Lysozyme adsorption at a silica surface using simulation and experiment : effects of pH on protein layer structure

Hen Egg White Lysozyme (HEWL) is a widely used exemplar to study protein adsorption on surfaces and interfaces. Here we use fully atomistic Molecular Dynamics (MD) simulations, Multi-Parametric Surface Plasmon Resonance (MP-SPR), contact angle and zeta potential measurements to study HEWL adsorption at a silica surface. The simulations provide a detailed description of the adsorption mechanism and indicate that at pH7 the main adsorption driving force is electrostatics, supplemented by weaker hydrophobic forces. Moreover, they reveal the preferred orientation of the adsorbed protein and show that its structure is only slightly altered at the interface with the surface. This provides the basis for interpreting the experimental results, which indicate the surface adsorbs a close-packed monolayer at about pH10 where the surface has a large negative zeta potential and the HEWL is positively charged. At higher pH, the adsorption amount of the protein layer is greatly reduced due to the loss of charge on the protein. At lower pH, the smaller zeta potential of the surface leads to lower HEWL adsorption. These interpretations are complemented by the contact angle measurements that show how the hydrophobicity of the surface is greatest when the surface coverage is highest. The simulations provide details of the hydrophobic residues exposed to solution by the adsorbed HEWL, completing the picture of the protein layer structure

Haar expectations of ratios of random characteristic polynomials

We compute Haar ensemble averages of ratios of random characteristic polynomials for the classical Lie groups K = O(N), SO(N), and USp(N). To that end, we start from the Clifford-Weyl algebera in its canonical realization on the complex of holomorphic differential forms for a C-vector space V. From it we construct the Fock representation of an orthosymplectic Lie superalgebra osp associated to V. Particular attention is paid to defining Howe's oscillator semigroup and the representation that partially exponentiates the Lie algebra representation of sp in osp. In the process, by pushing the semigroup representation to its boundary and arguing by continuity, we provide a construction of the Shale-Weil-Segal representation of the metaplectic group. To deal with a product of n ratios of characteristic polynomials, we let V = C^n \otimes C^N where C^N is equipped with its standard K-representation, and focus on the subspace of K-equivariant forms. By Howe duality, this is a highest-weight irreducible representation of the centralizer g of Lie(K) in osp. We identify the K-Haar expectation of n ratios with the character of this g-representation, which we show to be uniquely determined by analyticity, Weyl group invariance, certain weight constraints and a system of differential equations coming from the Laplace-Casimir invariants of g. We find an explicit solution to the problem posed by all these conditions. In this way we prove that the said Haar expectations are expressed by a Weyl-type character formula for all integers N \ge 1. This completes earlier work by Conrey, Farmer, and Zirnbauer for the case of U(N).Comment: LaTeX, 70 pages, Complex Analysis and its Synergies (2016) 2:

Cascading reaction of arginase and urease on a graphene-based FET for ultrasensitive, real-time detection of arginine

Herein, a biosensor based on a reduced graphene oxide field effect transistor (rGO-FET) functionalized with the cascading enzymes arginase and urease was developed for the detection of L-arginine. Arginase and urease were immobilized on the rGO-FET sensing surface via electrostatic layer-by-layer assembly using polyethylenimine (PEI) as cationic building block. The signal transduction mechanism is based on the ability of the cascading enzymes to selectively perform chemical transformations and prompt local pH changes, that are sensitively detected by the rGO-FET. In the presence of L-arginine, the transistors modified with (PEI/urease(arginase)) multilayers showed a shift in the Dirac point due to the change in the local pH close to the graphene surface, produced by the catalyzed urea hydrolysis. The transistors were able to monitor L-arginine in the 10–1000 μM linear range with a LOD of 10 μM, displaying a fast response and a good long-term stability. The sensor showed stereospecificity and high selectivity in the presence of non-target amino acids. Taking into account the label-free, real-time measurement capabilities and the easily quantifiable, electronic output signal, this biosensor offers advantages over state-of-the-art L-arginine detection methods.Fil: Berninger, Teresa. Austrian Institute Of Technology; AustriaFil: Bliem, Christina. Austrian Institute Of Technology; AustriaFil: Piccinini, Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Azzaroni, Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Knoll, Wolfgang. Austrian Institute Of Technology; Austri

