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  • 1.
    Aili, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Baltzer, Lars
    Division of Organic Chemistry, Department of Biochemistry and Organic Chemistry, BMC, Box 576, Uppsala UniVersity, SE-751 23 Uppsala, Sweden.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Assembly of Polypeptide-Functionalized Gold Nanoparticles through a Heteroassociation- and Folding-Dependent Bridging2008In: Nano letters (Print), ISSN 1530-6984, E-ISSN 1530-6992, Vol. 8, no 8, p. 2473-2478Article in journal (Refereed)
    Abstract [en]

    Gold nanoparticles were functionalized with a synthetic polypeptide, de novo-designed to associate with a charge complementary linker polypeptide in a folding-dependent manner. A heterotrimeric complex that folds into two disulphide-linked four-helix bundles is formed when the linker polypeptide associates with two of the immobilized peptides. The heterotrimer forms in between separate particles and induces a rapid and extensive aggregation with a well-defined interparticle spacing. The aggregated particles are redispersed when the disulphide bridge in the linker polypeptide is reduced.

  • 2.
    Aili, Daniel
    et al.
    Linköping University, The Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, The Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Rydberg, Johan
    Linköping University, The Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Nesterenko, Irina
    Linköping University, The Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Björefors, Fredrik
    Linköping University, The Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Baltzer, Lars
    Division of Organic Chemistry, Department of Biochemistry and Organic Chemistry, BMC, Box 599, Uppsala University, SE-751 24 Uppsala, Sweden..
    Liedberg, Bo
    Linköping University, The Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Controlled Assembly of Gold Nanoparticles using De Novo Designed Polypeptide Scaffolds2008In: Proceedings SPIE, Vol. 6885, Photonic Biosensing and Microoptics, 2008, p. 688506-1-688506-8Conference paper (Refereed)
    Abstract [en]

    Heterodimerization between designed helix-loop-helix polypeptides was utilized in order to assemble gold nanoparticles on planar substrates. The peptides were designed to fold into four-helix bundles upon dimerization. A Cys-residue in the loop region was used to immobilize one of the complementary peptides on a maleimide containing SAM on planar gold substrates whereas the second peptide was immobilized directly on gold nanoparticles. Introducing the peptide decorated particles over a peptide functionalized surface resulted in particle assembly. Further, citrate stabilized particles were assembled on amino-silane modified glass and silicon substrates. By subsequently introducing peptides and gold nanoparticles, particle-peptide hybrid multi layers could be formed.

  • 3.
    Aili, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Rydberg, Johan
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Nesterenko, Irina
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Björefors, Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Baltzer, Lars
    Department of Biochemistry and Organic Chemistry, BMC, Box 599, Uppsala UniVersity, SE-751 24 Uppsala, Sweden.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Folding Induced Assembly of Polypeptide Decorated Gold Nanoparticles2008In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 130, no 17, p. 5780-5788Article in journal (Refereed)
    Abstract [en]

    Reversible assembly of gold nanoparticles controlled by the homodimerization and folding of an immobilized de novo designed synthetic polypeptide is described. In solution at neutral pH, the polypeptide folds into a helix–loop–helix four-helix bundle in the presence of zinc ions. When immobilized on gold nanoparticles, the addition of zinc ions induces dimerization and folding between peptide monomers located on separate particles, resulting in rapid particle aggregation. The particles can be completely redispersed by removal of the zinc ions from the peptide upon addition of EDTA. Calcium ions, which do not induce folding in solution, have no effect on the stability of the peptide decorated particles. The contribution from folding on particle assembly was further determined utilizing a reference peptide with the same primary sequence but containing both D and L amino acids. Particles functionalized with the reference peptide do not aggregate, as the peptides are unable to fold. The two peptides, linked to the nanoparticle surface via a cysteine residue located in the loop region, form submonolayers on planar gold with comparable properties regarding surface density, orientation, and ability to interact with zinc ions. These results demonstrate that nanoparticle assembly can be induced, controlled, and to some extent tuned, by exploiting specific molecular interactions involved in polypeptide folding.

  • 4.
    Aili, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Tai, Feng-I
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Baltzer, Lars
    Department of Biochemistry andOrganic Chemistry Uppsala University, BMC, Box 576, 75123 Uppsala, Sweden.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Self-Assembly of Fibers and Nanorings from Disulfide-Linked Helix–Loop–Helix Polypeptides2008In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 47, no 30, p. 5554-5556Article in journal (Refereed)
  • 5.
    Aili, Daniel
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Enander, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Synthetic de novo designed polypeptides for control of nanoparticle assembly and biosensing2007In: Bionanotechnology; from self-assembly to cellbiology,2007, London: Biochemical Society Transactions , 2007, p. 532-Conference paper (Refereed)
    Abstract [en]

         

  • 6.
    Aili, Daniel
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Enander, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Towards novel functional materials and sensors using de novo designed polypeptides on gold nanoparticles2006In: Europtrode VIII,2006, 2006Conference paper (Other academic)
    Abstract [en]

        

  • 7.
    Aili, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Rydberg, Johan
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics . Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Aggregation-Induced Folding of a de novo Designed Polypeptide Immobilized on Gold Nanoparticles2006In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, no 7, p. 2194 -2195Article in journal (Refereed)
    Abstract [en]

    This communication reports the first steps in the construction of a novel, nanoparticle-based hybrid material for biomimetic and biosensor applications. Gold nanoparticles were modified with synthetic polypeptides to enable control of the particle aggregation state in a switchable manner, and particle aggregation was, in turn, found to induce folding of the immobilized peptides.

  • 8.
    Aili, Daniel
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Enander, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Rydberg, Johan
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Baltzer, Lars
    Uppsala University.
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Alpha helix-inducing dimerization of synthetic polypeptide scaffolds on gold - a model system for receptor mimicking and biosensing2004In: 8th World Congress on Biosensors,2004, 2004Conference paper (Other academic)
  • 9.
    Aili, Daniel
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Enander, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Rydberg, Johan
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Baltzer, Lars
    Uppsala University.
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Folding-induced aggregation of polypeptide-decorated gold nanoparticles - an nano-scale Lego for the construction of complex hybrid materials2004In: 5th International Conference on Biological Physics,2004, 2004Conference paper (Other academic)
  • 10.
    Aili, Daniel
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Enander, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Rydberg, Johan
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Baltzer, Lars
    Uppsala University.
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Immobilization and heterodimerisation of helix-loop-helix polypeptides on gold surfaces - a model system for peptide-surface interactions2003In: 1st World congress on Synthetic Receptors,2003, 2003Conference paper (Other academic)
  • 11.
    Aili, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Selegård, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Baltzer, Lars
    Division of Organic Chemistry, Department of Biochemistry and Organic Chemistry, BMC, Box 576, Uppsala University, SE-751 23 Uppsala, Sweden.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Colorimetric Protein Sensing by Controlled Assembly of Gold Nanoparticles Functionalized with Synthetic Receptors2009In: Small, ISSN 1613-6810, Vol. 5, no 21, p. 2445-2452Article in journal (Refereed)
    Abstract [en]

