RESUMO
Serum calcium isotopes (δ44/42Ca) have been suggested as a non-invasive and sensitive Ca balance marker. Quantitative δ44/42Ca changes associated with Ca flux across body compartment barriers relative to the dietary Ca and the correlation of δ44/42CaSerum with bone histology are unknown. We analyzed Ca and δ44/42Ca by mass-spectrometry in rats after two weeks of standard-Ca-diet (0.5%) and after four subsequent weeks of standard- and of low-Ca-diet (0.25%). In animals on a low-Ca-diet net Ca gain was 61 ± 3% and femur Ca content 68 ± 41% of standard-Ca-diet, bone mineralized area per section area was 68 ± 15% compared to standard-Ca-diet. δ44/42Ca was similar in the diets, and decreased in feces and urine and increased in serum in animals on low-Ca-diet. δ44/42CaBone was higher in animals on low-Ca-diet, lower in the diaphysis than the metaphysis and epiphysis, and unaffected by gender. Independent of diet, δ44/42CaBone was similar in the femora and ribs. At the time of sacrifice, δ44/42CaSerum inversely correlated with intestinal Ca uptake and histological bone mineralization markers, but not with Ca content and bone mineral density by µCT. In conclusion, δ44/42CaBone was bone site specific, but mechanical stress and gender independent. Low-Ca-diet induced marked changes in feces, serum and urine δ44/42Ca in growing rats. δ44/42CaSerum inversely correlated with markers of bone mineralization.
Assuntos
Calcificação Fisiológica , Cálcio , Animais , Densidade Óssea , Cálcio/análise , Isótopos de Cálcio , Cálcio da Dieta , Dieta , RatosRESUMO
Timely and accurate diagnosis of osteoporosis is essential for adequate therapy. Calcium isotope ratio (δ44/42Ca) determination has been suggested as a sensitive, noninvasive, and radiation-free biomarker for the diagnosis of osteoporosis, reflecting bone calcium balance. The quantitative diagnostic is based on the calculation of the δ44/42Ca difference between blood, urine, and bone. The underlying cellular processes, however, have not been studied systematically. We quantified calcium transport and δ44/42Ca fractionation during in vitro bone formation and resorption by osteoblasts and osteoclasts and across renal proximal tubular epithelial cells (HK-2), human vein umbilical endothelial cells (HUVECs), and enterocytes (Caco-2) in transwell systems and determined transepithelial electrical resistance characteristics. δ44/42Ca fractionation was furthermore quantified with calcium binding to albumin and collagen. Calcified matrix formed by osteoblasts was isotopically lighter than culture medium by -0.27 ± 0.03 within 5 days, while a consistent effect of activated osteoclasts on δ44/42Ca could not be demonstrated. A transient increase in δ44/42Ca in the apical compartment by 0.26 occured across HK-2 cells, while δ44/42Ca fractionation was small across the HUVEC barrier and absent with Caco-2 enterocytes, and with binding of calcium to albumin and collagen. In conclusion, δ44/42Ca fractionation follows similar universal principles as during inorganic mineral precipitation; osteoblast activity results in δ44/42Ca fractionation. δ44/42Ca fractionation also occurs across the proximal tubular cell barrier and needs to be considered for in vivo bone mineralization modeling. In contrast, the effect of calcium transport across endothelial and enterocyte barriers on blood δ44/42Ca should be low and is absent with physiochemical binding of calcium to proteins.