A facile molecularly imprinted polymer-based fluorometric assay for detection of histamine

Histamine is a biogenic amine naturally present in many body cells. It is also a contaminant that is mostly found in spoiled food. The consumption of foods containing high levels of histamine may lead to an allergy-like food poisoning. Analytical methods that can routinely screen histamine are thus urgently needed. In this paper, we developed a facile and cost-effective molecularly imprinted polymer (MIP)-based fluorometric assay to directly quantify histamine. Histamine-specific MIP nanoparticles (nanoMIPs) were synthesized using a modified solid-phase synthesis method. They were then immobilized in the wells of a microplate to bind the histamine in aqueous samples. After binding, o-phthaldialdehyde (OPA) was used to label the bound histamine, which converted the binding events into fluorescent signals. The obtained calibration curve of histamine showed a linear correlation ranging from 1.80 to 44.98 μM with the limit of detection of 1.80 μM. This method was successfully used to detect histamine in spiked diary milk with a recovery rate of more than 85%

Interaction of lecithin:cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I-derived peptides

LCAT is an enzyme responsible for the formation of cholesteryl esters from unesterified cholesterol (UC) and phospholipid (PL) molecules in HDL particles. However, it is poorly understood how LCAT interacts with lipoproteins and how apoA-I activates it. Here we have studied the interactions between LCAT and lipids through molecular simulations. In addition, we studied the binding of LCAT to apoA-I-derived peptides, and their effect on LCAT lipid association-utilizing experiments. Results show that LCAT anchors itself to lipoprotein surfaces by utilizing nonpolar amino acids located in the membrane-binding domain and the active site tunnel opening. Meanwhile, the membrane-anchoring hydrophobic amino acids attract cholesterol molecules next to them. The results also highlight the role of the lid-loop in the lipid binding and conformation of LCAT with respect to the lipid surface. The apoA-I-derived peptides from the LCAT-activating region bind to LCAT and promote its lipid surface interactions, although some of these peptides do not bind lipids individually. The transfer free-energy of PL from the lipid bilayer into the active site is consistent with the activation energy of LCAT. Furthermore, the entry of UC molecules into the active site becomes highly favorable by the acylation of SER181.Peer reviewe

Spin coated chitin films for biosensors and its analysis are dependent on chitin-surface interactions

Chitin, abundant in nature, is a renewable resource with many possible applications in bioengineering. Biosensors, capable of label-free and in-line evaluation, play an important role in the investigation of chitin synthesis, degradation and interaction with other materials. This work presents a comparative study of the usefulness of a chitin surface preparation, either on gold (Au) or on polystyrene (PS). In both cases the most common method to dissolve chitin was used, followed by a simple spin-coating procedure. Multi-parametric surface plasmon resonance (MP-SPR), modeling of the optical properties of the chitin layers, scanning electron microscopy, and contact angle goniometry were used to confirm: the thickness of the layers in air and buffer, the refractive indices of the chitin layers in air and buffer, the hydrophobicity, the binding properties of the chitin binding domain (CBD) of Bacillus circulans, and the split-intein capture process. Binding of the CBD differed between chitin on Au versus chitin on PS in terms of binding strength and binding specificity due to a less homogenous structured chitin-surface on Au in comparison to chitin on PS, despite a similar thickness of both chitin layers in air and after running buffer over the surfaces. The use of the simple method to reproduce chitin films on a thin polystyrene layer to study chitin as a biosensor and for chitin binding studies was obvious from the SPR studies and the binding studies of CBD as moiety of chitinases or as protein fusion partner. In conclusion, stable chitin layers for SPR studies can be made from chitin in a solution of dimethylacetamide (DMA) and lithium chloride (LiCl) followed by spin-coating if the gold surface is protected with PS.Peer reviewe