    A strategy for colorimetric sensing of proteins, based on the induced assembly of polypeptide-functionalized gold nanoparticles, is described. Recognition was accomplished using a polypeptide sensor scaffold designed to specifically bind the model analyte, human carbonic anhydrase II (HCAII). The extent of particle aggregation, induced by the Zn2+-triggered dimerization and folding of a second polypeptide also present on the surface of the gold nanoparticle, gave a readily detectable colorimetric shift that was dependent on the concentration of the target protein. In the absence of HCAII, particle aggregation resulted in a major redshift of the plasmon peak whereas analyte binding prevented formation of dense aggregates, significantly reducing the magnitude of the redshift. The limit of detection of HCAII was estimated to be around 15 nM. The versatility of the technique was demonstrated using a second model system based on the recognition of a peptide sequence from the tobacco mosaic virus coat protein (TMVP by a recombinant antibody fragment. This strategy is proposed as a generic platform for robust and specific protein analysis that can be further developed for monitoring a wide range of target proteins.

  • 12.
    Aili, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Selegård, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Baltzer, Lars
    Uppsala University .
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Colorimetric sensing: Small 21/20092009In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 5, no 21Article in journal (Other academic)
    Abstract [en]

    The cover picture illustrates a novel concept for colorimetric protein sensing based on the controllable assembly of polypeptide-functionalized gold nanoparticles. Recognition of the analyte is accomplished by polypeptide-based synthetic receptors immobilized on gold nanoparticles. Also present on the particle surface is a de novo-designed helix-loop-helix polypeptide that homodimerizes and folds into four-helix bundles in the presence of Zn2+, resulting in particle aggregation. Analyte binding interferes with the folding-induced aggregation, giving rise to a clearly detectable colorimetric response.

  • 13.
    Andersson, Olof
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Nikkinen, Henrik
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Enander, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Towards affinity arrays for the detection of protein analytes2008In: Europtrode IX,2008, 2008Conference paper (Other academic)
  • 14.
    Andersson, Olof
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Nikkinen, Henrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Kanmert, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    A multiple-ligand approach to extending the dynamic range of analyte quantification in protein microarrays2009In: Biosensors and bioelectronics, ISSN 0956-5663, Vol. 24, no 8, p. 2458-2464Article in journal (Refereed)
    Abstract [en]

    This work describes a concept for extending the dynamic range of quantification in an affinity biosensor assay by using a set of ligands with different affinities toward a common analyte. For a demonstration of the principle, three synthetic, biotinylated polypeptides capable of binding a model protein analyte with different affinities (10-9 M ≤ Kd ≤ 10-7 M) were immobilized in a microarray format on a gold slide covered with an oligo(ethylene glycol)-containing alkane thiolate self-assembled monolayer. For controllable immobilization, coupling was mediated by the biotinneutravidin interaction. A five-element affinity array, comprising single-peptide spots as well as spots where peptides were immobilized in mixtures, was realized by means of piezodispensation. Imaging surface plasmon resonance was used to study binding of the analyte to the different spots. The lower limit of quantification was ~3 nM and the corresponding upper limit was increased by more than an order of magnitude compared to if only the highest-affinity ligand would have been used. Affinity array sensors with multiple ligands for each analyte are particularly interesting for omitting dilution steps and providing highly accurate data in assays where several analytes such as disease biomarkers with extremely variable concentrations are quantified in parallel.

  • 15.
    Andersson, Theresa
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Dolphin, Gunnar T.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Nilsson, Jonas W.
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    The binding of human Carbonic Anhydrase II by functionalized folded polypeptide receptors2005In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 12, no 11, p. 1245-1252Article in journal (Refereed)
    Abstract [en]

    Several receptors for human carbonic anhydrase II (HCAII) have been prepared by covalently attaching benzenesulfonamide carboxylates via aliphatic aminocarboxylic acid spacers of variable length to the side chain of a lysine residue in a designed 42 residue helix-loop-helix motif. The sulfonamide group binds to the active site zinc ion of human carbonic anhydrase II located in a 15 Å deep cleft. The dissociation constants of the receptor-HCAII complexes were found to be in the range from low micromolar to better than 20 nM, with the lowest affinities found for spacers with less than five methylene groups and the highest affinity found for the spacer with seven methylene groups. The results suggest that the binding is a cooperative event in which both the sulfonamide residue and the helix-loop-helix motif contribute to the overall affinity.

  • 16.
    Aronsson, Christopher
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Dånmark, Staffan
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Zhou, Feng
    Nanyang Technology University, Singapore.
    Öberg, Per
    Linköping University, Department of Electrical Engineering, Vehicular Systems. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Su, Haibin
    Nanyang Technology University, Singapore.
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Self-sorting heterodimeric coiled coil peptides with defined and tuneable self-assembly properties2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, no 14063Article in journal (Refereed)
    Abstract [en]

    Coiled coils with defined assembly properties and dissociation constants are highly attractive components in synthetic biology and for fabrication of peptide-based hybrid nanomaterials and nanostructures. Complex assemblies based on multiple different peptides typically require orthogonal peptides obtained by negative design. Negative design does not necessarily exclude formation of undesired species and may eventually compromise the stability of the desired coiled coils. This work describe a set of four promiscuous 28-residue de novo designed peptides that heterodimerize and fold into parallel coiled coils. The peptides are non-orthogonal and can form four different heterodimers albeit with large differences in affinities. The peptides display dissociation constants for dimerization spanning from the micromolar to the picomolar range. The significant differences in affinities for dimerization make the peptides prone to thermodynamic social self-sorting as shown by thermal unfolding and fluorescence experiments, and confirmed by simulations. The peptides self-sort with high fidelity to form the two coiled coils with the highest and lowest affinities for heterodimerization. The possibility to exploit self-sorting of mutually complementary peptides could hence be a viable approach to guide the assembly of higher order architectures and a powerful strategy for fabrication of dynamic and tuneable nanostructured materials.