Assuntos
Isótopos de Cálcio/química , Cálcio/química , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Transporte Biológico , Células CACO-2 , Cálcio/metabolismo , Linhagem Celular , Células Endoteliais da Veia Umbilical Humana , Humanos , Túbulos Renais Proximais/citologia , Ligação ProteicaRESUMO
BACKGROUND: The Special AT-rich Sequence Binding Protein 1 (SATB1) regulates the expression of many genes by acting as a global chromatin organizer. While in many tumor entities SATB1 overexpression has been observed and connected to pro-tumorigenic processes, somewhat contradictory evidence exists in brain tumors with regard to SATB1 overexpression in glioblastoma and its association with poorer prognosis and tumor progression. On the functional side, initial data indicate that SATB1 may be involved in several tumor cell-relevant processes. METHODS: For the detailed analysis of the functional relevance and possible therapeutic potential of SATB1 inhibition, we employ transient siRNA-mediated knockdown and comprehensively analyze the cellular and molecular role of SATB1 in glioblastoma. RESULTS: In various cell lines with different SATB1 expression levels, a SATB1 gene dose-dependent inhibition of anchorage-dependent and -independent proliferation is observed. This is due to cell cycle-inhibitory and pro-apoptotic effects of SATB1 knockdown. Molecular analyses reveal SATB1 knockdown effects on multiple important (proto-) oncogenes, including Myc, Bcl-2, Pim-1, EGFR, ß-catenin and Survivin. Molecules involved in cell cycle, EMT and cell adhesion are affected as well. The putative therapeutic relevance of SATB1 inhibition is further supported in an in vivo tumor xenograft mouse model, where the treatment with polymeric nanoparticles containing SATB1-specific siRNAs exerts antitumor effects. CONCLUSION: Our results demonstrate that SATB1 may represent a promising target molecule in glioblastoma therapy whose inhibition or knockdown affects multiple crucial pathways.
Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Animais , Apoptose , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Ciclo Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/antagonistas & inibidores , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Nus , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The accurate determination of the maximum turnover number and Michaelis constant for membrane enzymes remains challenging. Here, this problem has been solved by observing in parallel the hydrolysis of thousands of individual fluorescently labeled immobilized liposomes each processed by a single phospholipase A2 molecule. The release of the reaction product was tracked using total internal reflection fluorescence microscopy. A statistical analysis of the hydrolysis kinetics was shown to provide the Michaelis-Menten parameters with an accuracy better than 20% without variation of the initial substrate concentration. The combined single-liposome and single-enzyme mode of operation made it also possible to unravel a significant nanoscale dependence of these parameters on membrane curvature.
Assuntos
Lipídeos de Membrana/metabolismo , Fosfolipases A2/metabolismo , Biocatálise , Corantes/química , Humanos , Hidrólise , Cinética , Lipossomos/metabolismo , Lipídeos de Membrana/química , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfolipases A2/líquido cefalorraquidianoRESUMO
SATB1 (special AT-rich binding protein 1) is a global chromatin organizer regulating the expression of a large number of genes. Overexpression has been found in various solid tumors and positively correlated with prognostic and clinicopathological properties. In colorectal cancer (CRC), SATB1 overexpression and its correlation with poor differentiation, invasive depth, TNM (tumor, nodes, metastases) stage and prognosis have been demonstrated. However, more detailed studies on the SATB1 functions in CRC are warranted. In this article, we comprehensively analyze the cellular and molecular role of SATB1 in CRC cell lines with different SATB1 expression levels by using RNAi-mediated knockdown. Using siRNAs with different knockdown efficacies, we demonstrate antiproliferative, cell cycle-inhibitory and proapoptotic effects of SATB1 knockdown in a SATB1 gene dose-dependent manner. Tumor growth inhibition is confirmed in vivo in a subcutaneous tumor xenograft mouse model using stable knockdown cells. The in-depth analysis of cellular effects reveals increased activities of caspases-3, -7, -8, -9 and other mediators of apoptotic pathways. Similarly, the analysis of E- and N-cadherin, slug, twist, ß-catenin and MMP7 indicates SATB1 effects on epithelial-mesenchymal transition (EMT) and matrix breakdown. Our results also establish SATB1 effects on receptor tyrosine kinases and (proto-)oncogenes such as HER receptors and Pim-1. Taken together, this suggests a more complex molecular interplay between tumor-promoting and possible inhibitory effects in CRC by affecting multiple pathways and molecules involved in proliferation, cell cycle, EMT, invasion and cell survival.
