Potential and utilization of water extracts from spruce bark

The aim of this thesis was to investigate potential ways to utilize water extracts from spruce bark. Spruce bark has been found to contain three main stilbene glucosides (astringin, isorhapontin, polydatin), which have potential in the treatment of skin aging and cosmetics applications. Since stilbene glucosides are water soluble, they are dissolved into the debarking process waters of the pulp and paper industry. These waters are not fully utilized at the moment, and being considered as a waste stream, which adds to the costs of the mill by increasing COD levels of the waste water. This waste stream could potentially be transformed to a source of stilbene glucosides, to provide additional revenue to the mill. In the experimental part, temperature effect on hot water extraction yields of industrial spruce bark was studied. Mechanical pressing of heated spruce bark was performed with a laboratory scale mechanical press to study the effect on the COD levels. Finally, bark press waters obtained from the mill were characterized, with partial purification of stilbene glucosides by ultrafiltration with 2 and 5 kDa filters. The yields were calculated by gravimetric and spectroscopic analysis. Two dimensional (2D) solution-state 1H–13C correlation NMR spectroscopy provided the structural verification of the stilbene glucosides and other compounds present in the samples. Extraction yield of industrial spruce bark was increasing 1.4 times every 20 ◦C, on average, reaching yield of 5.0% at 80 ◦C. The mechanical pressing was able to press out 26±5% of the total mass of the sample. COD level was 75,100 mg/L for 80 ◦C sample. This was two times higher compared to bark press sample of taken from a paper mill, which had COD level of 37,500 mg/L. Ultrafiltration could remove 90% of polyphenols and permeated 14% of the stilbenes of the feed. With 2D-HSQC NMR, all three major stilbene glucosides were identified from the filtrate and the feed

Development of fast, reliable and automated isolation and fractionation methods for nanosized subpopulations of human biomacromolecules

This doctoral thesis describes the development of fast, reliable and automated isolation and fractionation methods for nanosized subpopulations of human biomacromolecules. The focus of the study was on subpopulations of lipoproteins and extracellular vesicles (EVs) that are important in the detection of different diseases, such as atherosclerotic cardiovascular diseases and cancer, and may even possess therapeutic potential. In the thesis, immunoaffinity chromatography (IAC) with selective antibodies immobilized on the monolithic disk columns were utilized for the selective isolation of biomacromolecules from human plasma, while asymmetrical flow field-flow fractionation (AsFlFFF or AF4) was able to fractionate relevant subpopulations of biomacromolecules (e.g., small dense low-density lipoproteins, exomeres, and exosomes) from the isolates. Continuous flow quartz crystal microbalance (QCM) and partial filling affinity capillary electrophoresis (PF-ACE) were employed to study the affinity of the interactions between the antibody and lipoproteins. The first step was to develop a method to study interactions between antibody and lipoproteins to select a high affinity antibody useful for the isolation of lipoprotein subpopulations by IAC. The interaction data obtained with PF-ACE was analyzed to determine the heterogeneity of the interactions with adsorption energy distribution calculations, while the QCM data was processed with interaction maps. The affinity constants obtained with QCM and PF-ACE agreed well with each other. Next, the IAC methods were developed to capture EVs of different cellular origins from human plasma using anti-CD9 monoclonal antibody (mAb), while anti-CD61 mAb was exploited to capture platelet-derived EVs. The anti-apolipoprotein B-100 (anti-apoB-100) mAb was exploited to immunocapture apoB-100 containing lipoproteins. The anti-apoB-100 mAb was also characterized by the PF-ACE and QCM studies. Appropriate elution conditions were found for the IAC methods, which has often been an issue with magnetic beads-based immunoaffinity methods. Since IAC allowed selective isolation of EVs and lipoproteins, a size-based separation to their subpopulations with AsFlFFF was introduced as a successive step. This enabled additional characterization of subpopulations by nanoparticle tracking analysis, western blotting, electron microscopy, capillary electrophoresis coupled with laser-induced fluorescent detection, zeta potential measurements, as well as free amino acids and glucose analysis with hydrophilic interaction liquid chromatography-tandem mass spectrometry. Finally, IAC was successfully on-line coupled to AsFlFFF, resulting in quick and automated isolation and fractionation of the subpopulations of EVs and lipoproteins. The constructed IAC-AsFlFFF system was able to process reliably 18–38 samples in 24 h with only minor operator involvement, resulting in highly reproducible and gentle fractionation of EV subpopulations in the size range of exomeres and exosomes. Polymeric monolithic disk columns were utilized for the first time for the IAC-based isolation of EVs and their subpopulations from human plasma, and for the detection of exomeres in CD9+ EVs and CD61+ platelet-derived EVs from human plasma samples. The results demonstrated that CD61+ EVs are potentially taking part in gluconeogenesis based on free amino acids and glucose present as cargo.Väitöskirjassa kehitettiin nopeita, luotettavia ja automatisoituja eristämis- ja fraktiointimenetelmiä ihmisen biomakromolekyylien nanokokoisille alaryhmille. Tutkimuksen painopiste oli lipoproteiinien ja solunulkoisten vesikkelien (EV) alaryhmissä. Näiden biomakromolekyylien alaryhmien tutkiminen on tärkeää eri sairauksien, kuten ateroskleroottisten sydän- ja verisuonitautien ja syöpien havaitsemisessa. Biomakromolekyylien eristämiseen ihmisen veriplasmasta käytettiin ensimmäistä kertaa immunoaffiniteettikromatografiaa, jossa oli selektiivisiä monoklonaalisia vasta-aineita immobilisoituna polymeerisille monoliittisille kiekkopylväille. Eristettyjen biomakromolekyylien jatkokäsittely epäsymmetrisen virtauskenttävirtausfraktioinnin (AsFlFFF tai AF4) avulla erotteli niiden alaryhmät toisistaan koon perusteella (esimerkiksi eksomerit ja eksosomit). Kvartsikidemikrovaakaa (QCM) ja kapillaarielektroforeesia (PF-ACE) käytettiin monoklonaalisen vasta-aineen ja lipoproteiinien välisen vuorovaikutuksen affiniteetin tutkimiseksi. Lopuksi immunoaffiniteettikromatografia liitettiin yhteen epäsymmetriseen virtauskenttävirtausfraktiointiin automatisoiduksi järjestelmäksi. Näin mahdollistettiin lipoproteiinien ja solunulkoisten vesikkelien alaryhmien nopea, toistettava ja automaattinen eristäminen veriplasmasta. Järjestelmä kykeni käsittelemään 18–38 näytettä 24 tunnissa vähäisellä operaattorin osallistumisella. Ensimmäistä kertaa oli mahdollista eristää ja fraktioida veriplasmanäytteistä erittäin toistettavasti ja hellävaraisesti solunulkoisten vesikkelien alaryhmiä eksomerien ja eksosomien kokoluokassa. Alaryhmien jatkotutkimuksissa paljastui myös, että verihiutaleiden solunulkoisten vesikkelien alaryhmät osallistuvat mahdollisesti glukoneogeneesiin

Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor

Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of <50 nm exomeres and 50-80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography - asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants Kd ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63+ EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV - ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1.Peer reviewe

Raman spectroscopy combined with comprehensive gas chromatography for label-free characterization of plasma-derived extracellular vesicle subpopulations

Raman spectroscopy together with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOFMS) was employed to characterize exomere- (<50 nm) and exosome-sized (50-80 nm) EVs isolated from human plasma by the novel on-line immunoaffinity chromatography - asymmetric flow field-flow fractionation method. CD9(+), CD63(+), and CD81(+) EVs were selected to represent general EV subpopulations secreted into plasma, while CD61(+) EVs represented the specific EV subset derived from platelets. Raman spectroscopy could distinguish EVs from non-EV particles, including apolipoprotein B-100-containing lipoproteins, signifying its potential in EV purity assessment. Moreover, platelet-derived (CD61(+)) EVs of both exomere and exosome sizes were discriminated from other EV subpopulations due to different biochemical compositions. Further investigations demonstrated composition differences between exomere- and exosome-sized EVs, confirming the applicability of Raman spectroscopy in distinguishing EVs, not only from different origins but also sizes. In addition, fatty acids that act as building blocks for lipids and membranes in EVs were studied by GCxGC-TOF-MS. The results achieved highlighted differences in EV fatty acid compositions in both esterified (membrane lipids) and non-esterified (free fatty acids) fractions, indicating possible differences in membrane structures, biological functions, and roles in cell-to-cell communications of EV subpopulations.Peer reviewe

Reliable Strategy for Analysis of Complex Biosensor Data

When using biosensors, analyte biomolecules of several different concentrations are percolated over a chip with immobilized ligand molecules that form complexes with analytes. However, in many cases of biological interest, e.g., in antibody interactions, complex formation steady-state is not reached. The data measured are so-called sensorgram, one for each analyte concentration, with total complex concentration vs time. Here we present a new four-step strategy for more reliable processing of this complex kinetic binding data and compare it with the standard global fitting procedure. In our strategy, we first calculate a dissociation graph to reveal if there are any heterogeneous interactions. Thereafter, a new numerical algorithm, AIDA, is used to get the number of different complex formation reactions for each analyte concentration level. This information is then used to estimate the corresponding complex formation rate constants by fitting to the measured sensorgram one by one. Finally, all estimated rate constants are plotted and clustered, where each cluster represents a complex formation. Synthetic and experimental data obtained from three different QCM biosensor experimental systems having fast (close to steady-state), moderate, and slow kinetics (far from steady-state) were evaluated using the four-step strategy and standard global fitting. The new strategy allowed us to more reliably estimate the number of different complex formations, especially for cases of complex and slow dissociation kinetics. Moreover, the new strategy proved to be more robust as it enables one to handle system drift, i.e., data from biosensor chips that deteriorate over time.Peer reviewe

Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool. (C) 2016 Elsevier Inc. All rights reserved.Peer reviewe

Electrokinetic characterization of extracellular vesicles with capillary electrophoresis : A new tool for their identification and quantification

