| 1. |
- Andersson, Margaretha, et al.
(författare)
-
Characterization of Surface-Modified Nanoparticles for in Vivo Biointeraction. A Sedimentation Field Flow Fractionation Study
- 2005
-
Ingår i: Analytical Chemistry. ; 77, s. 5488-5493
-
Tidskriftsartikel (refereegranskat)abstract
- Sedimentation field flow fractionation (SdFFF) is an emerging high-performance analytical tool for separation and determination of size and adsorption characteristics of colloidal particles. This study demonstrates how SdFFF can be used to characterize nanoparticles prepared for in vivo applications including (1) the quantification of polymer uptake on nanoparticles where surface coverage is crucial and (2) the coupling of cell adhesive peptides containing the Arg-Gly-Asp motif (RGD). Quantitative information about polymer adhesion in order to prepare a bioinert surface and an accurate determination of ligand uptake are both of obvious importance for the understanding of, for example, relations between the number of attached molecules for biointeraction and an observed therapeutic effect. In addition, the present work highlights the necessity to perform careful characterization of commercially available particulate starting materials, in terms of size and polydispersity, prior to biological experimentation.
|
|
| 2. |
|
|
| 3. |
- Fromell, Karin, et al.
(författare)
-
A particulate platform for bioluminescent immunosensing
- 2007
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:22, s. 8601-8607
-
Tidskriftsartikel (refereegranskat)abstract
- The present study examines pyruvate kinase-conjugated antibodies for potential use in EUSA applications. The conjugates had an acceptable stability, and the coupling inflicted only minor impairment on the kinase activity. To mimic the setup of an immunoassay under development, a test antigen (BSA) was attached to polystyrene nanoparticles. This arrangement was found to be suitable as solid support for presentation of antigens in sensitive bioluminescence assays. The nanoparticles were well characterized in terms of protein surface load and were used to establish the number of conjugate complexes needed to generate a detectable signal. Under the biochemical conditions employed here, the detection limit of the pyruvate kinase conjugate lies in the femtomole range.
|
|
| 4. |
- Fromell, Karin, et al.
(författare)
-
Nanoparticle decorated surfaces with potential use in glycosylation analysis
- 2005
-
Ingår i: Colloids and Surfaces B: Biointerfaces. ; 46, s. 84–91-
-
Tidskriftsartikel (refereegranskat)abstract
- A majority of all biologically active proteins are glycosylated and various diseases have proven to correlate with alterations in protein glycosylation. Sensitive identification of different glycoprotein glycoforms is therefore of great diagnostic value. Here we describe a method with potential for glycoprotein profiling, based on lectins as capture probes immobilized on particulate substrates in the nm-range. The nanoparticles present high concentrations of attachment sites for specific ligands and cause minimal steric hindrance to binding. In the present model study the mannose-binding lectin ConA has been coupled to polystyrene nanoparticles via a poly(ethyleneoxide) linker which protects the protein conformation and activity and prevents unspecific protein adsorption. The ConA-coated particles are accommodated at different spots on the analytical surface via oligonucleotide linkage. This attachment, which relies on the hybridization of complementary oligonucleotides, allows firm fixation of the particles at specific positions. The ConA attached to the particles has retained conformation and activity and binds selectively to a series of different glycoproteins. The results indicate the potential for using a multi-lectin nanoparticle array in glycoprotein mapping.
|
|
| 5. |
|
|
| 6. |
- Fromell, Karin, 1972-
(författare)
-
Nanoscale Reaction Systems
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in this thesis describes the use of polystyrene nanoparticles as model surfaces for bioanalytical work. Nanoparticles constitute convenient platforms for the attachment of bioactive agents, and receptor coated particles offer high local concentration of binding sites for specific ligands with minimal steric hindrance. However, it is not only the amount of bound protein that matters, the proteins must also be immobilized at the surface in such ways that they fully retain their activity, while at the same time protecting the surface from unspecific uptake of undesired components. The present work relates to the controlled immobilization of multiple types of active biomolecules onto nanoparticle surfaces to make them multifunctional. The surface expansion offered by the nanoparticles, in combination with the closeness between the reactants co-immobilized on the same particle, enables coupled reactions to be carried at a higher rate than otherwise possible. Thus, particle-decorated surfaces of this kind are highly suitable for miniaturized bioanalytical systems. Sensitive microarray systems are under development, including lectin-coated nanoparticles for glycoprotein mapping and a diagnostic device for Point-of-Care testing with a nanoparticle-based detection system.The full evaluation of protein attachment to nanoparticles requires precise analytical techniques for particle characterization, both in bare and coated form. The mass-sensitive SdFFF technique occupies a prominent position for particle characterization, as it offers both accurate determination of particle size and a quantification of adsorbed layers on small particles, whether of synthetic or biopolymeric nature. Here, this analytical technique is developed to precisely characterize nanoparticles that are sequentially coated with different layers, each rendering the particles a specific functionality. The thesis demonstrates how precise mass uptakes can be determined for each specific layer, and how control over the exact surface composition of the modified particles can be established for optimization of biological activity.