Assuntos
Apoptose , Ciclo Celular , Proliferação de Células , Neoplasias do Colo/patologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Animais , Neoplasias do Colo/metabolismo , Transição Epitelial-Mesenquimal , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/antagonistas & inibidores , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Nus , RNA Interferente Pequeno/genética , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Imaging of individual lipid vesicles is used to track single-enzyme kinetics of phospholipid hydrolysis. The method is employed to quantify the catalytic activity of phospholipase A2 (PLA2) in both pure and complex biological fluids. The measurements are demonstrated to offer a subpicomolar limit of detection (LOD) of human secretory PLA2 (sPLA2) in up to 1000-fold-diluted cerebrospinal fluid (CSF). An additional new feature provided by the single-enzyme sensitivity is that information about both relative concentration variations of active sPLA2 in CSF and the specific enzymatic activity can be simultaneously obtained. When CSF samples from healthy controls and individuals diagnosed with Alzheimer's disease (AD) are analyzed, the specific enzymatic activity is found to be preserved within 7% in the different CSF samples whereas the enzyme concentration differs by up to 56%. This suggests that the previously reported difference in PLA2 activity in CSF samples from healthy and AD individuals originates from differences in the PLA2 expression level rather than from the enzyme activity. Conventional ensemble averaging methods used to probe sPLA2 activity do not allow one to obtain such information. Together with an improvement in the LOD of at least 1 order of magnitude compared to that of conventional assays, this suggests that the method will become useful in furthering our understanding of the role of PLA2 in health and disease and in detecting the pharmacodynamic effects of PLA2-targeting drug candidates.
Assuntos
Fosfolipases A2 Secretórias/líquido cefalorraquidiano , Fosfolipídeos/líquido cefalorraquidiano , Doença de Alzheimer/líquido cefalorraquidiano , Biocatálise , Estudos de Casos e Controles , Corantes Fluorescentes , Humanos , Hidrólise , Cinética , Limite de Detecção , Microscopia de Fluorescência , Fosfolipases A2 Secretórias/química , Fosfolipídeos/químicaRESUMO
Using tethered sub-100 nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature, we have in this work designed a total internal reflection fluorescence microscopy-based single vesicle assay to investigate how an antiviral amphipathic α-helical (AH) peptide interacts with lipid membranes to induce membrane curvature-dependent pore formation and membrane destabilization. Based on a combination of statistics from single vesicle imaging, binding kinetics data, and theoretical analysis, we propose a mechanistic model that is consistent with the experimentally observed peptide association and pore formation kinetics at medically relevant peptide concentrations (10 nM to 1 µM) and unusually low peptide-to-lipid (P/L) ratio (~1/1000). Importantly, the preference of the AH peptide to selectively rupture virions with sub-100 nm diameters appears to be related to membrane strain-dependent pore formation rather than to previously observed nanoscale membrane curvature facilitated binding of AH peptides. Compared to other known proteins and peptides, the combination of low effective P/L ratio and high specificity for nm-sized membrane curvature lends this particular AH peptide great potential to serve as a framework for developing a highly specific and potent antiviral agent for prophylactic and therapeutic applications while avoiding toxic side effects against host cell membranes.
Assuntos
Antivirais/farmacologia , Lipídeos/química , Nanotecnologia/métodos , Biotina/química , Membrana Celular/metabolismo , Fluoresceínas/química , Cinética , Lipídeos de Membrana/química , Nanopartículas/química , Peptídeos/química , Fosfatidilcolinas/química , Estrutura Secundária de Proteína , Rodaminas/química , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície , Propriedades de SuperfícieRESUMO
Parkinson's disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-synuclein. There is increasing evidence suggesting that the aggregation of α-synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and Förster resonance energy transfer, we report the direct observation of α-synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α-synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α-synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross-ß-sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo.