  • 17.
    Carlsson, Jenny
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics . Linköping University, The Institute of Technology.
    Gullstrand, Camilla
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences.
    Westermark, Gunilla
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Ludvigsson, Johnny
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Paediatrics in Linköping.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    An indirect competitive immunoassay for insulin autoantibodies based on surface plasmon resonance2008In: Biosensors and Bioelectronics, ISSN 0956-5663, Vol. 24, no 4, p. 876-881Article in journal (Refereed)
    Abstract [en]

    We have developed a sensitive and specific method based on surface plasmon resonance (SPR) for detection of insulin autoantibodies (IAA) in serum samples from individuals at high risk of developing type 1 diabetes (T1D). When measuring trace molecules in undiluted sera with label-free techniques like SPR, non-specific adsorption of matrix proteins to the sensor surface is often a problem, since it causes a signal that masks the analyte response. The developed method is an indirect competitive immunoassay designed to overcome these problems. Today, IAA is mainly measured in radio immunoassays (RIAs), which are time consuming and require radioactively labeled antigen. With our SPR-based immunoassay the overall assay time is reduced by a factor of >100 (4 days to 50 min), while sensitivity is maintained at a level comparable to that offered by RIA.

  • 18.
    Carlsson, Jenny
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Gullstrand, Camilla
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Westermark, Gunilla
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Ludvigsson, Johnny
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Paediatrics in Linköping.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Determination of insulin autoantibodies using surface plasmon resonance: A screening study of newly diagnosed type 1 diabetes patients2008Manuscript (preprint) (Other academic)
    Abstract [en]

    We have investigated the screening potential of a surface plasmon resonance (SPR)-based indirectcompetitive immunoassay for quantification of insulin autoantibodies (IAA) in sera from childrennewly diagnosed with type 1 diabetes (T1D), using a radioimmunoassay (RIA) as reference technique.The two methods agreed well with respect to sample classification of 54 sera from newly diagnosedT1D children and 32 reference sera from non-diabetic children. Interestingly, five samples from newlydiagnosed T1D patients classified as IAA-negative according to RIA were IAA-positive with the SPRbasedassay, suggesting that the SPR-based assay might provide a higher sensitivity than the referenceRIA. However, 14 percent of the analyzed samples (five samples from non-diabetics and seven fromnewly diagnosed T1D patients) gave rise to anomalously high and easily distinguishable responses withthe SPR-based method, precluding IAA-quantification. A considerable part of the paper is devoted to adiscussion of possible causes of these anomalous responses. They were not due to temporary changesin the status of the patients, such as infections at the time of sampling, and also not related tocomplement activation. It is speculated whether a plausible explanation should instead be sought in theexistence of anti-idiotypic antibodies to IAA.

  • 19.
    Choulier, Laurence
    et al.
    University of Strasbourg.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Environmentally Sensitive Fluorescent Sensors Based on Synthetic Peptides2010In: SENSORS, ISSN 1424-8220, Vol. 10, no 4, p. 3126-3144Article, review/survey (Refereed)
    Abstract [en]

    Biosensors allow the direct detection of molecular analytes, by associating a biological receptor with a transducer able to convert the analyte-receptor recognition event into a measurable signal. We review recent work aimed at developing synthetic fluorescent molecular sensors for a variety of analytes, based on peptidic receptors labeled with environmentally sensitive fluorophores. Fluorescent indicators based on synthetic peptides are highly interesting alternatives to protein-based sensors, since they can be synthesized chemically, are stable, and can be easily modified in a site-specific manner for fluorophore coupling and for immobilization on solid supports.

  • 20.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Folded polypeptide scaffolds for biosensor and biochip applications: design, synthesis, functionalisation and characterisation2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis describes the design, synthesis and evaluation of functional molecular units intended for use in biosensor and microarray applications. A flexible, synthetic helix-loop-helix polypeptide that dimerises to form four-helix bundles was used as a scaffold and was modified with affinity ligands and fluorescent probes to specifically bind a target biomolecule and report on this event in an integrated process. The well-characterised binding of carbonic anhydrase by its benzenesulphonamide inhibitor was employed as a model interaction, and the emission intensity of the probe(s) was found to correlate with carbonic anhydrase concentration. A molecular array, spanning two orders of magnitude in affmity and useful for one-step target quantification, was designed by varying the spacer of the benzenesulphonamide derivative. The scaffold itself was found to contribute to binding, expanding the parameters available for affmity modulation. In a separate study focused on the interaction model system, it was revealed that a destabilising point mutation distant from the carbonic anhydrase active site resulted in faster dissociation rates of the benzenesulphonamide ligand. and that this effect was mediated by increased molecular dynamics caused by destabilisation.

    The fluorescence intensity difference displayed by free and target-bound peptides was found to be critically dependent on the position of the probe(s) in the scaffold, showing that the polypeptide fold, providing directionality of incorporated moieties, contributed considerably to peptide function. Dual labelling of the scaffold with different probes in positions where they displayed increased intensity in the corresponding single-probe peptides resulted in a synergistic emission increase upon target protein binding, significantly enhancing sensitivity. The peptides were shown to bind the target protein as monomers, and the molecular basis for sensing was a combination of specific peptide-protein interactions and dimer dissociation. The photochemical crosstalk between the probes was interrupted upon expulsion of one of the monomers upon binding.

    Strategies for thiol-dependent attachment of the peptides to modified gold surfaces were explored, and folding of immobilised scaffolds was demonstrated in the case of a model system with controllable dirnerisation properties. Results indicating that the sensing ability was retained upon peptide immobilisation were encouraging and prompted future studies on the relation between peptide structure and function, aiming at successful sensor surface and rnicroarray designs for the identification, quantification and characterisation of a wide variety of target biomolecules.

