| 1. |
- Shafaat, Atefeh
(författare)
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Development of Wireless Biosensors Integrated into the Radio Frequency Antenna for Chipless and Battery-less Monitoring of Biological Reactions
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Development of wireless sensors and biosensors is currently experiencing a rapid progress with a substantial focus directed toward highlighting their potential applications as non-invasive wearables, implants, and highly mobile point-of-care devices. Integration of wireless biosensors into the Internet of Things (IoT) is widely acknowledged as a technological advancement with the potential to significantly change daily life. To maximize this potential, simple integration of biosensors with wireless communication elements would be advantageous. In this regard, systems functioning in chipless, and battery-less modes outperform integrated circuit (IC) based and battery-powered wireless biosensors. Nevertheless, the accessibility of these wireless designs is still limited. In this study, we present a novel approach where incorporating silver nanoparticles(AgNPs) as a part of the radio frequency (RF) tag antenna enables the realization of simple, chipless, and battery-less wireless sensing of biological oxidation and reduction reactions. We exemplified the mechanism of operation in such systems by electronic wiring of enzymes through direct electron transfer (DET) and microorganisms through mediated electron transfer (MET) to the redox conversion of Ag/AgCl. The wiring was designed to facilitate the transformation of metallic AgNPs into AgCl (Ag → AgCl) or the conversion of AgCl particles back into metallic AgNPs (AgCl → Ag) when the enzymatic/microorganism based electron transfer reactions were present. These reactions occurring on the biosensor RF tag antenna strongly changed the impedance of the tag, which was wirelessly monitored by a radio frequency identification (RFID) reader. The functionality of the proposed setup in direct electron transfer coupling of the enzymatic reactions to the redox conversion of the Ag/AgCl was demonstrated by wireless detection of glucose in whole blood samples and hydrogen peroxide penetrated through the skin membrane using the enzymes glucose dehydrogenase(GDH) and horseradish peroxidase (HRP). Additionally, the capability of the proposed configuration in mediated electron transfer wiring of microorganisms to the Ag/AgCl electrochemistry was shown by wireless monitoring of medically relevant microbial biofilms in simulated wound fluid. Generalizing, the results of this work, for the first time, demonstrated that exploiting Ag/AgCl as a part of the tag antenna allows simple, chipless, and battery-less wireless sensing of biological oxidation and reduction reactions.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- Blasi Romero, Anna
(författare)
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Bioactive nanocellulose materials for the treatment of chronic wounds
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chronic wounds represent a burden for the healthcare system and significantly affect the quality of life of the patients. There is currently a lack of efficient treatments but new, improved therapeutic approaches are under development. Suggested innovative wound care therapies consist on the topical administration of bioactive compounds aimed at restoring the balance in the wound environment and promoting the healing. However, their effectiveness is limited due to the highly oxidative and proteolytic environment in the chronic wound. In the work presented in this thesis, a series of bioactive nanocellulose-based materials were developed with the aim of addressing some of the present demands in chronic wound care. Wood-derived cellulose nanofibrils (CNFs) were functionalized with selected bioactive molecules expected to endow CNFs with the ability to modulate the chronic wound environment. Different chemical approaches were explored to combine CNFs with the following biomolecules: the amino acid cysteine, the peptide oligoproline and the host defense peptide KR-12. Materials were characterized in terms of chemical structure, degree of substitution and bioactivity.The immobilization of cysteine onto CNFs (cys-CNF) provided the material with radical oxygen species (ROS) scavenging properties and the ability to inhibit protease activity, properties that were related to the presence of free thiol groups on the nanofibers. Storage conditions in an inert atmosphere or in the form of aerogel were proposed to assure the long-term activity of the cys-CNF material. Investigations on the use of the ROS-sensitive oligoproline to crosslink CNFs provided optimized protocols to maximize peptide substitution and the degree of crosslinking. The oligoproline-CNF materials were sensitive to ROS-mediated cleavage and provided a protective effect to cells exposed to oxidative conditions. Moreover, the feasibility of preparing ROS-responsive drug delivery hydrogels based on the oligoproline-CNF was demonstrated, with indications that tuning the length of the oligoproline peptide could be exploited to tailor the release rate of small proteins. CNF materials with antibacterial properties and the ability to modulate the response of pro-inflammatory macrophages were obtained by immobilizing KR-12 derivatives onto CNFs. This study highlighted the importance in the selection of the conjugation chemistry to preserve the activity of the peptide once immobilized. To conclude, this work has contributed with valuable strategies to develop bioactive CNF-based materials with the potential of paving the way for advanced solutions in the field of chronic wound care.
