is one commonly used surface-sensitive method, reaching sensitivities down to the level of single molecules, but it requires fluorescent labeling of at least one of the interaction partners and is often also hampered by photo bleaching processes. In this thesis, we introduce a new wide-field surface-sensitive microscopy platform, based on a nanofabricated planar optical waveguide design that is capable of label-free evanescent-field microscopy of biological nanoparticles well below 100 nm in diameter. The waveguide generates an evanescent-field at the interface between the core of the waveguide and an aqueous solution, providing a thin sheet of illumination that offers imaging with low background disturbance. The device is presented in two designs, being compatible with either upright or inverted microscopes. The work presented demonstrates how simultaneous monitoring of fluorescence and scattering signals can offer new information about the relation between scattering intensity, refractive index and lipid content of biological nanoparticles, such as exosomes. Further, the microfluidic design allowed not only for convenient liquid handling with dead volumes of a few microliter, it is also showed to aid label-free investigations of the interaction between proteins and individual lipid vesicles, with the latter serving as cell-membrane mimic. With the device also being compatible with formation of fluid supported lipid bilayers, preliminary results suggest that the design will open up a possibility to simultaneously determine the size, scattering intensity and fluorescence emission at the level of individual biological nanoparticles. With this realized, we foresee a broad applicability of the microscopy platform as multidimensional characterization tool for biological nanoparticles and beyond.
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| 11. |
- Ranjbari, Elias, et al.
(författare)
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Direct Measurement of Total Vesicular Catecholamine Content with Electrochemical Microwell Arrays
- 2020
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:16, s. 11325-11331
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Tidskriftsartikel (refereegranskat)abstract
- We have designed and fabricated a microwell array chip (MWAC) to trap and detect the entire content of individual vesicles after disruption of the vesicular membrane by an applied electrical potential. To understand the mechanism of vesicle impact electrochemical cytometry (VIEC) in microwells, we simulated the rupture of the vesicles and subsequent diffusion of entrapped analytes. Two possibilities were tested: (i) the vesicle opens toward the electrode, and (ii) the vesicle opens away from the electrode. These two possibilities were simulated in the different microwells with varied depth and width. Experimental VIEC measurements of the number of molecules for each vesicle in the MWAC were compared to VIEC on a gold microdisk electrode as a control, and the quantified catecholamines between these two techniques was the same. We observed a prespike foot in a significant number of events (similar to 20%) and argue this supports the hypothesis that the vesicles rupture toward the electrode surface with a more complex mechanism including the formation of a stable pore intermediate. This study not only confirms that in standard VIEC experiments the whole content of the vesicle is oxidized and quantified at the surface of the microdisk electrode but actively verifies that the adsorbed vesicle on the surface of the electrode forms a pore in the vicinity of the electrode rather than away from it. The fabricated MWAC promotes our ability to quantify the content of vesicles accurately, which is fundamentally important in bioanalysis of the vesicles.
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| 12. |
- Rupert, Deborah, 1986, et al.
(författare)
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Effective Refractive Index and Lipid Content of Extracellular Vesicles Revealed Using Optical Waveguide Scattering and Fluorescence Microscopy
- 2018
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 34:29, s. 8522-8531
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) are generating a growing interest because of the key roles they play in various biological processes and because of their potential use as biomarkers in clinical diagnostics and as efficient carriers in drug-delivery and gene-therapy applications. Their full exploitation, however, depends critically on the possibility to classify them into different subpopulations, a task that in turn relies on efficient means to identify their unique biomolecular and physical signatures. Because of the large heterogeneity of EV samples, such information remains rather elusive, and there is accordingly a need for new and complementary characterization schemes that can help expand the library of distinct EV features. In this work, we used surface-sensitive waveguide scattering microscopy with single EV resolution to characterize two subsets of similarly sized EVs that were preseparated based on their difference in buoyant density. Unexpectedly, the scattering intensity distribution revealed that the scattering intensity of the high-density (HD) population was on an average a factor of three lower than that of the low-density (LD) population. By further labeling the EV samples with a self-inserting lipid-membrane dye, the scattering and fluorescence intensities from EVs could be simultaneously measured and correlated at the single-particle level. The labeled HD sample exhibited not only lower fluorescence and scattering intensities but also lower effective refractive index (n approximate to 1.35) compared with the LD EVs (n approximate to 1.38), indicating that both the lipid and protein contents were indeed lower in the HD EVs. Although separation in density gradients of similarly sized EVs is usually linked to differences in biomolecular content, we suggest based on these observations that the separation rather reflects the ability of the solute of the gradient to penetrate the lipid membrane enclosing the EVs, that is, the two gradient bands are more likely because of the differences in membrane permeability than to differences in biomolecular content of the EVs.
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| 13. |
- Saavedra, Valeria, 1985, et al.
(författare)
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Extending charge separation lifetime and distance in patterned dye-sensitized SnO2–TiO2 µm-thin films
- 2017
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Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 19:34, s. 22684-22690
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Tidskriftsartikel (refereegranskat)abstract
- A simple method for the preparation of patterned dye-sensitized SnO2–TiO2 thin films, designed to prolongthe lifetime of the interfacial charge separated state is presented. Using microfluidic technology,the thin films were sensitized with the organic sensitizer D35 such that they contain SnO2–TiO2 areaswith dye and SnO2 dye-free areas at which injected electrons can be accumulated. Single wavelengthtransient absorption spectroscopy confirmed significantly extended charge separation lifetime at thedye-semiconductor interface. Sufficiently high density of injected electrons results in substantialdecrease of charge recombination rate constants (kcr); a factor of B50 compared to dye-sensitized TiO2thin films and a factor of B2000 compared to dye-sensitized SnO2 thin films. Furthermore, the potentialof this approach was confirmed by photoinduced conduction band mediated electron transfer from thedye to a model electron acceptor, Co protoporphyrin IX, which was adsorbed to the SnO2-only regions.
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| 14. |
- Sjöberg, Mattias, 1991, et al.
(författare)
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Time-Resolved and Label-Free Evanescent Light-Scattering Microscopy for Mass Quantification of Protein Binding to Single Lipid Vesicles
- 2021
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 21:11, s. 4622-4628
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Tidskriftsartikel (refereegranskat)abstract
- In-depth understanding of the intricate interactions between biomolecules and nanoparticles is hampered by a lack of analytical methods providing quantitative information about binding kinetics. Herein, we demonstrate how label-free evanescent light-scattering microscopy can be used to temporally resolve specific protein binding to individual surface-bound (∼100 nm) lipid vesicles. A theoretical model is proposed that translates protein-induced changes in light-scattering intensity into bound mass. Since the analysis is centered on individual lipid vesicles, the signal from nonspecific protein binding to the surrounding surface is completely avoided, offering a key advantage over conventional surface-based techniques. Further, by averaging the intensities from less than 2000 lipid vesicles, the sensitivity is shown to increase by orders of magnitude. Taken together, these features provide a new avenue in studies of protein-nanoparticle interaction, in general, and specifically in the context of nanoparticles in medical diagnostics and drug delivery.
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