|
|
| 7. |
- Hansson, Lars-Olof, et al.
(författare)
-
Comparison between Chicken and Rabbit Antibody Based Particle Enhanced Cystatin C Reagents for Immunoturbidimetry
- 2008
-
Ingår i: Journal of immunoassay & immunochemistry. - : Informa UK Limited. - 1532-1819 .- 1532-4230. ; 29:1, s. 1-9
-
Tidskriftsartikel (refereegranskat)abstract
- We have compared three commercial particle enhanced cystatin C reagents. One of the reagents utilizes chicken antibodies and the other two reagents are rabbit antibody based. We show that the chicken antibody based reagent yields a higher delta absorbance when reacting with the antigen. IgY coupled to latex particles show a strong scatter response even at high antigen concentrations in contrast to the steep decline in scatter previously reported for IgY antibodies in solution. The reagent also showed a low CV for duplicate samples. Laying hens thus seems as an interesting source of antibodies for particle-enhanced immunoassays.
|
|
| 8. |
- ter Veen, Rik, et al.
(författare)
-
Shifts in polystyrene particle surface charge upon adsorption of the Pluronic F108 surfactant
- 2005
-
Ingår i: Journal of Colloid and Interface Science. ; 288:1, s. 124-128
-
Tidskriftsartikel (refereegranskat)abstract
- Electrical field-flow fractionation (ElFFF) and sedimentation field-flow fractionation (SdFFF) were used in combination to study the adsorption of the triblock polymeric surfactant, Pluronic F108 [(EO)129-(PO)56-(EO)129] to 200 nm polystyrene (PS) latex spheres. The SdFFF technique allowed an accurate determination of the mass of surfactant adsorbed on each particle from a solution of given concentration. To complement this isotherm study, we show that ElFFF can be used to measure fractional coverages of the formed electrically neutral surfactant layers on the charged PS particles. Through a combination of the two techniques it is possible to gain information about the structure of the adsorbate layer. Thus, when Pluronic F108 is taken up by the PS surface from solutions of low concentration, all three blocks appear to adhere to the surface as long as there is free space available. As the solution concentration increases and the fractional coverage reaches approximately 20%, the surface turns crowded enough to let the strongly adsorbing PPO blocks competitively displace the weakly adherent PEO blocks, which gradually rise to extend into the aqueous phase until the surface is fully saturated.
|
|
| 9. |
|
|
| 10. |
- Arnell, Robert, et al.
(författare)
-
Biotechnological Approach to the Synthesis of 9α-Hydroxylated Steroids
- 2007
-
Ingår i: Preparative Biochemistry & Biotechnology. - : Informa UK Limited. - 1082-6068 .- 1532-2297. ; 37:4, s. 309-321
-
Tidskriftsartikel (refereegranskat)abstract
- The steroid 9α-hydroxylase gene has been cloned from Mycobacterium smegmatis into Escherichia coli BL21. Progesterone added to bioreactors was subjected to in vivo transformation into 9α-hydroxyprogesterone. In 7 days, 43.6 mg9α-hydroxyprogesterone was formed from 53.8 mg/L progesterone. The enzyme also has shown evidence of processing 4-androstene-3,17-dione in vivo. An extensive analytical method development, including LLE, HPLC-DAD, MS, andNMR was performed to verify the product and to enable a quantitative analysis. Protocols for analytical and preparative separation have been developed, using binaphtol as internal standard. Both the growth pattern and the bioconversion ratewere unaffected by the presence of binaphtol in the bioreactor. The enzyme was purified by immobilised metal affinity and ion exchange chromatography, resulting in low in vitro activity.
|
|
| 11. |
|
|
| 12. |
- Belew, M., et al.