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| 6. |
- Naeimipour, Sajjad, 1987-
(författare)
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Modular Enzyme-Responsive Polysaccharide-Based Hydrogels for Biofabrication
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Engineered human tissue and disease models can decrease the cost and time of developing new drugs and treatments, facilitate personalized medicine, and eliminate the need for animal models that poorly represent the human body and are ethically problematic. However, the current conventional cell expansion methods using 2D culture flasks cannot enable the development of such complex multi-cellular 3D models. In general, hydrogels are considered promising materials that can make the biofabrication of tissue models possible. Hydrogels are highly hydrated materials comprised of either synthetic or naturally derived polymers, or a combination of both, and can form an environment mimicking the biomacromolecular network surrounding cells in the body. This network of biopolymers, known as extracellular matrix (ECM), is comprised of proteins such as collagen, laminin, fibronectin, and polysaccharides such as hyaluronan (HA), heparan, keratan, and chondroitin sulfate. The design of hydrogels representing the physical and biochemical properties of the ECM and which can be used for biofabrication is challenging but of increasing interest due to the rapid progress in the development of 3D and 4D bioprinting techniques. As the ECM properties differ between various tissues and disease conditions and change over time, a dynamic modular hydrogel system is needed to that can be optimized for each cell and tissue type. This thesis aims to develop modular enzyme-responsive polysaccharide-based hydrogels for 3D cell culture and biofabrication. The natural polysaccharides, hyaluronic acid (HA) and alginate (Alg) were used as the main backbone in the hydrogels developed in this thesis. HA was modified by conjugating bicyclo[6.1.0]non-4-yne (BCN) to the backbone to form HA-BCN-based hydrogels by a bioorthogonal strain-promoted alkyne-azide cycloaddition. The click reaction between BCN and azide groups allowed for modulating the biochemical and mechanical properties of the HA-BCN hydrogels. HA-BCN was further decorated with peptides to explore peptide folding and dimerization-mediated dynamic cross-linking and biofunctionalization. This system was further used to explore possibilities to dynamically alter the properties of 3D bioprinted structures, mimicking the biomineralization process in bone tissue. In a different study, a tumor model comprising fibroblast and breast cancer cells (MCF7) was bioprinted using HA-BCN cross-linked by matrix metalloporotease (MMP) cleavable and PEG-diazide MMP-resistant cross-linkers, demonstrating the synergistic relationship between hydrogel degradability and cancer cell growth, intensified by the presence of fibroblasts. The possibility of incorporating a conductive module into this hydrogel system was explored using the enzyme-assisted polymerization of ETE-S to form an interpenetrating conductive network inside HA-BCN hydrogel. The in situ and user-triggered polymerization of conductive ETE-S was demonstrated after 3D printing HA-BCN bioink containing ETE-S monomers into a lattice shape structure. We also demonstrated that cellulose nanofibrils (CNF) improved the printability of HA-BCN bioinks, and this hybrid bioink was used for printing self-standing cell-laden 3D structures. Besides these studies, a novel enzymatically triggered thiol-based chemistry was developed to address the unwanted oxidation of thiol-containing hydrogels and decrease the off-target thiol reactions during hydrogel synthesis and formation. Alginate containing sulfhydryl moieties, protected by an enzyme-labile Phacm group (AlgCP), was treated with penicillin G acylase and subsequently formed a disulfide cross-linked hydrogel. We studied the gelation kinetics and rheological properties of AlgCP and different modes of cross-linking by reversible disulfide bonds, a thiol-maleimide Micheale-type addition reaction, and ionic interactions between alginate and Ca2+ ions. MCF7 breast cancer cells cultured in the AlgCP hydrogels formed spheroids that could be harvested by GSH dissolution of the hydrogels. Finally, this novel chemistry enabled bioprinting of multi-material 3D structures with control over the printed structure's physiochemical properties, including the type and density of cross-linkers. Bioprinted fibroblasts formed extended morphology, and MCF7 cells formed spheroids in the bioprinted lattice structures. The hydrogel systems developed and explored in this thesis are modular and exhibit dynamic and tunable properties, and are applicable for a wide range of 3D cell culture and bioprinting applications. The hydrogels were either formed in response to the activity of an enzyme or remodeled by enzymes. Both enzyme-responsive HA-BCN and AlgCP hydrogel systems are promising bioinks for generating more elaborate and spatially defined cell-laden 3D structures whose features can be altered post-printing by cell-secreted and extrinsic reagents. These hydrogel-based toolkits can play a vital role in developing tissue and disease models that can make the drug discovery process faster, cheaper, and animal-free.
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