Assuntos
Bicamadas Lipídicas/química , alfa-Sinucleína/química , Transferência Ressonante de Energia de Fluorescência , Bicamadas Lipídicas/metabolismo , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Mutação , Polimerização , Estrutura Secundária de Proteína , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Despite many experimental studies on cooperative effects during protein adsorption events, this phenomenon is still poorly characterized and subject of much controversy. In this study, we address the topic of cooperativity using two distinct experimental approaches, namely, kinetic analysis and surface imaging, both based on supercritical angle fluorescence (SAF) microscopy. Several model systems comprising the two proteins BSA and fibrinogen, two different ionic strength conditions and varying pH environments were investigated. The combination of the experimental information obtained from kinetic analysis and from real-time in situ scan images unravel a clear correlation between cooperative adsorption and a heterogeneous protein layer build-up. We propose a mechanistic model of protein adsorption based on an overlap of classical Langmuir-type adsorption on unoccupied surface areas and an additional cooperative adsorption pathway near preadsorbed proteins which is consistent with the experimental observations. Moreover, the growth of two-dimensional surface clusters as an often assumed element of cooperativity could be excluded for the studied systems. The model includes the often observed phenomenon that the adsorption rate decelerates abruptly above a certain coverage limit. Furthermore, the observed evolution of the heterogeneous protein distribution on the surface is in good agreement with the proposed model.
Assuntos
Fibrinogênio/química , Soroalbumina Bovina/química , Adsorção , Animais , Bovinos , Concentração de Íons de Hidrogênio , Modelos Biológicos , Propriedades de SuperfícieRESUMO
We demonstrate that a recently developed coating comprised of silicone nanofilaments can be selectively functionalized to yield well defined superhydrophobic, superhydrophilic, superoleophobic or superoleophilic domains on a single substrate, constituting a simple and versatile toolbox for surface scientists to create and study surfaces with extreme wetting properties.
RESUMO
Animal models are essential tools to understand the mechanisms underlying the development and progression of renal disease and to study potential therapeutic approaches. Recently, interventional models suitable to induce acute and chronic kidney disease in the mouse have become a focus of interest due to the wide availability of genetically engineered mouse lines. These models differ by their damaging mechanism (cell toxicity, immune mechanisms, surgical renal mass reduction, ischemia, hypertension, ureter obstruction etc.), functional and histomorphological phenotype and disease evolution. The susceptibility to a damaging mechanism often depends on strain and gender. The C57BL/6 strain, the most commonly used genetic background of transgenic mice, appears to be relatively resistant against developing glomerulosclerosis, proteinuria and hypertension. This review serves to provide a comprehensive overview of interventional mouse models of acute and chronic kidney disease.
Assuntos
Modelos Animais de Doenças , Nefropatias/fisiopatologia , Animais , Predisposição Genética para Doença , Nefropatias/genética , CamundongosRESUMO
Lead is a neurotoxin that has been documented to affect many forms of wildlife, and has been identified as a limiting factor in a population of California Condors in Northern Arizona. The Arizona Game and Fish Department provides vouchers for free nonlead ammunition to hunters selected to hunt within the distribution of California Condors, with the intention of having fewer lead-laden offal piles available to California Condors. Although wildlife agencies may reasonably assume voucher programs motivate hunters into choosing nonlead ammunition, the lead reduction efforts attributable to the voucher program has not been empirically quantified. Our intention was to compare a control group of hunters to a treatment group of hunters within California Condor occupied areas. Both groups received educational materials regarding the deleterious effects of lead, but the treatment group also received a voucher for a free initial box of ammunition. About half of the control group used nonlead ammunition, compared to about three-fourths of the treatment group. Prominent barriers to adoption of nonlead ammunition included a general difficulty of obtaining it, obtaining it in the desired caliber, and its costliness. Frequently mentioned motivations for using nonlead was the exhortation to use it by the Department, and the desire to aid California Condor recovery by hunters. The disparate compliance rates found herein confirm and quantify the success of nonlead ammunition voucher programs, but underscore the importance of working to increase the supply of nonlead ammunition with the end of facilitating its procurement and reducing its cost.
