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- Martinsson, Erik, 1983-
(författare)
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Nanoplasmonic Sensing using Metal Nanoparticles
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In our modern society, we are surrounded by numerous sensors, constantly feeding us information about our physical environment. From small, wearable sensors that monitor our physiological status to large satellites orbiting around the earth, detecting global changes. Although, the performance of these sensors have been significantly improved during the last decades there is still a demand for faster and more reliable sensing systems with improved sensitivity and selectivity. The rapid progress in nanofabrication techniques has made a profound impact for the development of small, novel sensors that enables miniaturization and integration. A specific area where nanostructures are especially attractive is biochemical sensing, where the exceptional properties of nanomaterials can be utilized in order to detect and analyze biomolecular interactions. The focus of this thesis is to investigate plasmonic nanoparticles composed of gold or silver and optimize their performance as signal transducers in optical biosensors. Metal nanoparticles exhibit unique optical properties due to excitation of localized surface plasmons, which makes them highly sensitive probes for detecting small, local changes in their surrounding environment, for instance the binding of a biomolecule to the nanoparticle surface. This is the basic principle behind nanoplasmonic sensing based on refractometric detection, a sensing scheme that offers real-time and label-free detection of molecular interactions. This thesis shows that the sensitivity for detecting local refractive index changes is highly dependent on the geometry of the metal nanoparticles, their interaction with neighboring particles and their chemical composition and functionalization. An increased knowledge about how these parameters affects the sensitivity is essential when developing nanoplasmonic sensing devices with high performance based on metal nanoparticles.
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| 2. |
- Selegård, Robert, 1934-
(författare)
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Polypeptide functionalized gold nanoparticles for bioanalytical applications
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Detection strategies that allow for simple, rapid, cost efficient and sensitive monitoring of proteins and their interactions with biomolecules are of great importance in drug development and diagnostics. This thesis describes the development of bioanalytical applications based on the tunable self-assembly of gold nanoparticles functionalized with a de novo designed polypeptide. Strategies for protein affinity sensing and for detection of several fundamentally important biological processes have been investigated, including Zn2+-mediated coordination between polypeptides and low molecular weight chelants and protease and phosphatase activity.A Zn2+ responsive synthetic polypeptide designed to fold into a helix-loop-helix motif and dimerize into a four-helix bundle has been used to control the stability and self-assembly of gold nanoparticles. This polypeptide has a high negative net charge at neutral pH as a consequence of its many glutamic acid residues, efficiently preventing folding and dimerization due to charge repulsion. Zn2+ coordination provides a means to trigger folding and dimerization at neutral pH. The polypeptide can be readily attached to gold nanoparticles via a cysteine residue in the loop region, retaining its folding properties and responsiveness to Zn2+. The polypeptide functionalized gold nanoparticles display excellent colloidal stability but aggregate reversibly after addition of millimolar concentrations of Zn2+. Aggregates are dense with a defined interparticle distance corresponding to the size of the four-helix bundle, resulting in a distinct red shift of the localized surface plasmon resonance band.Three completely different strategies for colorimetric biosensing have been developed, all being based on the same responsive hybrid nanomaterial. In the first strategy a synthetic receptor was co-immobilized on the gold nanoparticles together with the Zn2+ responsive polypeptide. Protein analyte binding to the receptor could be detected as this interaction sterically prevented aggregation induced by Zn2+. In the second strategy the reduction in colloidal stability caused by specific proteolytic cleavage of the immobilized polypeptide was exploited to monitor the enzymatic activity. The third strategy utilized the sensitivity of the system to small variations in Zn2+ concentration. The presence of low molecular weight chelants was found to influence the mode of aggregation, both by sequestering Zn2+ and through the formation of ternary complexes involving the polypeptides, which prevented dimerization and thus aggregation. This approach was further developed into a generic concept for phosphatase detection exploiting the different affinity of enzyme substrates and reaction products for Zn2+.The flexibility of the different detection schemes enables detection of a large number of analytes by exploiting the tunable stability of the nanoparticles and the possibilities to effectively decouple the recognition event and the nanoparticle stability modulation.
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| 3. |
- Utterström, Johanna, 1993-
(författare)
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Design and Optimization of Membrane Active Peptides and Lipid Vesicles for Triggered Release
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Liposomes can reduce toxic side effects and improve the efficacy of drugs and several liposome-based drug formulations are approved for clinical use. The therapeutic effect is dependent on the bioavailability of the drug and a slow drug release from liposomes can reduce their efficacy. Multiple strategies have been proposed to control the release of drugs from liposomes using both external stimuli such as light, heat and ultrasound, and endogenous factors such as changes in pH or enzymatic activity. However, because of the difficulties to efficiently modulate lipid membrane permeability and the challenges to trigger drug release in the target tissue, no stimuli responsive liposomes have so far been approved. There is consequently a great need for new means to tune lipid membrane integrity for liposome cargo release to improve the development of new advanced drug delivery systems for better and safer treatment of patients. The aim of this thesis was to design and explore synthetic membrane active peptides for triggered release from liposomes and to expand the knowledge on how peptide-lipid conjugation strategies and lipid properties affect the membrane activity of the peptides. This work was based on two different de novo designed cationic and amphipathic, conjugation-dependent membrane active peptides (CKV4 and JR2KC). Both peptides fold and adopt α-helical structures upon conjugation to liposomes, triggering lipid membrane destabilization. Addition of cholesterol in the lipid membrane greatly enhanced the release efficiency of JR2KC due to a peptide-triggered lipid phase separation, resulting in domains with high local peptide concentrations. Additionally, both peptide surface concentrations and lipid net charge were found to be important factors for efficient release. However, when the zeta potential decreased below -75 mV, conjugation-independent release mechanisms were triggered. Liposome size was shown to only have minor effects on the release kinetics for both sets of peptides while a mixture of saturated and unsaturated lipids was beneficial for the peptide-triggered membrane destabilization, possibly due to increased propensity for lipid phase separation. In addition to changing lipid properties, peptide-lipid conjugation strategies proved to highly affect the release kinetics, where the Michael addition reaction between a cysteine in the peptide and maleimide-lipids was much more efficient in causing peptide-triggered membrane destabilization than strain-promoted alkyne azide cycloaddition reactions using azide-modified peptides and DBCO-functionalized lipids. However, thiols tend to oxidize under ambient conditions which complicates peptide-lipid conjugation. This was addressed by synthesizing a peptide with a cysteine modified with an enzyme labile thiol protection group. Enzymatic deprotection allowed efficient peptide-lipid conjugation, reducing the risk of peptide oxidation. To further find means to tailor peptide-lipid interactions, we explored the effect of a competing peptide heterodimerization process on lipid membrane destabilization. Addition of a charge complementary peptide to CKV4 resulted in heterodimerization and folding into a coiled coil, which inhibited its membrane activity. However, when the two peptides were synthesized as a single sequence, the membrane activity was altered, most likely due to the induced preorganization increasing membrane affinity. The results presented in this thesis provide new understandings of the complex peptide-lipid interactions that govern peptide-induced release from liposomes and will facilitate further optimization in peptide design for the future development of advanced liposome-based drug delivery systems.
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