| 1. |
- Josefsson, Leila, et al.
(författare)
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Analysis of polyvinyl alcohol microbubbles in human blood plasma using capillary electrophoresis
- 2016
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Ingår i: Journal of Separation Science. - : Wiley-VCH Verlagsgesellschaft. - 1615-9306 .- 1615-9314. ; 39:8, s. 1551-1558
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Tidskriftsartikel (refereegranskat)abstract
- Recently, a new type of ultrasound contrast agent that consists of air-filled microbubbles stabilized with a shell of polyvinyl alcohol was developed. When superparamagnetic nanoparticles of iron oxide are incorporated in the polymer shell, a multimodal contrast agent can be obtained. The biodistribution and elimination pathways of the polyvinyl alcohol microbubbles are essential to investigate, which is limited with today's techniques. The aim of the present study was, therefore, to develop a method for qualitative and quantitative analysis of microbubbles in biological samples using capillary electrophoresis with ultraviolet detection. The analysis parameters were optimized to a wavelength at 260 nm and pH of the background electrolyte ranging between 11.9 and 12. Studies with high-intensity ultrasonication degraded microbubbles in water showed that degraded products and intact microbubbles could be distinguished, thus it was possible to quantify the intact microbubbles solely. Analysis of human blood plasma spiked with either plain microbubbles or microbubbles with nanoparticles demonstrated that it is possible to separate them from biological components like proteins in these kinds of samples.
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| 2. |
- Josefsson, Leila
(författare)
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Bioanalysis using capillary electrophoresis and mass spectrometry : Applied on proteins, protein nanofibrils and polyvinyl alcohol microbubbles
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The sequencing of the genome of various species, including the human species, have led to increased understanding about how a protein structure is generated, and how specific structures are related to the proteins’ functionality. In paper I and II of this thesis, the folding of proteins in vitro to form hierarchical nanostructures, which in vivo often have a pathological effect, have been studied. Protein isolates from soybean and potato, that are byproducts from oil and starch production, respectively, were used as a starting material for protein nanofibril (PNF) formation, and mass spectrometry was used to identify the building blocks that are included in the formed PNF. The five peptides identified in soybean PNF and the six peptides identified in potato PNF originated from the major seed storage proteins for the respective crop.The use of ionic liquids has increased for improvement of the performance of different separation techniques due to their adjustable properties, and good solvating ability. In paper III, an ionic liquid and water mixture was used as background electrolyte in capillary electrophoresis for protein separation. The system showed high reproducibility at basic conditions, and could potentially be used for routine control analysis.Many diseases and injuries require clinical diagnosis techniques e.g. ultrasound imaging, to be detected, and for the physician to be able to decide the correct therapy. To increase the resolution of such imaging techniques, contrast agents can be used. In paper IV-VI, a newly developed contrast agent consisting of air-filled microbubbles stabilized with a shell of polyvinyl alcohol (PVA-MBs) was studied. Development of a capillary electrophoretic method for analysis of the PVA-MBs with the intentions to be used for clinical diagnosis is performed, where different detectors such as a UV detector, a UV area imaging detector and an in-house constructed microscope are used to increase the sensitivity of detection for the PVA-MBs. The developed method could be used for quantification of the contrast agent, since individual PVA-MBs were visible using the imaging detectors. Findings regarding the mobility of the PVA-MBs in human blood plasma and in water implies that a protein corona was formed around the MBs.
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| 3. |
- Josefsson, Leila
(författare)
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Bioanalytical separation using capillary electrophoresis : Applications with microbubbles and proteins
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis the possibilities of using capillary electrophoresis as a separation technique for analysis of proteins and microbubbles is presented.A complete analytical process consists of five necessary steps of which one is the actual analysis step. For this step a suitable analytical technique is needed. Capillary electrophoresis (CE) is one of the common analytical separation techniques used for analysis of a diversity of analytes, and can be both used in routine analysis and for research purposes. The reason for using CE, compared to other liquid-based separation techniques, is mainly short analysis time, high resolution, and negligible sample volumes and solvent waste. Depending on the characteristics of the analytes, and the sample matrix, different modes of CE can be used, where capillary zone electrophoresis (CZE) is the most employed one. The basic principle of CZE is separation of the analytes due to differences in total mobility, which is dependent on the charge and size of the analytes, and the electroosmotic flow (EOF). The EOF can be controlled by several parameters e.g. choice of background electrolyte (BGE), and the optimization of the parameters has been discussed throughout the thesis.To improve the properties of the BGE, an ethylammonium nitrate (EAN) water solution was used as BGE for CE analysis in Paper I. The precision of the EOF with this method was determined by adjusting the pH of the BGE, the concentration of EAN in the BGE, and the electric field. Model proteins were thereafter analysed using the optimal parameters yielding a precision sufficient for routine control.One example of the applications of CE is separation of novel contrast agents, which consist of polyvinyl alcohol microbubbles (PVA-MBs). In Paper II, a method for analysis of PVA-MBs in biological samples using CE with UV-detection was developed. It was also established that intact PVA-MBs could be distinguished from ultrasound degraded PVA-MBs in the same set-up.
