Concentrating Membrane Proteins Using Asymmetric Traps and AC Electric Fields

Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol.2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery2006, 5, 993). However, the structure-function relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structure-function determination of these species (Poo et al. Nature1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a “nested trap” and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins

The enzyme mechanism of copper-containing nitrite reductase from Alcaligenes faecalis and its application in biosensor-like devices

Copper-containing nitrite reductases (NiRs) are enzymes that efficiently reduce nitrite to nitric oxide in potent denitrifying bacteria. There has been an interest in their application in amperometric biosensors for monitoring nitrite levels in natural and waste waters. NiRs have a complex enzyme mechanism and depend on nitrite concentration and pH. Although the mechanism has been intensively studied, it is still controversial. In this thesis, a combined fluorescence and electrochemical method is used to simultaneously monitor the nitrite turn-over rate of a NiR from Alcaligenes faecalis S-6 and the oxidation state of the type-1 copper electron transfer site inside the enzyme. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labelled enzyme molecules immobilised on modified gold, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by F6rster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. Here, a determining role of internal electron transfer is found in NiR's mechanism. Moreover, the heterogeneous interfacial electron transfer to adsorbed NiRs is observed. The electro- activity and binding of labelled and unlabelled NiRs on gold modified with different self-assembled monolayers (SAMs) are studied to understand the effect of NiR labelling on the protein-electrode interactions. Here, electro-active and well-ordered biofilms of NiRs are found on electrodes with SAMs carrying the positive charge (negative NiRs) or when NiRs are modified with fluorophores that help orienting them on SAM-modified gold. Attempts have also been made to implement fluorescently labelled NiRs into the biosensor-like device with a fluorescence output using gold electrodes modified with tethered lipid bilayers (tBLMs) or conducting polymers (CPs). In spite of being unsuccessful, these studies give a better understanding of potential-dependent polymer dynamics and a control over protein immobilisation on functionalised lipid bilayer platforms.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Piezoresistive Load Sensing and Percolation Phenomena in Portland Cement Composite Modified with In-Situ Synthesized Carbon Nanofibers

Carbon nanofibers (CNFs) were directly synthesized on Portland cement particles by chemical vapor deposition. The so-produced cements contained between 2.51–2.71 wt% of CNFs; depending on the production batch. Several mortar mixes containing between 0 and 10 wt% of the modified cement were produced and the electrical properties at various ages and the load sensing capabilities determined. The percolation threshold related to the electrical conductivity was detected and corresponded to the amount of the present CNFs, 0.271, 0.189, 0.135 and 0.108 wt%. The observed threshold depended on the degree of hydration of the Portland cement. The studied mortars showed a strong piezoresistive response to the applied compressive load reaching a 17% change of the electrical resistivity at an applied load of 3.5 MPa and 90% at 26 MPa. This initial study showed that the studied material is potentially suitable for future development of novel fully integrated monitoring systems for concrete structures.Validerad;2019;Nivå 2;2019-04-15 (oliekm)</p

Sensing mechanisms of nanomodified Portland cement composites

Mortar sensors were fabricated as beams incorporating different amounts of carbon nanofibers (CNFs) synthesized in-situ on cement particles. Changes in electrical resistivity were measured and compared to recorded changes in compressive stress, temperature, and humidity. Sensing mechanisms and corresponding models were developed. The findings of the study indicate that the piezoresistive effect is influenced by the critical concentration of CNFs inside the composite matrix and the tunneling effect. In addition, water absorption and desorption, as well as the amount of chemically bound water played an important role in humidity sensing. Thermal fluctuation-induced tunneling conduction was dominant for the temperature sensitivity.Validerad;2024;Nivå 2;2024-06-03 (joosat);Funder: Skanska Sverige;Full text: CC BY License;</p

Novel humidity sensors based on nanomodified Portland cement

Commonly used humidity sensors are based on metal oxides, polymers or carbon. Their sensing accuracy often deteriorates with time, especially when exposed to higher temperatures or very high humidity. An alternative solution based on the utilization of Portland cement-based mortars containing in-situ grown carbon nanofibers (CNFs) was evaluated in this study. The relationship between the electrical resistivity, CNF content and humidity were determined. The highest sensitivity was observed for samples containing 10 wt.% of the nanomodified cement which corresponded to 0.27 wt.% of CNFs. The highest calculated sensitivity was approximately 0.01024 per 1% change in relative humidity (RH). The measured electrical resistivity is a linear function of the RH in the humidity range between 11% and 97%. The percolation threshold value was estimated to be at around 7 wt.% of the nanomodified cement, corresponding to ~0.19 wt.% of CNFs.Validerad;2021;Nivå 2;2021-04-19 (alebob)</p

Load Sensing Capability of Cementitious Matrixes—Nanomodified Cement Versus Carbon Nanotube Dispersion

A cement-based matrix incorporating conductive materials such as carbon nanotubes and carbon nanofibers can have self-sensing capability. Both nanomaterials are characterized by excellent physical, mechanical and electrical properties. A disadvantage is that due to their hydrophobic nature it is very difficult to ensure uniform dispersion throughout the cementitious matrix. To overcome this problem a new nanomodified cement containing in-situ attached CNFs was developed leading to a very homogenous and conductive binder matrix. This study aimed to compare the piezoresistive responses of two types of matrixes, one based on the nanomodified cement and the second containing multi-walled carbon nanotubes. Several mortars were prepared containing either MWCNTs or the nanomodified cement, which partially replaced the untreated cement. The effective amount of the carbon nanomaterials was the same for both types of mixes and ranged from 0 wt.% to 0.271 wt.%, calculated by the all binder weight. Changes in the electrical properties were determined while applying compressive load. The results showed that the binders based on the nanomodified cement have significantly better load sensing capabilities and are suitable for applications in monitoring systems

Monitoring temperature and hydration by mortar sensors made of nanomodified Portland cement

Mortar beams incorporating carbon nanofibers (CNFs), which were synthesized in situ on Portland cement particles, were used to produce nanomodified Portland cement sensors (SmartCem sensors). SmartCem sensors exhibited an electrical response comparable to a thermistor with a temperature coefficient of resistivity of − 0.0152/ °C. The highest temperature sensing was obtained for the SmartCem sensor, which contained ~ 0.271 wt.% of CNFs. The calculated temperature sensitivity was approximately 11.76% higher in comparison with the mortar beam containing only unmodified Portland cement. SmartCem sensors were used to monitor the cement hydration in large-scale self-compacting concrete beams. The measurements were conducted after casting for 7 days. Additionally, commercially available thermocouple and humidity sensors were used as references. The results showed that changes in electrical resistivity measured by the SmartCem sensor were well aligned with the ongoing hydration processes.Validerad;2023;Nivå 2;2023-12-04 (joosat);Full text license: CC BYThis article has previously appeared as a manuscript in a thesis.</p