(författare)
-
Purification of Recombinant Human Serum Albumin (rHSA) Produced by Genetically Modified Pichia Pastoris
- 2008
-
Ingår i: Separation science and technology (Print). - : Informa UK Limited. - 0149-6395 .- 1520-5754. ; 43:11-12, s. 3134-3153
-
Tidskriftsartikel (refereegranskat)abstract
- Recombinant human serum albumin (rHSA) was produced by genetically transformed Pichia pastoris yeast. The cell-culture supernatant (CCS) contained 8–12 g/l rHSA that was purified in a three-step procedureinvolving (1) a capture step using the newly developed cation exchanger CaptoTM MMC; (2) an intermediate step using Phenyl SepharoseTM and, (3) a polishingstep using Aminobutyl SepharoseTM 6 FF. The total recovery was 25–35% and the product fulfils the purity criteria of the European Pharmacopeia. Purified rHSA and plasma-derived HSA were essentially identical judging bySDS- or native-PAGE, and the pigment level (expressed as A 350/A280) in the rHSA was 0.03 or less and was strongly dependent on the quality of the CCS.Dimers and polymers in the final product were less than that found in purified plasma-derived HSA. The molar mass of the purified rHSA, as well as of its natural counterpart, is 67 000 Daltons by MALDI-ToF mass spectrometry, while the iso-electric points of both recombinant and natural HSA ranged between pH 5.42–5.55 when determined in 8M urea. The stability profiles of both proteins after heat treatment were identical as determined by differential scanning calorimetry (DSC). The results obtained here suggest the purified rHSA to be a homogeneous protein identical to its natural counterpart.
|
|
| 13. |
- Caldwell, Karin D., et al.
(författare)
-
The Origin of Sepahdex
- 2006
-
Ingår i: GIT Laboratory Journal: Europe. ; 10:5, s. 18-20
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
|
|
| 14. |
|
|
| 15. |
- Feiler, Adam A., et al.
(författare)
-
Adsorption and viscoelastic properties of fractionated mucin (BSM) and bovine serum albumin (BSA) studied with quartz crystal microbalance (QCM-D)
- 2007
-
Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 0021-9797 .- 1095-7103. ; 315:2, s. 475-481
-
Tidskriftsartikel (refereegranskat)abstract
- The adsorption profile and viscoelastic properties of bovine submaxillary gland mucin (BSM) and bovine serum albumin (BSA), extracted from a commercial mucin preparation, adsorbing to polystyrene surfaces has been studied using quartz crystal microbalance with dissipation monitoring (QCM-D). A significant difference in the adsorption properties of the different proteins was detected; with the BSA adsorbing in a flat rigid layer whilst the mucin adsorbed in a diffuse, highly viscoelastic layer. Subsequent addition of BSA to the preadsorbed mucin layer resulted in stiffening of the protein layer which was attributed to complexation of the mucin by BSA. In contrast, a preadsorbed layer of BSA prevented mucin adsorption altogether. Combined mixtures of mucin and BSA in well defined ratios revealed intermediate properties between the two separate protein species which varied systematically with the protein ratios. The results shed light on the synergistic effects of complexation of lower molecular weight biomolecular species with mucin. The possibility to selectively control protein uptake and tailor the physical properties of the adsorbed layer makes mucin an attractive option for application in biomaterial coatings.
|
|
| 16. |
- Gullberg, Elisabet, et al.