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| 4. |
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| 5. |
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| 6. |
- Josefsson, Leila, et al.
(författare)
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Implementation of a ultraviolet area imaging detector for analysis of polyvinyl alcohol microbubbles by capillary electrophoresis
- 2020
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1619
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Tidskriftsartikel (refereegranskat)abstract
- Contrast agents are widely used to enhance the image quality in clinical imaging using e.g. ultrasound. The contrast agents used for ultrasound imaging are mainly microbubbles (MBs) with a soft or hard shell encapsulating a core of gas. In the present study, MBs with a hard shell of polyvinyl alcohol (PVA), and a core of air were analysed in a capillary electrophoretic system using a UV area imaging detector. The detector was operating at 3 wavelengths; 214 nm, 255 nm and 525 nm, and the highest absorbance for individual PVA-MBs were obtained at 214 nm. Two detection windows and a vertical loop capillary position enabled tracking of the PVA-MBs both in an upward and a downward flow direction, where PVA-MBs had different flow distributions and slightly higher average flow velocity upwards, attributed to temperature differences in the capillary that was part within the instrument and part outside. The tracking also allowed counting and quantification of the PVA-MBs. Separation of PVA-MBs from proteins present in human blood plasma was achieved, with multi-wavelength imaging showing best contrast at 525 nm. The PVA-MBs absolute values of negative zeta potential and anionic mobility when injected from plasma in the pH 12 background electrolyte are higher than those obtained for MBs injected from buffer, consistent with their increased negative charge due to a protein corona coating of the PVA-MBs.
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| 7. |
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| 8. |
- Josefsson, Leila, et al.
(författare)
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Potato Protein Nanofibrils Produced from a Starch Industry Sidestream
- 2020
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Ingår i: ACS Sustainable Chemistry and Engineering. - : AMER CHEMICAL SOC. - 2168-0485. ; 8:2, s. 1058-1067
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Tidskriftsartikel (refereegranskat)abstract
- Protein nanofibrils have emerged as promising building blocks in functional bio/nanomaterials as well as in food products. We here demonstrate that nanofibrils with amyloid-like properties can be produced from potato protein isolate, a major sidestream from the starch industry. Methods for solubilization of potato proteins are evaluated, and a protocol for the assembly of protein nanofibrils is presented. Characterization of the nanofibrils shows that they are rich in beta-sheet structure and display the cross-beta X-ray fiber diffraction pattern, which is a hallmark of amyloid-like fibrils. Atomic force microscopy shows that the fibrils are ca. 4-5 nm in diameter with a nanoscale morphology that displays a high degree of curvature. Using mass spectrometry we identify four peptides that constitute the core building blocks of the nanofibrils and show that they originate from two different classes of proteins. The structural characteristics of these peptides are distinct from previously studied plant protein nanofibrils and thereby reveal new knowledge about the formation of protein nanostructures from agricultural resources.
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| 9. |
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| 10. |
- Josefsson, Leila, et al.
(författare)
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Structural basis for the formation of soy protein nanofibrils
- 2019
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Ingår i: RSC Advances. - : ROYAL SOC CHEMISTRY. - 2046-2069. ; 9:11, s. 6310-6319
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid-like protein nanofibrils (PNFs) can assemble from a range of different proteins including disease-associated proteins, functional amyloid proteins and several proteins for which the PNFs are neither related to disease nor function. We here examined the core building blocks of PNFs formed by soy proteins. Fibril formation at pH 2 and 90 degrees C is coupled to peptide hydrolysis which allows isolation of the PNF-forming peptides and identification of them by mass spectrometry. We found five peptides that constitute the main building blocks in soy PNFs, three of them from the protein b-conglycinin and two from the protein glycinin. The abilities of these peptides to form PNFs were addressed by amyloid prediction software and by PNF formation of the corresponding synthetic peptides. Analysis of the structural context in the native soy proteins revealed two structural motifs for the PNF-forming peptides: (i) so-called b-arches and (ii) helical segments involved in quaternary structure contacts. However, the results suggest that neither the native structural motifs nor the protein of origin defines the morphology of the PNFs formed from soy protein isolate.
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