    List of papers
    1. Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor
    Open this publication in new window or tab >>Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor
    Show others...
    2001 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 296, no 2, p. 188-196Article in journal (Refereed) Published
    Abstract [en]

    The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s−1. For the mutants, dissociation rates were faster (0.022–0.025 s−1), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-25364 (URN)10.1006/abio.2001.5301 (DOI)9807 (Local ID)9807 (Archive number)9807 (OAI)
    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13
    2. Designed, folded polypeptide scaffolds that combine key biosensing events of recognition and reporting
    Open this publication in new window or tab >>Designed, folded polypeptide scaffolds that combine key biosensing events of recognition and reporting
    Show others...
    2002 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 67, no 9, p. 3120-3123Article in journal (Refereed) Published
    Abstract [en]

    No abstract available.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-42053 (URN)10.1021/jo010954n (DOI)59995 (Local ID)59995 (Archive number)59995 (OAI)
    Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
    3. A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis
    Open this publication in new window or tab >>A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis
    Show others...
    2004 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 10, no 10, p. 2375-2385Article in journal (Refereed) Published
    Abstract [en]

    A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix–loop–helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02–3 μM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-42052 (URN)10.1002/chem.200305391 (DOI)59994 (Local ID)59994 (Archive number)59994 (OAI)
    Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
    4. Designed, functionalized helix-loop-helix motifs that bind human carbonic anhydrase II: a new class of synthetic receptor molecules
    Open this publication in new window or tab >>Designed, functionalized helix-loop-helix motifs that bind human carbonic anhydrase II: a new class of synthetic receptor molecules
    2004 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 14, p. 4464-4465Article in journal (Refereed) Published
    Abstract [en]

    Polypeptides designed to fold into helix−loop−helix motifs and to dimerize to form four-helix bundles were functionalized by the introduction of a sulfonamide derivative known to bind human carbonic anhydrase II (HCAII) and one or both of the dansyl- and methoxycoumarin fluorescent probes. The 42-residue sequence DC that carries all three substituents in solvent-exposed positions was found to bind HCAII with a dissociation constant of 5 nM in aqueous solution at pH 7. At 2 μM concentration, DC was mainly dimeric in aqueous solution but bound HCAII as a monomer. Upon addition of a large excess of a helix−loop−helix motif without a high-affinity ligand, KE2-Q, a ternary complex was formed between HCAII, DC, and KE2-Q. Hydrophobic interactions between DC and HCAII and coordination of the sulfonamide group to the zinc ion of HCAII contributed cooperatively to binding in a demonstration of the usefulness of folded polypeptide−small organic molecule chimera as novel protein receptors. The DC homodimer was found to be a very sensitive biosensor component due to intermolecular quenching of its fluorescence that was inhibited upon binding to HCAII.

    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-45770 (URN)10.1021/ja038799c (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
    5. Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold
    Open this publication in new window or tab >>Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold
    Show others...
    2005 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, no 6, p. 2480-2487Article in journal (Refereed) Published
    Abstract [en]

    Designed, synthetic polypeptides that assemble into four-helix bundles upon dimerization in solution were studied with respect to folding on planar gold surfaces. A model system with controllable dimerization properties was employed, consisting of negatively and positively charged peptides. Circular dichroism spectroscopy and surface plasmon resonance based measurements showed that at neutral pH, the peptides were able to form heterodimers in solution, but unfavorable electrostatic interactions prevented the formation of homodimers. The dimerization propensity was found to be both pH- and buffer-dependent. A series of infrared absorption−reflection spectroscopy experiments of the polypeptides attached to planar gold surfaces revealed that if the negatively charged peptide was immobilized from a loading solution where it was folded, its structure was retained on the surface provided it had a cysteine residue available for anchoring to gold. If it was immobilized as random coil, it remained unstructured on the surface but was able to fold through heterodimerization if subsequently exposed to a positively charged polypeptide. When the positively charged peptide was immobilized as random coil, heterodimerization could not be induced, probably because of high-affinity interactions between the charged primary amine groups and the gold surface. These observations are intended to pave the way for future engineering of functional surfaces based on polypeptide scaffolds where folding is known to be crucial for function.

    Place, publisher, year, edition, pages
    ACS Publications, 2005
    National Category
    Other Basic Medicine
    Identifiers
    urn:nbn:se:liu:diva-15115 (URN)10.1021/la048029u (DOI)
    Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2018-01-12Bibliographically approved
  • 21.
    Enander, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Strategies for improved target quantification in protein microarrays2008In: 2nd Label-Free Protein Array Workshop,2008, 2008Conference paper (Other academic)
  • 22.
    Enander, Karin
    et al.
    Division of Organic Chemistry, Uppsala University.
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Division of Organic Chemistry, Department of Chemistry, BMC, Uppsala University, Uppsala, Sweden.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold2005In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, no 6, p. 2480-2487Article in journal (Refereed)
    Abstract [en]

    Designed, synthetic polypeptides that assemble into four-helix bundles upon dimerization in solution were studied with respect to folding on planar gold surfaces. A model system with controllable dimerization properties was employed, consisting of negatively and positively charged peptides. Circular dichroism spectroscopy and surface plasmon resonance based measurements showed that at neutral pH, the peptides were able to form heterodimers in solution, but unfavorable electrostatic interactions prevented the formation of homodimers. The dimerization propensity was found to be both pH- and buffer-dependent. A series of infrared absorption−reflection spectroscopy experiments of the polypeptides attached to planar gold surfaces revealed that if the negatively charged peptide was immobilized from a loading solution where it was folded, its structure was retained on the surface provided it had a cysteine residue available for anchoring to gold. If it was immobilized as random coil, it remained unstructured on the surface but was able to fold through heterodimerization if subsequently exposed to a positively charged polypeptide. When the positively charged peptide was immobilized as random coil, heterodimerization could not be induced, probably because of high-affinity interactions between the charged primary amine groups and the gold surface. These observations are intended to pave the way for future engineering of functional surfaces based on polypeptide scaffolds where folding is known to be crucial for function.

  • 23.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Andersson, Linda
    Göteborg universitet.
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Designed, folded polypeptides for bioanalytical purposes - molecular scaffolds that combine recognition and reporting2001In: 4th International Conference on Structural Molecular Biology,2001, 2001Conference paper (Other academic)
    Abstract [en]

      

  • 24.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Choulier, L
    Université Louis Pasteur.
    Olsson, Linnéa
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Yushchenko, Dmitry
    Université Louis Pasteur.
    Kanmert, Daniel
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Klymchenko, Andrey
    Université Louis Pasteur.
    Demchenko, A
    Palladin Institute of Biochemistry.
    Mély, Yves
    Université Louis Pasteur.
    Altschuh, Danièle
    Université Louis Pasteur.
    Development of peptide-based ratiometric biosensor constructs for direct fluorescence detection of protein analytes2007In: VII European Symposium of the Protein Society,2007, 2007Conference paper (Other academic)
    Abstract [en]

        

  • 25.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Choulier, Laurence
    Université Louis Pasteur.
    Selegård, Linnéa
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Yushchenko, Dmitry
    Université Louis Pasteur.
    Kanmert, Daniel
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Klymchenko, Andrey
    Université Louis Pasteur.
    Demchenko, Alexander
    Palladin Institute of Biochemistry.
    Mély, Yves
    Université Louis Pasteur.
    Altschuh, Danièle
    Université Louis Pasteur.
    A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte2008In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 19, no 9, p. 1864-1870Article in journal (Refereed)
    Abstract [en]

    We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore (IN*/IT*) upon binding of Fab57P. When the peptide was labeled in the C terminus, the IN*/I T* ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats. © 2008 American Chemical Society.