(författare)
-
Identification of Cell Adhesion Molecules in the Human Follicle-Associated Epithelium That Improve Nanoparticle Uptake into the Peyer's Patches
- 2006
-
Ingår i: Journal of Pharmacology and Experimental Therapeutics. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0022-3565 .- 1521-0103. ; 319:2, s. 632-639
-
Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to identify cell adhesion molecules that could serve as targets of the human follicle-associated epithelium (FAE) overlying Peyer's patches and to assess nanoparticle uptake levels across this epithelium. We first studied the expression of the mouse M-cell marker beta(1)-integrin and used a model of human FAE derived from intestinal epithelial Caco-2 cells and Raji B-cells to identify additional potential targets by cDNA array. The protein expression of potential targets in the model FAE and in human ileal FAE tissues was quantified by immunofluorescence. Integrin targeting was studied by investigating the transport of Arg-Gly-Asp (RGD)-coated (integrin- binding), Arg-Gly-Glu (RGE)-coated (nonintegrin-binding), and uncoated nanoparticles across ileal specimens mounted in Ussing chambers. Both beta(1)-integrin and the cell adhesion molecule CD9 were more abundantly expressed in the model and human FAE compared with the Caco-2 control cells or villus epithelium (VE). Uncoated nanoparticles were not taken up across either FAE or VE. General integrin targeting with RGD improved the nanoparticle transport dramatically across the FAE and to a lower extent across the VE. Compared with RGE, RGD improved transport 4-fold across the FAE. There was no difference in the transport of RGD- and RGE-coated nanoparticles across the VE. In conclusion, beta(1)-integrin and CD9 were identified as targets in human FAE. The difference in RGD- and RGE-mediated transport across the FAE, but not the VE, suggests that a specific integrin interaction was the dominating mechanism for improved nanoparticle uptake across the FAE., whereas charge interaction contributed substantially to the improved VE uptake.
|
|
| 17. |
- Lundin, Maria, et al.
(författare)
-
Comparison of the adsorption kinetics and surface arrangement of "as received" and purified bovine submaxillary gland mucin (BSM) on hydrophilic surfaces
- 2009
-
Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 0021-9797 .- 1095-7103. ; 336:1, s. 30-39
-
Tidskriftsartikel (refereegranskat)abstract
- The effect of bovine serum albumin (BSA) as impurity in a commercial bovine submaxillary gland mucin preparation (BSM; Sigma M3895) on the adsorption of BSM to hydrophilic surfaces (mica and silica) has been Studied in terms of adsorption kinetics, amount and structure of the formed adlayer. The Surface Force Apparatus (SFA) was used to gain information about the extended and compressed structure of adsorbed "as received" BSM, purified BSM, BSA extracted from the "as received" BSM and mixtures of the latter Purified proteins. The adsorbed amount was estimated using a combination of X-ray Photoelectron Spectroscopy (XPS), Enzyme-Linked Immuno Sorbent Assay (ELISA), Enzyme-Linked Lectin Assay (ELLA), Dual Polarization Interferometry (DPI) and Quartz Crystal Microbalance (QCM-D) measurements. Under the used conditions, purified BSM showed very low affinity for silica and only small amounts were found to adsorb on mica. Initially, the BSM molecules adopted an extended conformation on the mica surface with tails extending into the bulk phase. These tails were irreversibly compressed into a very thin (10 A) layer upon applying a high load. "As received" BSM formed considerably thicker Compressed layers (35 A); however, the extended layer structure was qualitatively the same. When Mixtures of purified BSM and BSA were coadsorbed on mica, a 9 wt-% albumin content gave a comparable layer thickness as the "as received" BSM and from XPS data we draw the conclusion that the albumin content in the layer adsorbed from "as received" BSM was approximately 5 wt-%. Adsorption from an equal amount of BSM and BSA revealed that even though the amount of BSM is scarce in the mixed layer, the few BSM molecules have a drastic effect on the adsorbed thickness and Structure. Clearly, this study shows the importance of characterizing the mucin used since differences in purity give rise to different adsorption behaviours in terms of both adsorbed amount and layer Structure. (C) 2009 Elsevier Inc. All rights reserved.
|
|
| 18. |
- Margreiter, Gerd, et al.
