| 1. |
- T. Tegler, Lotta, et al.
(författare)
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A highly selective polypeptide binder for human Acetylcholine esterase
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A highly selective high-affinity polypeptide conjugate binder for human Acetylcholine Esterase (hAChE) has been obtained by coupling a derivative of acridine, a known medium-affinity inhibitor of hAChE, to each member of a 16-membered set of 42-residue polypeptide scaffolds. The best candidate, 4-C10L17-Ac, was identified to have a KD of 10 nM or less in an assay where each polypeptide conjugate was titrated with hAChE in 50 mM phosphate buffer at pH 7.0 and 298K. It was found in a sandwich ELISA to have high selectivity for hAChE in cerebrospinal fluid. Targeting the active site of hAChE by a polypeptide conjugate binder presents a special problem as it is buried deep inside the protein in a cavity that is approximately 20 Å deep. In order to permit simultaneous and cooperative binding of the acridine and the polypeptide to the active site and the AChE surface a fourteen atom spacer was needed. The 9-aminoacridine group was linked to the side chain of a lysine residue in each polypeptide via a spacer prepared from two aminohexanoic acid fragments. The results reinforce the impression that polypeptide conjugates are excellent alternatives to currently used protein binder technologies in diagnostic and therapeutic applications and that the conjugation of enzyme inhibitors to polypeptide scaffolds is a strategy of general applicability in the design of high-affinity binders for enzymes.
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| 2. |
- Andersson, Per Ola, et al.
(författare)
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A Novel ATR-FTIR Approach for Characterisation and Identification of Ex Situ Immobilised Species
- 2007
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Ingår i: ChemPhysChem. - : Wiley. - 1439-4235 .- 1439-7641. ; 8:5, s. 712-722
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a novel method to analyse ex situ prepared protein chips by attenuated total reflection Fourier IR spectroscopy (ATR-FTIR), which circumvents tedious functionalisation steps of internal reflection elements (IREs), and simultaneously allows for complementary measurements by other analytical techniques. This concept is proven by utilising immobilised metal affinity capture (IMACTM) chips containing about 10 m thick films of copolymers coated with nitrilotriacetic acid (NTA) groups, which originally was manufactured for surface enhanced laser desorption ionisation (SELDI) spectrometry. Three immobilisation steps were analysed by ATR-FTIR spectroscopy: 1) NTA complexation with nickel(II) ions 2) binding of two histidine (His)-tagged synthetic peptides of 25 (25-His6) and 48 (48-His6) amino acids to the NTA-groups and 3) attachment of a ligand, mesyl amide, to the surface-bound 48-His6. Despite interference from H2O, both amide I and II were well resolved. Utilising peptide adsorption in the thick copolymer matrix yields a high saturation peptide concentration of 100 mg mL-1 and a dissociation constant of 116±11 M, as determined by a detailed analysis of the Langmuir adsorption isotherm. The mesyl amide ligand was directly seen in the raw ATR-FTIR spectrum with specific peaks in the fingerprint region at 1172 and 1350 cm-1. Several aspects of the fine structure of the amide I band of the peptide were analysed: influences from secondary structure, amino side chains and competing contamination product. We believe that this approach has great potential as a stand-alone or complementary analytical tool for determination of the chemical composition of functionalised surfaces. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and applied in other experimental set-ups.
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| 3. |
- Ausen, Dag, et al.
(författare)
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Foresight Biomedical Sensors
- 2007
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- The foresight study on biomedical sensors has addressed different approaches with future use of biomedical sensors in the health care sector, like: How will biomedical sensors shape the healthcare systems of the future? How can they impact the quality and cost of healthcare and what are the business opportunities in the Nordic region?
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| 4. |
- Cai, Yixiao, et al.