  • 26.
    Enander, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Dolphin, Gunnar
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Andersson, Linda
    Department of Organic Chemistry Göteborg University.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Designed, folded polypeptide scaffolds that combine key biosensing events of recognition and reporting2002In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 67, no 9, p. 3120-3123Article in journal (Refereed)
    Abstract [en]

    No abstract available.

  • 27.
    Enander, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Dolphin, Gunnar
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Designed, functionalized helix-loop-helix motifs that bind human carbonic anhydrase II: a new class of synthetic receptor molecules2004In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 14, p. 4464-4465Article in journal (Refereed)
    Abstract [en]

    Polypeptides designed to fold into helix−loop−helix motifs and to dimerize to form four-helix bundles were functionalized by the introduction of a sulfonamide derivative known to bind human carbonic anhydrase II (HCAII) and one or both of the dansyl- and methoxycoumarin fluorescent probes. The 42-residue sequence DC that carries all three substituents in solvent-exposed positions was found to bind HCAII with a dissociation constant of 5 nM in aqueous solution at pH 7. At 2 μM concentration, DC was mainly dimeric in aqueous solution but bound HCAII as a monomer. Upon addition of a large excess of a helix−loop−helix motif without a high-affinity ligand, KE2-Q, a ternary complex was formed between HCAII, DC, and KE2-Q. Hydrophobic interactions between DC and HCAII and coordination of the sulfonamide group to the zinc ion of HCAII contributed cooperatively to binding in a demonstration of the usefulness of folded polypeptide−small organic molecule chimera as novel protein receptors. The DC homodimer was found to be a very sensitive biosensor component due to intermolecular quenching of its fluorescence that was inhibited upon binding to HCAII.

  • 28.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Baltzer, Lars
    Uppsala universitet.
    Structure-dependent signalling in fluorescent biosensor units based on designed, folded polypeptide scaffolds2004In: 19:e Organikerdagarna,2004, 2004Conference paper (Other academic)
    Abstract [en]

      

  • 29.
    Enander, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Dolphin, Gunnar
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis2004In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 10, no 10, p. 2375-2385Article in journal (Refereed)
    Abstract [en]

    A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix–loop–helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02–3 μM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry.

  • 30.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    De novo designed helix-loop-helix polypeptides - a structure-function-based design strategy for microarray and biosensor applications2003In: 1st World Congress on Synthetic Receptors,2003, 2003Conference paper (Refereed)
  • 31.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Designed, folded polypeptides as functional units in biosensor applications2002In: 18:e Organikerdagarna,2002, 2002Conference paper (Other academic)
  • 32.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Löfdahl, Mikael
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Designed, folded polypeptides as functional units in surface-based biosensors - versatile scaffolds connecting recognition and reporting2002In: Europtrode VI,2002, 2002Conference paper (Refereed)
  • 33.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Löfdahl, Mikael
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Helix-loop-helix polypeptides as scaffolds for designed biosensing surfaces2002In: 6th World Congress on Biosensors,2002, 2002Conference paper (Other academic)
  • 34.
    Ericsson, Emma
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Bui, Lan
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Oriented Protein Immobilization by Chelate Associated PhotochemistryManuscript (preprint) (Other academic)
    Abstract [en]

    We demonstrate herein the synthesis, characterization and application of a novel chelateassociated photochemistry (CAP) for oriented and robust attachment of biomolecular ligandsto sensing surfaces. The chelation agent is nitrilotriacetic acid (NTA) which is capable ofcoordinating two histidine (His) molecules in the presence of Nickel. Therefore a ligandmodified with a His-sequence can be attached to NTA to form an oriented assembly ofligands on the sensor surface. The ligand is then covalently bound to the surface via a nearbyphotolabile benzophenone (BP) which attacks C-H bonds upon UV light activation. Theligand is then available for analyte interaction. The synthesized compounds used in this studyare based on the well-known organosulphur surface chemistry for proper attachment to goldsurfaces. Besides the two BP and NTA alkane thiols/disulphides we also synthesized a fillermolecule with an oligo (ethylene glycol) (OEG) tail to fine tune the surface composition andto reduce non-specific binding. Results from surface plasmon resonance (SPR) measurementsusing a Biacore 3000 instrument indicate that up to 55% larger analyte response is obtainedwith CAP as compared to the response obtained with the random orientation achieved byphotoimmobilization alone.

  • 35.
    Ericsson, Emma
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Bui, Lan
    Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, 52074 Aachen, Germany.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Controlled Orientation and Covalent Attachment of Proteins on Biosensor Surfaces by Chelation Assisted Photoimmobilization2013Manuscript (preprint) (Other academic)
    Abstract [en]

    This report presents a novel method for uniform orientation and covalent attachment of proteins to sensing surfaces, termed Chelation Assisted Photoimmobilization (CAP). Alkanethiols terminated with either nitrilotriacetic acid (NTA), benzophenone (BP) or oligo(ethylene glycol) were synthesized and mixed self-assembled monolayers (SAMs) were prepared on gold and thoroughly characterized by infrared reflection absorption spectroscopy (IRAS), ellipsometry and contact angle goniometry. In the process of CAP, NTA chelates Ni2+ and the complex coordinates a His-tagged ligand in an oriented assembly. The ligand is then photoimmobilized via BP, which forms covalent bonds upon UV light activation. The CAP concept was demonstrated using human IgG-Fc modified with C-terminal hexahistidine tags (His-IgGFc) as the ligand and protein A as the analyte.

    In the development of affinity biosensors, uniform orientation of ligand molecules where all analyte binding sites are accessible is often preferred to random orientation. In order to monitor the effect of ligand orientation on analyte response, the ligand-analyte interaction was quantified by surface plasmon resonance analysis, both in the case of CAP and when the ligand was attached by conventional amine coupling on surfaces presenting NTA. Responses were adjusted for differences in ligand immobilization level using IRAS. The normalized analyte response with randomly oriented ligand was 2.5 times higher than that with ligand immobilized by CAP, probably due to molecular crowding effects on the surface and the fact that His-IgGFc is bivalent for protein A. This is a reminder that many other factors than orientation alone may play a decisive role in analyte binding on biosensor surfaces.