(författare)
-
Size characterization of inclusion bodies by sedimentation field-flow fractionation
- 2008
-
Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 138:3-4, s. 67-73
-
Tidskriftsartikel (refereegranskat)abstract
- Sedimentation field-flow fractionation (sedFFF) was evaluated to characterize the size of Delta(4-23)TEM-beta-lactamase inclusion bodies (IBs) overexpressed in fed-batch cultivations of Escherichia coli. Heterologous Delta(4-23)TEM-beta-lactamase protein formed different sizes of IBs, depending upon the induction conditions. In the early phases of recombinant protein expression, induced with low concentrations of IPTG (isopropyl-beta-d-thiogalactoside), IB masses were larger than expected and showed heterogeneous size distributions. During cultivation, IB sizes showed a Gaussian distribution and reached a broad range by the end of the fed-batch cultivations. The obtained result proved the aptitude of sedFFF to rapidly assess the size distribution of IBs in a culture.
|
|
| 19. |
|
|
| 20. |
- Samuelsson, Jörgen, 1971-
(författare)
-
Development of Methods for Phase System Characterization in Liquid Chromatography
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this thesis is first and foremost to improve the fundamental knowledge of nonlinear and preparative separation theory by focusing on some of the remaining “white spots” on the theoretical chromatographic map. Secondly, the acquired knowledge is used to develop, validate and execute new methods for phase characterization in liquid chromatography. The methodology used in this thesis is a combination of experiments, fundamental nonlinear theory and systematic computer simulations. A fundamental knowledge of the molecular interactions between the compounds to be separated and the separation media requires the determination of adsorption isotherms over a broad concentration range to give a complete picture of all interactions in the separation system - weak as well as strong. In addition, such adsorption data is essential for optimization in preparative chromatography. For the first time, it has been experimentally shown that the injected molecules are not present in the detected peak when a small excess of molecules are injected into a chromatographic system equilibrated with a constant stream of identical molecules. Several experimental procedures for this method were developed such as (i) the optimal injection strategy and (ii) different labeling methods for visualizing the injected molecules. Remarkable phenomena in the single-component case, such as invisible peak deformation and deformed (invisible) frontal chromatograms, are reported, investigated, and explained. This phenomenon has asides from its future practical implementation, also a large didactic value. The accuracy of the ECP method is experimentally improved, and used to characterize the separation of protolytic compounds at different pH on modern commercially available silica and hybrid silica column packing materials. That investigation enables us to answer why basic compounds give a much more compact preparative peak profile at pH 11 than they yields at lower pH.
|
|
| 21. |
- Sandberg, Tomas, 1973-
(författare)
-
On the Development of Mucin-based Biomaterial Coatings
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Owing to their key role in mucosal functioning as surface barriers with biospecific interaction potentials, the mucins are interesting candidates for use as surface modifiers in biomaterials applications. In this work, “mild” fractionation procedures were used to prepare mucins of bovine (BSM), porcine (PGM), and human (MG1) origin. Biophysicochemical analysis showed the prepared mucins to differ in size, charge, conformation, and composition. In turn, these factors were shown to govern mucin adsorption on hydrophilic and hydrophobic model surfaces. To enable for detailed coating analysis, methods for the qualitative and quantitative analysis of mucin-based coatings were developed. Of particular interest, a method for the determination of the fraction of surface-exposed, presumed bioactive proteins in a complex mucin coating was described. It was shown, using microscopy and activation assays, that mucin precoating effectively suppresses the neutrophil response towards a polymeric model biomaterial. Under optimal coating conditions, all mucins performed equally well, thus indicating them to be functionally similar. Coating analysis suggested that efficient mucin surface-shielding is critical for good mucin coating performance. Following a study on the complexation of albumin with preadsorbed mucin, we investigated the effect of mucin precoating on the conformation and neutrophil-activating properties of adsorbed host proteins. We found that mucin precoating greatly reduces the strong immune-response normally caused by adsorbed proinflammatory proteins (IgG and sIgA). Detailed coating analysis revealed that the fraction of surface-exposed protein in the mucin-protein composite influences the neutrophil response. Unexpectedly low neutrophil activation for composites containing near-monolayer concentrations of exposed IgG, suggested IgG to act synergistically with mucin on the surface. Conformational analysis supported this by showing that a preadsorbed mucin layer could stabilize adsorbed IgG through complexation. Our findings link well to the complex in vivo situation and suggest that functional mucosal mimics can be created in situ for improved biomaterials performance.
|
|
| 22. |
- Sandberg, Tomas, et al.