Multi-parametric surface plasmon resonance platform for studying liposome-serum interactions and protein corona formation

When nanocarriers are administered into the blood circulation, a complex biomolecular layer known as the “protein corona” associates with their surface. Although the drivers of corona formation are not known, it is widely accepted that this layer mediates biological interactions of the nanocarrier with its surroundings. Label-free optical methods can be used to study protein corona formation without interfering with its dynamics. We demonstrate the proof-of-concept for a multi-parametric surface plasmon resonance (MP-SPR) technique in monitoring the formation of a protein corona on surface-immobilized liposomes subjected to flowing 100 % human serum. We observed the formation of formulation-dependent “hard” and “soft” coronas with distinct refractive indices, layer thicknesses, and surface mass densities. MP-SPR was also employed to determine the affinity (KD) of a complement system molecule (C3b) with cationic liposomes with and without polyethylene glycol. Tendency to create a thick corona correlated with a higher affinity of opsonin C3b for the surface. The label-free platform provides a fast and robust preclinical tool for tuning nanocarrier surface architecture and composition to control protein corona formation.When nanocarriers are administered into the blood circulation, a complex biomolecular layer known as the "protein corona" associates with their surface. Although the drivers of corona formation are not known, it is widely accepted that this layer mediates biological interactions of the nanocarrier with its surroundings. Label-free optical methods can be used to study protein corona formation without interfering with its dynamics. We demonstrate the proof-ofconcept for a multi-parametric surface plasmon resonance (MP-SPR) technique in monitoring the formation of a protein corona on surface-immobilized liposomes subjected to flowing 100 % human serum. We observed the formation of formulation-dependent "hard" and "soft" coronas with distinct refractive indices, layer thicknesses, and surface mass densities. MP-SPR was also employed to determine the affinity (K-D) of a complement system molecule (C3b) with cationic liposomes with and without polyethylene glycol. Tendency to create a thick corona correlated with a higher affinity of opsonin C3b for the surface. The label-free platform provides a fast and robust preclinical tool for tuning nanocarrier surface architecture and composition to control protein corona formation.Peer reviewe