  • 36.
    Ericsson, Emma
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Bui, Lan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Biosensors and Bioelectronics. Linköping University, The Institute of Technology.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Site-Specific and Covalent Attachment of His-Tagged Proteins by Chelation Assisted Photoimmobilization: A Strategy for Microarraying of Protein Ligands2013In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 29, no 37, p. 11687-11694Article in journal (Refereed)
    Abstract [en]

    A novel strategy for site-specific and covalent attachment of proteins has been developed, intended for robust and controllable immobilization of histidine (His)-tagged ligands in protein microarrays. The method is termed chelation assisted photoimmobilization (CAP) and was demonstrated using human IgG-Fc modified with C-terminal hexahistidines (His-IgGFc) as the ligand and protein A as the analyte. Alkanethiols terminated with either nitrilotriacetic acid (NTA), benzophenone (BP); or oligo(ethylene glycol) were synthesized and mixed self-assembled monolayers (SAMs) were prepared on gold and thoroughly characterized by infrared reflection absorption spectroscopy (IRAS), ellipsometry, and contact angle goniometry. In the process of CAP, NTA chelates Ni2+ and the complex coordinates the His-tagged ligand in an oriented assembly. The ligand is then photoimmobilized via BP, which forms covalent bonds upon UV light activation. In the development of affinity biosensors and protein microarrays, site-specific attachment of ligands in a fashion where analyte binding sites are available is often preferred to random coupling. Analyte binding performance of ligands immobilized either by CAP or by standard amine coupling was characterized by surface plasmon resonance in combination with IRAS. The relative analyte response with randomly coupled ligand was 2.5 times higher than when site-specific attachment was used. This is a reminder that also when immobilizing ligands via residues far from the binding site, there are many other factors influencing availability and activity. Still, CAP provides a valuable expansion of protein immobilization techniques since it offers attractive microarraying possibilities amenable to applications within proteomics.

  • 37.
    Ericsson, Emma M
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Bui, Lan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.
    Controlled orientation and covalent attachment of proteins on biosensor surfaces by Chelation Assisted Photoimmobilization2013Conference paper (Other academic)
    Abstract [en]

    In the context of surface chemistry for affinity biosensor chips, it is widely accepted that uniform orientation of the immobilized recognition element (ligand) is preferred over random orientation. However, this assumption has often been based on studies where differences in ligand immobilization level have not been taken into account. In this contribution, we present a novel two-step method for homogenous orientation and covalent attachment of proteins to sensing surfaces, called Chelation Assisted Photoimmobilization (CAP). Careful quantification of the effect of ligand orientation on analyte responses was performed by comparing this strategy to immobilization by conventional amine coupling.

     In CAP, the chelation agent is nitrilotriacetic acid (NTA) which chelates Ni2+. A His-tagged ligand forms an oriented assembly when binding Ni2+-NTA and is then covalently bound to the surface via photolabile benzophenone (BP), which attacks C-H bonds upon UV light activation. We relied on a surface chemistry based on self-assembled monolayers (SAMs) of oligo(ethylene glycol) (OEG)-containing alkanethiolates on gold. Alkanethiols terminated with either NTA, BP or OEG were synthesized and mixed SAMs were characterized by infrared reflection absorption spectroscopy (IRAS), ellipsometry and contact angle goniometry. IRAS was also used to quantify ligand immobilization levels obtained either by CAP or by amine coupling via the carboxyl groups of an NTA-presenting surface. The model ligand was human IgG-Fc modified with a C-terminal 6xHis-tag and the analyte was Protein A. The ligand-analyte interaction was quantified by a surface plasmon resonance biosensor.

     Analyte responses were normalized with respect to the ligand amounts obtained by the two immobilization strategies. Interestingly, the normalized analyte response with randomly oriented ligand was >2 times higher than that with ligand immobilized by CAP. This shows that oriented ligand immobilization is not necessarily a means of increasing the sensitivity of a biosensor. Factors that may influence performance include the valency of the ligand and constraints related to the surface chemistry used for orientation.

  • 38.
    Halling Linder, Cecilia
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Chemistry.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Magnusson, Per
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
    Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I2016In: Calcified Tissue International, ISSN 0171-967X, E-ISSN 1432-0827, Vol. 98, no 3, p. 284-293Article in journal (Refereed)
    Abstract [en]

    Bone is a biological composite material comprised primarily of collagen type I and mineral crystals of calcium and phosphate in the form of hydroxyapatite (HA), which together provide its mechanical properties. Bone alkaline phosphatase (ALP), produced by osteoblasts, plays a pivotal role in the mineralization process. Affinity contacts between collagen, mainly type II, and the crown domain of various ALP isozymes were reported in a few in vitro studies in the 1980s and 1990s, but have not attracted much attention since, although such interactions may have important implications for the bone mineralization process. The objective of this study was to investigate the binding properties of human collagen type I to human bone ALP, including the two bone ALP isoforms B1 and B2. ALP from human liver, human placenta and E. coli were also studied. A surface plasmon resonance-based analysis, supported by electrophoresis and blotting, showed that bone ALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. Further, the B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform. Human bone and liver ALP (with identical amino acid composition) displayed pronounced differences in binding, revealing that post-translational glycosylation properties govern these interactions to a large extent. In conclusion, this study presents the first evidence that glycosylation differences in human ALPs are of crucial importance for protein–protein interactions with collagen type I, although the presence of the ALP crown domain may also be necessary. Different binding affinities among the bone ALP isoforms may influence the mineral-collagen interface, mineralization kinetics, and degree of bone matrix mineralization, which are important factors determining the material properties of bone.

  • 39.
    Kanmert, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Brorsson, Ann-Christin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    In Vitro Amyloid Fibril Formation of Human IgG-FcManuscript (preprint) (Other academic)
    Abstract [en]

    Both light and heavy chains of human antibodies are known to be associated with immunoglobulin related amyloidosis, but in vitro formation of amyloid fibrils has previously only been reported for light chain sequences. Here we show that fibrillation of the Fc fragment of human IgG of all subclasses can be induced by heating to at least 75°C at neutral pH and physiological salt concentration. The observed protein assemblies share key properties with those constituting amyloid, i.e. they are thioflavinophilic and congophilic and have a typical fibril appearance in the transmission electron microscope. This study of the amyloidogenic properties of human IgG-Fc, comprising the CH2 and CH3 domains of the IgG heavy chain, is important for increasing the understanding of which parts of IgG that could be involved in amyloid formation in vivo.