(författare)
-
Potential use of mucins as biomaterial coatings. I. Fractionation, characterization, and model adsorption of bovine, porcine, and human mucins
- 2009
-
Ingår i: Journal of Biomedical Materials Research Part A. - : Wiley. - 1549-3296 .- 1552-4965. ; 91A:3, s. 762-772
-
Tidskriftsartikel (refereegranskat)abstract
- Previously, we presented evidence that mucins have potential as biomaterial coatings. Here, we reveal substantial batch-to-batch variations for a frequently used commercial bovine salivary mucin preparation (BSM) and stress the importance of standardizing mucins intended for comparative purposes. "Mild" fractionation strategies, aiming at preserving natural mucin functions, were used to prepare two more defined BSM fractions as well as three mucin fractions from porcine gastric (PGM) and human salivary (MG1) sources. While the BSM and PGM were highly purified and mainly adopted random coil conformations in solution, the MG1 contained mucin-bound components (1.6 wt% albumin) and appeared compact. Average molar masses and root-mean-square radii for the predominant BSM, PGM, and MG1 species spanned 0.8-4.2 MDa and 46-86 nm, respectively. An ellipsometric evaluation, using hydrophilic and hydrophobic silica, showed the mucin adsorption to be slow and related to mucin charge, size, conformation, and compositional complexity. The mass uptakes on hydrophobic silica averaged 2.6, 2.6, and 5.0 mg/m(2), for BSM, PGM, and MG1, respectively. Finally, we find that stable mucin coatings can be formed on polymers of different wettability. The reported mucin preparations serve as platforms for a series of studies on the biocompatibility of mucin coatings.
|
|
| 23. |
- Sandberg, Tomas, et al.
(författare)
-
Potential use of mucins as biomaterial coatings. II. Mucin coatings affect the conformation and neutrophil-activating properties of adsorbed host proteins – Towards a mucosal mimic
- 2009
-
Ingår i: Journal of Biomedical Materials Research: Part A. - : Wiley. - 1549-3296 .- 1552-4965. ; 91A:3, s. 773-785
-
Tidskriftsartikel (refereegranskat)abstract
- In continuation of our recent fractionation and characterization study on mucins of bovine salivary (BSM), porcine gastric (PGM), and human salivary (MG1) origin, this study evaluates the effect of mucin precoating on the conformation and neutrophil-activating properties of host proteins adsorbed to a polyethylene terephthalate-based model biomaterial. Microscopy combined with assays for the neutrophil releases of reactive oxygen species and human neutrophil lipocalin showed that mucin precoating greatly reduced the strong immune-response normally induced by adsorbed immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA), respectively. A similar finding was made for the proinflammatory fibrinogen. Although the total uptakes of these proteins depended on the mucin surface concentration, a detailed composite analysis suggested the fraction Of surface-exposed protein to be a stronger determinant of coating performance. The unexpectedly low neutrophil activation showed by composites containing near-monolayer concentrations of exposed IgG and sIgA, respectively, suggested that these act synergistically with mucin on the surface. In support of this hypothesis, quartz crystal microbalance with dissipation monitoring measurements revealed that a preadsorbed BSM layer stabilizes IgG through complexation on a polymeric model surface. Our findings link well to the complex in vivo situation and suggest that functional mucosal mimics can be created in situ for improved biomaterials performance.
|
|
| 24. |
- Sandberg, Tomas, et al.
(författare)
-
Surface analysis of pure and complex mucin coatings
- 2009
-
Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 0021-9797 .- 1095-7103. ; 333:1, s. 180-187
-
Tidskriftsartikel (refereegranskat)abstract
- In the past, we introduced the idea of using mucin coatings to improve biomaterials performance. Here, we evaluate non-radioactive methods for the analysis of pure and human host protein-containing (complex) mucin coatings on a real-type substrate (Thermanox). A common protein quantification assay (mBCA) was combined with mass-calibrated, enzyme-amplified assays based on lectin (ELLA) and antibody (ELISA) recognition, to determine the total and specific amounts of surface-associated proteins. Model studies showed the mBCA assay to be of limited use at low mass loads, and steric effects to influence the ELLA at high surface layer densities. Non-specific responses due to substrate interaction were low for the ELLA and ELISAs. Cross-reactions were observed during ELLA analysis of analytes sharing high degree of O-glycosylation. Combined mBCA-ELLA-ELISA analysis suggested that mucin desorption was low upon protein addition and that low concentrations of ELISA-determined Protein for the complex coatings Could be explained in terms of low accessibility of proteins to the bulk environment. Specifically, a methodology is presented for the determination of the fraction of surface-exposed, presumed bioactive proteins in a complex mucin coating. Finally, X-ray photoelectron spectroscopy and infrared reflectance spectroscopy combined with multivariate data analysis were proven useful in the evaluation of mucin-based coatings.
|
|
| 25. |
- Xue, Bo, et al.