This work reports on the development of the first capillary electrophoresis methodology for the elucidation of extracellular vesicles' (EVs) electrokinetic distributions. The approach is based on capillary electrophoresis coupled with laser-induced fluorescent (LIF) detection for the identification and quantification of EVs after their isolation. Sensitive detection of these nanometric entities was possible thanks to an 'inorganic-species-free' background electrolyte. This electrolyte was made up of weakly charged molecules at very high concentrations to stabilize EVs, and an intra-membrane labelling approach was used to prevent EV morphology modification. The limit of detection for EVs achieved using the developed CE-LIF method reached 8 x 10(9) EV/mL, whereas the calibration curve was acquired from 1.22 x 10(10) to 1.20 x 10(11) EV/mL. The CE-LIF approach was applied to provide the electrokinetic distributions of various EVs of animal and human origins, and visualize different EV subpopulations from our recently developed high-yield EV isolation method. (C) 2020 Elsevier B.V. All rights reserved.Peer reviewe

Interpreting the biosensor data of biomolecular interactions

The literature part of this thesis reviewed the process of obtaining affinity information with quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) biosensors. Basic principles of these biosensors were also evaluated, along with the principles of data acquisition and finally the data processing. The raw data produced by QCM or SPR can be used to study biomolecular interactions qualitatively and quantitatively. These techniques are also powerful in obtaining kinetic and thermodynamic information of the biomolecular interactions. SPR and QCM can produce data easily, but data interpretation can be sometimes problematic. This is partly due to misconceptions on how the sensograms should be interpreted. Many of the interpretational problems can and should be avoided long before the modeling of the data takes place to obtain reliable affinity data. The literature part of this thesis also presents tools for developing good experimental design. Well-designed experimental set-up is the most important element for producing good biosensor data. One should also estimate from the sensogram shapes what kind of analysis is needed. This was explained in detail in the literature part, pointing out the key elements how sensograms with certain shape should be interpreted and further analyzed to obtain affinity constants. Data analysis part of the literature review provides also information how to use appropriate models (e.g. fitting equilibrium, kinetic or complex data) with extensive examples. Surface site distribution model will be also covered as the tool to analyze complex biomolecular interactions by QCM and SPR. In the experimental part, affinity of anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) towards different lipoproteins was studied with partially filling affinity capillary (PF-ACE) electrophoresis and QCM. PF-ACE with adsorption energy distribution (AED) calculations provided information on the heterogeneity of the interactions. For the first time, a modified surface site distribution model called Interaction map was utilized to model QCM data of lipoprotein interactions with anti-apoB-100 Mab. With the Interaction maps, it was possible to distinguish different kinetics of low-density lipoprotein (LDL) and anti-apoB-100 Mab interactions. Affinity constants obtained were used to evaluate thermodynamics of these interactions. Both methods were also used to evaluate interactions with other apoB-100 containing lipoproteins: intermediate-density lipoprotein (IDL) and very lowdensity lipoprotein (VLDL). It was found that the Interaction maps could distinguish two different kinetics from the mixture of IDL-VLDL with distinct affinity constants. Both methods agreed well with the affinity constants. It was found that the anti-apoB-100 Mab used in this study, had a high affinity towards apoB-100 containing lipoproteins. In the second part of the experimental, a convective interaction media (CIM) based LDL isolation platform was developed. In these studies, anti-apoB-100 Mab was immobilized on the CIM-disk and was used to isolate LDL from human plasma and serum samples. It was found that apolipoprotein based separation of LDL from plasma was possible, although not without difficulties, since apoB-100 is not only present in LDL, but also in VLDL and IDL. To circumvent this problem different antibodies (anti-apoE and anti-apoAI) were utilized to capture VLDL and IDL from the plasma before the interaction of LDL with the anti-apoB-100 CIM-disk. LDL was successfully isolated with this approach in a significantly reduced time compared to conventional ultracentrifugation method used for LDL isolation

Modern isolation and separation techniques for extracellular vesicles

Extracellular vesicles (EVs) are heterogenous membrane-bound vesicles released from various origins. EVs play a crucial role in cellular communication and mediate several physiological and pathological processes, highlighting their potential therapeutic and diagnostic applications. Due to the rapid increase in interests and needs to elucidate EV properties and functions, numerous isolation and separation approaches for EVs have been developed to overcome limitations of conventional techniques, such as ultracentrifugation. This review focuses on recently emerging and modern EV isolation and separation techniques, including size-, charge-, and affinity-based techniques while excluding ultracentrifugation and precipitation-based techniques due to their multiple limitations. The advantages and drawbacks of each technique are discussed together with insights into their applications. Emerging approaches all share similar features in terms of being time-effective, easy-to-operate, and capable of providing EVs with suitable and desirable purity and integrity for applications of interest. Combination and hyphenation of techniques have been used for EV isolation and separation to yield EVs with the best quality. The most recent development using an automated on-line system including selective affinity-based trapping unit and asymmetrical flow field-flow fractionation allows reliable isolation and fractionation of EV subpopulations from human plasma. (C) 2020 The Author(s). Published by Elsevier B.V.Peer reviewe