  • 40.
    Kanmert, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Brorsson, Ann-Christin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Thermal Induction of an Alternatively Folded State in Human IgG-Fc2011In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 6, p. 981-988Article in journal (Refereed)
    Abstract [en]

    We report the formation of a non-native, folded state of human IgG4-Fc induced by a high temperature at neutral pH and at a physiological salt concentration. This structure is similar to the molten globule state in that it displays a high degree of secondary structure content and surface-exposed hydrophobic residues. However, it is highly resistant to chemical denaturation. The thermally induced state of human IgG4-Fc is thus associated with typical properties of the so-called alternatively folded state previously described for murine IgG, IgG-Fab, and individual antibody domains (V(L), V(H), C(H)1, and C(H)3) under acidic conditions in the presence of anions. Like some of these molecules, human IgG4-Fc in its alternative fold exists as a mixture of different oligomeric structures, dominated by an equilibrium between monomeric and heptameric species. Heating further induces the formation of fibrous structures in the micrometer range.

  • 41.
    Kanmert, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Human IgG-Fc Forms an Alternatively Folded State at Low pHManuscript (preprint) (Other academic)
    Abstract [en]

    Anion-induced formation of so-called alternatively folded states has previously been described for murine IgG, IgG-Fab and several separate antibody domains, including CH3, at low pH. In this report, we confirm that this property can also be extended to the full Fc fragment of IgG. When incubating human IgG4-Fc at pH 2.0 in the presence of NaCl, the protein adopts a conformation that is characterized by a high degree of secondary structure but less well-defined tertiary structure compared to the native state. In contrast to both a classical molten globule and an acid-induced A state, however, the alternatively folded protein appears very stable toward chemical denaturation and unfolds in cooperative transitions. It further forms oligomeric assemblies that are primarily composed of dimers and dodecamers, and when incubated at physiologic temperatures with agitation, it displays prefibrillar structures that are both thioflavinophilic and congophilic.

  • 42.
    Kanmert, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Enocsson, Helena
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Science & Engineering.
    Wetterö, Jonas
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences.
    Kastbom, Alf
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Rheumatology in Östergötland.
    Skogh, Thomas
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Rheumatology in Östergötland.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Designed Surface with Tunable IgG Density as an in Vitro Model for Immune Complex Mediated Stimulation of Leukocytes2010In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 26, no 5, p. 3493-3497Article in journal (Refereed)
    Abstract [en]

    We present the design of an in vitro for immune-complex-mediated stimulation of leukocytes and its functional characteristics with respect to monocyte adhesion. The model was based on orientation-controlled immobilization of a humanized IgG1 monoclonal antibody (rituximab) via its interaction with a biotinylated peptide epitope derived from the CD20 marker. The peptide was linked to neutravidin covalently attached to it mixed self-assembled monolayer of carboxyl- and methoxy-terminated oligo(ethylene glycol) alkane thiolates on gold. The surface adhesion propensity of human monocytes (cell line U917) was highly dependent on the lateral IgG density and indicated that there exists a distance between IgG-Fc on the surface where interactions with Fc gamma receptors are optimal. This well-defined platform allows for a careful control of the size and orientation of artificial IgG immune complexes, it is easily made compatible with, for example, cellular imaging, and it will become useful for in vitro studies on the importance of Fc gamma receptor interactions in chronic immune-mediated diseases.

  • 43.
    Kanmert, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Kastbom, Alf
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Rheumatology in Östergötland.
    Almroth, Gunnel
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences.
    Skogh, Thomas
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Rheumatology in Östergötland.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Wetterö, Jonas
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences.
    IgG Rheumatoid Factor Against the Four Human Fc-gamma Subclasses in Early Rheumatoid Arthritis (the Swedish TIRA Project)Manuscript (preprint) (Other academic)
    Abstract [en]

    Rheumatoid factor (RF), i.e. a family of autoantibodies against the Fc part of IgG, is an important seromarker of rheumatoid arthritis (RA). Traditional particle agglutination without disclosing the antibody isotype remains the predominating diagnostic method in clinical routine. Although IgG-RF attracts pathogenic interest, its detection remains technically challenging. The present study aimed at developing a set of tests identifying IgG-RFs directed against the four IgG subclasses. IgG-RF against either subclass of human IgG-Fc were analyzed with four novel enzyme-linked immunosorbent assays (ELISAs) utilizing four recombinant human Fc-gamma fragments (hIgG1-4) as sources of antigen. Sera from 40 patients with recent-onset RA (20 seropositive and 20 seronegative by IgM-RF and IgA-RF-isotype specific ELISA) were analyzed. Sera from 20 healthy blood donors served as reference. Among the IgM-/IgA-RF positive RA-sera, IgG-RF was found directed against hIgG1, hIgG4, and most notably, with strikingly high reactivity against hIgG2, but not hIgG3. Significant correlations were seen between IgG-RF against hIgG2-Fc and IgA-RF (r = 0.513) and IgM-RF (r = 0.736) levels. Further prospective studies are warranted to elucidate any correlation to disease course and outcome.

  • 44.
    Kanmert, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Kastbom, Alf
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Rheumatology.
    Almroth, Gunnel
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences.
    Skogh, Thomas
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Rheumatology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Wetterö, Jonas
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences.
    IgG Rheumatoid Factors Against the Four Human Fc-gamma Subclasses in Early Rheumatoid Arthritis (The Swedish TIRA Project)2012In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 75, no 1, p. 115-119Article in journal (Refereed)
    Abstract [en]

    Rheumatoid factor (RF), i.e. a family of autoantibodies against the Fc part of IgG, is an important seromarker of rheumatoid arthritis (RA). Traditional particle agglutination without disclosing the antibody isotype remains the predominating diagnostic method in clinical routine. Although IgG-RF attracts pathogenic interest, its detection remains technically challenging. The present study aimed at developing a set of tests identifying IgG-RFs directed against the four IgG subclasses. IgG-RF against either subclass of human IgG-Fc were analysed with four novel enzyme-linked immunosorbent assays (ELISAs) utilizing four recombinant human Fc-gamma fragments (hIgG14) as sources of antigen. Sera from 40 patients with recent onset RA (20 seropositive and 20 seronegative by IgM-RF and IgA-RF-isotype-specific ELISA) were analysed. Sera from 20 healthy blood donors served as reference. Among the IgM-/IgA-RF-positive RA-sera, IgG-RF was found directed against hIgG1 and hIgG2, but not against hIgG3 or hIgG4. Significant correlations were seen between IgG-RF against hIgG2-Fc and IgM-RF (r = 0.666) levels. Further prospective studies are warranted to elucidate any correlation to disease course and outcome.