(författare)
-
Chromatographic and fluorometric study of interactions between thiophilic and hydrophobic ligands and tryptophan peptide homologues
- 2006
-
Ingår i: Journal of Chromatography A. ; 1107:1-2, s. 46-51
-
Tidskriftsartikel (refereegranskat)abstract
- The interactions of tryptophan and its peptide homologues with thiophilic ligands were studied in terms of their chromatographic retention and steady-state fluorescence under various conditions, and compared with non-polar structures typically regarded as pure hydrophobic ligands. The experimental results show that both non-polar and polar interactions are involved in what has been termed “thiophilic adsorption chromatography”.
|
|
| 26. |
- Åsberg, Peter, 1973-
(författare)
-
Hydrogels of conjugated polyelectrolytes for biosensor and biochip applications
- 2005
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the use of conjugated polyelectrolytes (CPEs) in biosensor devices. The method is based on non-covalent assembly of the biomolecule of interest and the CPE functioning as the reporter, in one case as a transducer, of biomolecular events. Devices of these assemblies on solid supports that can operate in liquid solutions have been the focus. Polythiophenes, both semiconducting and conducting, is the class of materials that has been used in this work. The semiconducting polythiophenes have ionic side chains which makes them water soluble. This ionic side chain is capable of both forming electrostatic and hydrogen bonds, and when paired with the hydrophobic backbone of the polymer a great number of interactions with biomolecules are possible. The highly conducting polythiophene derivative PEDOT -PSS, (PEDOT) doped with ionic and water soluble PSS polyelectrolyte, was used as the conducting material in 3D-electrode. Both the semiconducting and conducting polymers described above forms hydrogels on solid supports if crosslinked with the appropriate ion, biomolecule or polymer. Evaluation of the CPEs, both with and without biomolecules, was performed in liquid, solid and hydrogel state using a number of techniques. This was done to understand how the CPEs behave when exposed to different buffer systems and various biomolecules.Hydrogels of conjugated polyelectrolytes combined with biomolecules are attractive as biosensors. The advantage with the hydrogel format is the high water content, the porous structure and the large capacity of binding molecules. High water content is important to preserve the biomolecules by providing the correct buffered environment. In this thesis we demonstrated a hydrogel of the highly conducting PEDOT -PSS polymer that was crosslinked on a solid support together with horseradish peroxidase (HRP) enzyme, forming an enzyme-enhanced electrode. Further studies of hydrogels were done using in situ quartz crystal microbalance with dissipation (QCM-D). POWT is a CPE withproperties well suited for biochip applications and readily forms hydrogels when exposed to water-based buffer solutions or biomolecule solutions. Detection ofcomplementary DNA and rejection of non-complementary DNA in a POWT hydrogel was demonstrated. The interaction between POWT and DNAoligonucleotides was also evaluated using fluorescence resonance energy transfer (FRET) in solution. Labeled DNA oligonucleotides with energy accepting or donating fluorophores allowed us to determine distance and binding stoichiometry in the non-covalent POWT-DNA complex.Patterning and anchoring of biomolecules and non-covalent assembled CPE-biomolecule complexes to a chip surface was studied; in the adsorbed state these complexes are hydrogels. Our novel method is based on the modification of the surface energy of a hydrophilic substrate surface using hydrophobic poly(dimethylsiloxane) (PDMS) elastomer stamp containing a relief pattern. Different conformations in biomolecules could be detected using fluorescence microscopy, where the CPEs acts as reporters and the PDMS modified substrates as discriminator. Also, excellent enzyme activity in patterned CPE/Horseradish peroxidase (HRP) enzyme was shown.Distances between the individual molecules in solid state devices of conjugated polymers can be small. In luminescence devices, such as light emitting diodes or fluorescence biosensors, there is a chance of interaction between conjugated molecules especially if more than one type of molecule is present. Quenching of the light and fluorescence energy transfer can occur and a simple approach to study this was developed.
|
|