Development of MP-SPR biosensor analysis methology

Optical biosensors have gained high regard in the field of drug industry and clinical diagnostics owing to the high specificity of biological recognition elements as well as to the non-invasiveness of optical detection methods. The flexible and efficient use of optical biosensors, however, requires that the user understands their basic physical, chemical, and biological principles as well as the advantages and limitations of these devices. The literature review introduces the fundamentals of biosensing and biosensors, and describes the principles, advantages and challenges related to one type of surface plasmon resonance (SPR) device. In the experimental part of the thesis, a multiparametric SPR (MP-SPR)-system was used to measure the optical properties of samples in addition to the traditional adsorption of molecules. The concentrations and purity of protein solutions were determined by a UV-Vis spectrophotometer. The first experimental part showed the reproducibility and repeatability of a regenerable IgG-sensing surface prepared using switchavidin (a neutralized avidin mutant) and protein G molecules. Furthermore, the sensing surface has ultralow fouling ability against some common model proteins. The immobilization of IgG-molecules did not show, however, reliable results. Additionally, the thesis represents a mechanical model for the switchavidin adsorption that is based on the MP-SPR measurement data. Further research could concentrate on the measurement of more complex samples as well as on the development of automation that is based on the represented mechanical adsorption model. The second experimental part studied the applicability of a four-channel MP-SPR prototype into the measurement of fast association kinetics of small molecular weight (MW) drugs. A human carbonic anhydrase II enzyme was immobilized into a three-dimensional hydrogel matrix and the interaction kinetics of three sulfonamide inhibitors were compared with kinetic values in the literature. Only a single inhibitor (acetazolamide) gave satisfying results. In conclusion, the MP-SPR-prototype is capable of measuring the fast association kinetics of small MW drugs, but this requires the very accurate preparation of samples and buffers as well as the proper calibration of bulk responses. TIIVISTELMÄ MP-SPR biosensorimittausten analyysimenetelmien kehittäminen Optiset biosensorit ovat saaneet arvostusta lukuisissa käyttökohteissa, kuten lääketeollisuudessa ja kliinisessä diagnostiikassa, biologisen tunnistuselementin korkean spesifisyyden ja optisen detektiomenetelmän non-invasiivisuuden ansiosta. Optisen biosensorien tehokas ja joustava käyttö kuitenkin edellyttää, että käyttäjä ymmärtää näiden toiminnan fysikaalisia, kemiallisia ja biologisia perusperiaatteita sekä optisten menetelmien etuja ja rajoituksia. Tutkielman kirjallinen osa tutustuttaa biosensoreiden ja biosensoroinnin perusteisiin sekä esittelee erään optiseen pintaplasmoniresonanssiin (surface plasmon resonance, SPR) perustuvan laitteiston toimintaa, etuja ja haasteita. Tutkielman kokeellisissa osuuksissa käytettiin multiparametrista SPR-laitteistoa (MP-SPR), joka kykenee mittaamaan molekyylien adsorption lisäksi laajasti näytteen optisia ominaisuuksia. Proteiiniliuoksien konsentraatiot sekä niiden puhtausasteet määritettiin UV-Vis-spektrofotometrilla. Tutkielman ensimmäinen kokeellinen osuus osoitti switchavidiiniin (neutralisoitu avidiinimutantti) ja proteiini G molekyyleihin perustuvan regeneroitavan immunosensorointipinnan toistettavuuden ja vertailtavuuden. Lisäksi sensorointipinta osoitti, että proteiinien non-spesifinen sitoutuminen on erittäin vähäistä eräillä malliproteiineilla tutkittaessa. IgG-molekyylien immobisaation toimivuudesta ei kuitenkaan saatu luotettavia tuloksia. Lisäksi tutkielma esittää switchavidiinille MP-SPR mittausdataan perustuvan mekaanisen sitoutumismallin. Jatkotutkimuksissa voitaisiin keskittyä monimutkaisempien näytteiden mittaamiseen sekä kehittää esitettyyn sitoutumismalliin perustuvaa automaatiota. Tutkielman toinen kokeellinen osuus tutki nelikanavaisen MP-SPR-prototyypin soveltuvuutta nopeasti sitoutuvien pienlääkemolekyylien sitoutumisen mittaamiseen. Ihmisen hiilihappoanhydraasi II-entsyymiä immobilisoitiin kolmiulotteiseen hydrogeelimatriksiin ja kolmen sulfonamidi-inhibiittorin sitoutumiskinetiikkaa vertailtiin referenssiartikkelin tuloksiin. Tutkimuksessa onnistuttiin mittaamaan ja analysoimaan kineettiset vakiot vain yhdestä inhibiittorista (asetatsoliamidi). Johtopäätöksenä MP-SPR-prototyyppi kykenee mittaamaan pienmolekyylien nopeaa sitoutumiskinetiikkaa, mutta tämän edellytyksenä on erittäin tarkka näytteiden ja puskureiden valmistus sekä bulkkivasteiden oikeaoppinen kalibraatio

Groundwater resilience Nepal: preliminary findings from a case study in the Middle Hills