(författare)
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Changes in secondary structure of alpha-synuclein during oligomerization induced by reactive aldehydes
- 2015
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 464:1, s. 336-341
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Tidskriftsartikel (refereegranskat)abstract
- The oxidative stress-related reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) have been shown to promote formation of alpha-synuclein oligomers in vitro. However, the changes in secondary structure of alpha-synuclein and the kinetics of the oligomerization process are not known and were the focus of this study. Size exclusion chromatography showed that after 1 h of incubation, HNE induced the formation of an oligomeric alpha-synuclein peak with a molecular weight of about similar to 2000 kDa, which coincided with a decreasing similar to 50 kDa monomeric peak. With prolonged incubation (up to 24 h) the oligomeric peak became the dominating molecular species. In contrast, in the presence of ONE, a similar to 2000 oligomeric peak was exclusively observed after 15 min of incubation and this peak remained constant with prolonged incubation. Western blot analysis of HNE-induced alpha-synuclein oligomers showed the presence of monomers (15 kDa), SDS-resistant low molecular (30-160 kDa) and high molecular weight oligomers (>= 260 kDa), indicating that the oligomers consisted of both covalent and non-covalent protein. In contrast, ONE-induced alpha-synuclein oligomers only migrated as covalent cross-linked high molecular-weight material (>= 300 kDa). Both circular dichroism (CD) and Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy showed that the formation of HNE- and ONE-induced oligomers coincided with a spectral change from random coil to beta-sheet. However, ONE-induced alpha-synuclein oligomers exhibited a slightly higher degree of beta-sheet. Taken together, our results indicate that both HNE and ONE induce a change from random coil to beta-sheet structure that coincides with the formation of alpha-synuclein oligomers; albeit through different kinetic pathways depending on the degree of cross-linking. (C) 2015 Elsevier Inc. All rights reserved.
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| 5. |
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| 6. |
- Aghoutane, Youssra, et al.
(författare)
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Development of a molecularly imprinted polymer electrochemical sensor and its application for sensitive detection and determination of malathion in olive fruits and oils
- 2020
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Ingår i: Bioelectrochemistry. - : Elsevier. - 1567-5394 .- 1878-562X. ; 132
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Tidskriftsartikel (refereegranskat)abstract
- Malathion (MAL) is an organophosphorus (OP) insecticide. It is a cholinesterase inhibitor, 15 which can pose serious health and environmental problems. In this study, a sensitive and 16 selective molecular imprinted polymer (MIP) based on screen-printed gold electrodes (Au-17 SPE) for MAL detection in olive oils and fruits, was devised. The MIP sensor was prepared 18 using acrylamide as the functional monomer and MAL as the template. Subsequently, the 19 morphology of the electrode surface was studied by scanning electron microscopy (SEM) and 20 atomic force microscopy (AFM). The electrochemical characterization of the developed MIP 21 sensor was performed by cyclic voltammetry (CV), differential pulse voltammetry (DPV), 22 and electrochemical impedance spectroscopy (EIS) techniques. The operational repeatability 23 and stability of the sensor were studied. It was found to have a dynamic concentration range 24 of (0.1 pg mL-1-1000 pg mL-1) and a low limit of detection (LOD) of 0.06 pg mL-1. 25 Furthermore, the sensor was employed to determine MAL content in olive oil with a recovery 26 rate of 87.9% and a relative standard deviation of 8%. It was successfully applied for MAL 27 determination in real samples and promise to open new opportunities for the detection of OP 28 pesticides residues in various food products, as well as in environmental applications.
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| 7. |
- Ahlinder, Linnea, et al.