  • 45.
    Martinsson, Erik
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Mehdi Shahjamali, Mohammad
    Nanyang Technology University, Singapore .
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Boey, Freddy
    Nanyang Technology University, Singapore .
    Xue, Can
    Nanyang Technology University, Singapore .
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Local Refractive Index Sensing Based on Edge Gold-Coated Silver Nanoprisms2013In: The Journal of Physical Chemistry C, ISSN 1932-7447, E-ISSN 1932-7455, Vol. 117, no 44, p. 23148-23154Article in journal (Refereed)
    Abstract [en]

    Bulk and surface refractive index sensitivity for localized surface plasmon resonance (LSPR) sensing based on edge gold-coated silver nanoprisms (GSNPs) and gold nanospheres was investigated and compared with conventional surface plasmon resonance (SPR) sensing based on propagating surface plasmons. The hybrid GSNPs benefit from an improved stability since the gold frame protecting the unstable silver facets located at the silver nanoprisms (SNPs) edges and tips prevents truncation or rounding of their sharp tips or edges, maintaining a high refractive index sensitivity even under harsh conditions. By using layer-by-layer deposition of polyelectrolytes and protein adsorption, we found that GSNPs exhibit 4-fold higher local refractive index sensitivity in close proximity (andlt;10 nm) to the surface compared to a flat gold film in the conventional SPR setup. Moreover, the sensitivity was 8-fold higher with GSNPs than with gold nanospheres. This shows that relatively simple plasmonic nanostructures for LSPR-based sensing can be engineered to outperform conventional SPR, which is particularly interesting in the context of detecting low molecular weight compounds where a small sensing volume, reducing bulk signals, is desired.

  • 46.
    Petrone, Luigi
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Aldred, Nick
    Newcastle University, England.
    Emami, Kaveh
    Newcastle University, England.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.
    Ederth, Thomas
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.
    Clare, Anthony S.
    Newcastle University, England.
    Chemistry-specific surface adsorption of the barnacle settlement-inducing protein complex2015In: Interface Focus, ISSN 2042-8898, E-ISSN 2042-8901, Vol. 5, no 1, p. 20140047-Article in journal (Refereed)
    Abstract [en]

    Gregarious settlement in barnacle larvae (cyprids) is induced by a contact pheromone, the settlement-inducing protein complex (SIPC). The SIPC has been identified both in the cuticle of adult barnacles and in the temporary adhesive secretion (footprint) of cyprids. Besides acting as a settlement inducer, the presence of the SIPC in footprints points to its additional involvement in the adhesion process. SIPC adsorption behaviour was therefore investigated on a series of self-assembled monolayers (SAMs) by surface plasmon resonance at the pH of seawater (8.3). Fibrinogen and alpha(2)-macroglobulin (A2M) (blood complement protease inhibitors with which the SIPC shares 29% sequence homology) were used in the adsorption experiments as positive and negative standards, respectively. The mass uptake of the SIPC was comparable to that of fibrinogen, with adsorption observed even on the protein-resistant oligo(ethylene glycol) surface. Notably, on the positively charged SAM the SIPC showed a kinetic overshoot, indicating a metastable configuration causing the amount of adsorbed protein to temporarily exceed its equilibrium value. A2M adsorption was low or negligible on all SAMs tested, except for the positively charged surface, indicating that A2M adsorption is mainly driven by electrostatics. Evaluation of SIPC non-specific adsorption kinetics revealed that it adsorbed irreversibly and non-cooperatively on all surfaces tested.

  • 47. Riepl, M
    et al.
    Enander, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Schäfering, Michael
    Thermo Hybaid Interactiva GmbH.
    Kruschina, Margit
    Thermo Hybaid Interactiva GmbH.
    Ortigao, Flavio
    Thermo Hybaid Interactiva GmbH.
    Functionalized surfaces of mixed alkanethiols on gold as a platform for oligonucleotide microarrays2002In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 18, no 18, p. 7016-7023Article in journal (Refereed)
    Abstract [en]

    Mixed self-assembled monolayers of biotinylated- and ethylene glycol-terminated long-chain alkanethiols were prepared on gold surfaces in an attempt to develop a reliable protocol for immobilization of streptavidin. A broad range of surface analytical techniques including ellipsometry, atomic force microscopy, and infrared, fluorescence, and X-ray photoelectron spectroscopy were used to characterize the SAMs before and after immobilization of streptavidin. The first part of the work was focused on finding the mixing conditions that lead to optimum binding capacity of streptavidin. Mixed SAMs prepared from loading solutions containing 75-95% of the biotinylated alkanethiol resulted in high immobilization levels of functional streptavidin. The thin layers of streptavidin subsequently can be used for the immobilization of a broad spectrum of biotinylated biomolecules (e.g. oligonucleotides, cDNA, peptides, proteins, antibodies, and carbohydrates) and provides therefore an excellent platform for the fabrication of chips/arrays for biosensor and screening applications. This is successfully demonstrated by monitoring the hybridization between a biotinylated 24-mer capturing oligonucleotide and a labeled target 89-mer DNA using a fluorescence-based DNA-microarray detection system. Moreover, the DNA-microarray experiments also revealed (i) good selectivity when comparing the response of the complementary oligonucleotide with that of a random 24-mer capturing oligonucleotide and (ii) low levels of nonspecific binding to the streptavidin surface.

  • 48.
    Selegård, Robert
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.
    Generic Phosphatase Activity Detection using Zinc Mediated Aggregation Modulation of Polypeptide-Modified Gold Nanoparticles2014In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 6, no 23, p. 14204-14212Article in journal (Refereed)
    Abstract [en]

    A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme. Phosphatase activity generates inorganic phosphate that forms an insoluble complex with Zn2+. In a sample containing a preset concentration of Zn2+, phosphatase activity will markedly reduce the concentration of dissolved Zn2+ from the original value, which in turn affects the aggregation of gold nanoparticles functionalized with a designed Zn2+ responsive polypeptide. The change in nanoparticle stability thus provides a rapid and sensitive readout of the phosphatase activity. The assay is not limited to a particular enzyme or enzyme substrate, which is demonstrated using three completely different phosphatases and five different substrates, and thus constitutes a highly interesting system for drug screening and diagnostics.

  • 49.
    Svedhem, Sofia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Sjöbom, Hans
    Biacore AB, Uppsala, Sweden.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Löfås, Stefan
    Biacore AB, Uppsala, Sweden.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Sjöstrand, Sven-Erik
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Svensson, Stefan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 296, no 2, p. 188-196Article in journal (Refereed)
    Abstract [en]

    The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s−1. For the mutants, dissociation rates were faster (0.022–0.025 s−1), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.

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