Groundwater resources in the Middle Hills of Nepal perform a major role in supplying domestic and irrigation water and in regulating river flows. However, there has been little systematic study of groundwater within the region, making it difficult to evaluate how water supplies and river flows may change in response to climatic and anthropogenic change. To begin to build an evidence base, two catchments in the Middle Hills were investigated. The aim of the study was to characterise the hydrogeology of the catchments, assess water supplies and water usage and evaluate how resilient groundwater may be to change. Two contrasting sub-catchments within the Kali Gandaki River catchment were chosen: Ramche Village Development Committee (VDC), at an elevation of 2000 – 3000 m, with subsistence terraced farming and highly forested slopes, and Madanpokhara VDC which is largely below 1000 m, with expanding commercial agriculture. Groundwater sampling was undertaken during the post-monsoon season 2013 and pre-monsoon season 2014. Springs, tube wells and rivers across the two catchments were investigated using a combination of surveys, flow measurements, and sampling for inorganic chemistry, stable isotopes, groundwater residence time indicators (CFC and SF6) and noble gases. In addition, 12 months of weekly hydrological monitoring and monthly water usage surveys were undertaken at several sites. There is a heavy reliance on springs for water supply in Ramche. The springs are typically perennial but with significantly reduced flows during the winter and pre-monsoon season. The springs have bicarbonate groundwater chemistry and generally low overall mineralisation. Springs issuing from the higher slopes are reliant on seasonal monsoon rainfall and snow to sustain higher flows, but baseflows are sustained by groundwater storage within the weathered aquifer and will therefore have some inter-annual storage. Discrete springs issuing from lower slopes are most likely to be fed from groundwater storage within the fractured aquifer network. Groundwater residence time indicators (CFC and SF6) suggest a mean residence time of 10-20 years for pre-monsoon groundwater, implying inter-annual storage and therefore some built in resilience. However the general low storage of the groundwater environment suggests that none of the springs would be resilient to a long term reduction in precipitation. In the lower catchment of Madanpokhara where floodplain and outwash deposits are present, many hand-drilled shallow tubewells have been installed in the last 5-10 years, decreasing the reliance on springs. The development of groundwater resources has resulted in a thriving agricultural co-operative, inward migration and a growing population. These shallow tubewells have increased the resilience of the water supplies to change but are potentially vulnerable to over-exploitation as a result of the rapid increase in abstraction. Groundwater sampled in tubewells along the margin of the floodplain is modern (~20 yrs Mean Residence Time (MRT)) with bicarbonate groundwater chemistry and no significant water quality concerns. Groundwater sampled from tubewells towards the centre of the floodplain appears to be older (~50 yrs MRT) with elevated concentrations of iron, manganese, zinc and arsenic detected at some sites. With a growing recognition of the importance of groundwater storage in the Middle Hills there is significant potential to further advance the characterisation of groundwater systems and investigate the resilience of groundwater supplies to change. Systematic monitoring of groundwater, as springs flows, groundwater levels and chemistry would give a much better understanding of emerging trends. Likewise, monitoring current yields of springs and comparing to historic values at installation may allow some conclusions to be drawn about the trajectory of springflow. There are several groundwater-related initiatives underway within organisations in Nepal; the lessons learned from this current research, the methodologies used and the preliminary findings will be of value to these

Adsorpcja lizozymu – charakterystyka struktur białkowych z zastosowaniem metody mikrowagi kwarcowej z monitorowaniem dyssypacji energii oraz powierzchniowego rezonansu plazmonów

Lizozym (LSZ) odgrywa bardzo ważną rolę w układzie immunologicznym. Wykazuje silne działanie przeciwbakteryjne, dzięki czemu znalazł szerokie zastosowanie w medycynie i przemyśle farmaceutycznym. Poznanie mechanizmu adsorpcji LSZ ma istotne znaczenie dla określenia optymalnych warunków immobilizacji tego białka na powierzchni adsorpcyjnej, a ponadto określenia oddziaływania LSZ z wybranymi ligandami. Proces adsorpcji LSZ na powierzchni złota monitorowano, stosując dwie komplementarne metody: mikrowagę kwarcową z monitorowaniem dyssypacji energii (QCM-D) oraz wieloparametrowy powierzchniowy rezonans plazmonów (MP-SPR). pH silnie wpływa na efektywność procesu adsorpcji tego białka, a w konsekwencji na orientację cząsteczki LSZ na powierzchni adsorpcyjnej. Maksymalny stopień pokrycia otrzymano w pobliżu punktu izoelektrycznego białka, pH 10,0. Ponadto, na podstawie danych z QCM-D oraz MP-SPR, określono stopień hydratacji tworzonych warstw LSZ.Zeszyty Naukowe TDU