(författare)
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Graphene oxide nanoparticle attachment and its toxicity on living lung epithelial cells
- 2015
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Ingår i: RSC Advances. - : Royal Society of Chemistry. - 2046-2069. ; 5:73, s. 59447-59457
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Tidskriftsartikel (refereegranskat)abstract
- Since its discovery, graphene and its oxidized form, graphene oxide (GO), have attracted interest in a wide range of technical applications. Concerns about their potential toxicity calls for scrutinized studies, but hitherto conflicting results have been reported which partly may be due to variations of synthesis and exposure procedures. Here we report on the attachment and toxicity of contamination-free graphene oxide nanoparticles (GONP) in living lung epithelial cells. The synthesis of chemically pure GONP was made by an improvement of the Hummer's method based on graphene exfoliated from graphite using high-intensity ultrasonication, resulting in two dimensional sheets with a lateral dimension in the range 200 nm to 3 mu m and thickness of 0.9 nm. Confocal Raman spectroscopy combined with multivariate analysis was used to study the interaction of GONP and living cells. It is shown that overlapping Raman bands due to GONPs and biomolecules in the cells can clearly be separated with this approach. Orthogonal partial least squares discriminant analysis was used to compare spectral data collected from cells exposed to GONP with spectral data collected from non-exposed control cells, and spectral data from cells exposed to a surfactant known to induce apoptosis. Our analyses show that GONP readily attach to the cells, forming sheets which cover a large fraction of the cell surfaces, and induce small chemical changes. In particular, chemical modifications of proteins and lipids in lung epithelial cells are inferred. GONPs do not, however, decrease cell viability. In contrast, enhanced cell proliferation is observed. Our results shed new light on the interactions of GO, and in contrast to some previous reports, suggest that GO is not toxic. The hyperspectral Raman spectroscopy analysis employed here should be applicable for other fields in nanomedicine as a label-free non-perturbing analytical method.
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| 8. |
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| 9. |
- Ahlinder, Linnea, et al.
(författare)
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Large Uptake of Titania and Iron Oxide Nanoparticles in the Nucleus of Lung Epithelial Cells as Measured by Raman Imaging and Multivariate Classification
- 2013
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 105:2, s. 310-319
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Tidskriftsartikel (refereegranskat)abstract
- It is a challenging task to characterize the biodistribution of nanoparticles in cells and tissue on a subcellular level. Conventional methods to study the interaction of nanoparticles with living cells rely on labeling techniques that either selectively stain the particles or selectively tag them with tracer molecules. In this work, Raman imaging, a label-free technique that requires no extensive sample preparation, was combined with multivariate classification to quantify the spatial distribution of oxide nanoparticles inside living lung epithelial cells (A549). Cells were exposed to TiO2 (titania) and/or alpha-FeO(OH) (goethite) nanoparticles at various incubation times (4 or 48 h). Using multivariate classification of hyperspectral Raman data with partial least-squares discriminant analysis, we show that a surprisingly large fraction of spectra, classified as belonging to the cell nucleus, show Raman bands associated with nanoparticles. Up to 40% of spectra from the cell nucleus show Raman bands associated with nanoparticles. Complementary transmission electron microscopy data for thin cell sections qualitatively support the conclusions.
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| 10. |
- Ahlinder, Linnea, et al.
(författare)
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Noise Removal with Maintained Spatial Resolution in Raman Images of Cells Exposed to Submicron Polystyrene Particles
- 2016
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Ingår i: Nanomaterials. - : MDPI AG. - 2079-4991. ; 6:5
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Tidskriftsartikel (refereegranskat)abstract
- The biodistribution of 300 nm polystyrene particles in A549 lung epithelial cells has been studied with confocal Raman spectroscopy. This is a label-free method in which particles and cells can be imaged without using dyes or fluorescent labels. The main drawback with Raman imaging is the comparatively low spatial resolution, which is aggravated in heterogeneous systems such as biological samples, which in addition often require long measurement times because of their weak Raman signal. Long measurement times may however induce laser-induced damage. In this study we use a super-resolution algorithm with Tikhonov regularization, intended to improve the image quality without demanding an increased number of collected pixels. Images of cells exposed to polystyrene particles have been acquired with two different step lengths, i.e., the distance between pixels, and compared to each other and to corresponding images treated with the super-resolution algorithm. It is shown that the resolution after application of super-resolution algorithms is not significantly improved compared to the theoretical limit for optical microscopy. However, to reduce noise and artefacts in the hyperspectral Raman images while maintaining the spatial resolution, we show that it is advantageous to use short mapping step lengths and super-resolution algorithms with appropriate regularization. The proposed methodology should be generally applicable for Raman imaging of biological samples and other photo-sensitive samples.
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