Assuntos
Animais Selvagens , Monitoramento Ambiental , Poluentes Ambientais , Poluição Ambiental , Passatempos , Chumbo , Animais , Arizona , Conservação dos Recursos Naturais , Humanos , Inquéritos e QuestionáriosRESUMO
Advancement in the understanding of biomolecular interactions has benefited greatly from the development of surface-sensitive bioanalytical sensors. To further increase their broad impact, significant efforts are presently being made to enable label-free and specific biomolecule detection with high sensitivity, allowing for quantitative interpretation and general applicability at low cost. In this work, we have addressed this challenge by developing a waveguide chip consisting of a flat silica core embedded in a symmetric organic cladding with a refractive index matching that of water. This is shown to reduce stray light (background) scattering and thereby allow for label-free detection of faint objects, such as individual sub-20 nm gold nanoparticles as well as sub-100 nm lipid vesicles. Measurements and theoretical analysis revealed that light-scattering signals originating from single surface-bound lipid vesicles enable characterization of their sizes without employing fluorescent lipids as labels. The concept is also demonstrated for label-free measurements of protein binding to and enzymatic (phospholipase A2) digestion of individual lipid vesicles, enabling an analysis of the influence on the measured kinetics of the dye-labeling of lipids required in previous assays. Further, diffraction-limited imaging of cells (platelets) binding to a silica surface showed that distinct subcellular features could be visualized and temporally resolved during attachment, activation, and spreading. Taken together, these results underscore the versatility and general applicability of the method, which due to its simplicity and compatibility with conventional microscopy setups may reach a widespread in life science and beyond.
Assuntos
Técnicas Biossensoriais/métodos , Microscopia/métodos , Análise de Célula Única/métodos , Plaquetas/citologia , Células Cultivadas , Ouro/química , Humanos , Luz , Lipossomos/química , Lipossomos/metabolismo , Nanopartículas Metálicas/química , Espalhamento de RadiaçãoRESUMO
The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nano-structures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces.
Assuntos
Bicamadas Lipídicas/química , Nanoestruturas/química , RNA/química , Cátions/química , Recuperação de Fluorescência Após Fotodegradação , Bicamadas Lipídicas/metabolismo , Microscopia de Fluorescência , Fosfatidilcolinas/química , Técnicas de Microbalança de Cristal de Quartzo , RNA/metabolismo , Esfingosina/químicaRESUMO
The aggregation of α-synuclein (α-Syn) is believed to be one of the key steps driving the pathology of Parkinson's disease and related neurodegenerative disorders. One of the present hypotheses is that the onset of such pathologies is related to the rise of α-Syn levels above a critical concentration at which toxic oligomers or mature fibrils are formed. In the present study, we find that α-Syn aggregation in vitro is a spontaneous process arising at bulk concentrations as low as 1 nM and below in the presence of both hydrophilic glass surfaces and cell membrane mimicking supported lipid bilayers (SLBs). Using three-dimensional supercritical angle fluorescence (3D-SAF) microscopy, we observed the process of α-Syn aggregation in situ. As soon as α-Syn monomers were exposed to the surface, they started to adsorb and aggregate along the surface plane without a prior lag phase. However, at a later stage of the aggregation process, a second type of aggregate was observed. In contrast to the first type, these aggregates showed an extended structure being tethered with one end to the surface and being mobile at the other end, which protruded into the solution. While both types of α-Syn aggregates were found to contain amyloid structures, their growing mechanisms turned out to be significantly different. Given the clear evidence that surface-induced α-Syn aggregation in vitro can be triggered at bulk concentrations far below physiological concentrations, the concept of a critical concentration initiating aggregation in vivo needs to be reconsidered.
Assuntos
Nanotecnologia/métodos , alfa-Sinucleína/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microscopia de Fluorescência/métodos , Doença de Parkinson/metabolismo , Ligação Proteica/fisiologia , Propriedades de Superfície , alfa-Sinucleína/químicaRESUMO
Protein adsorption at solid surfaces plays a key role in many natural processes and has therefore promoted a widespread interest in many research areas. Despite considerable progress in this field there are still widely differing and even contradictive opinions on how to explain the frequently observed phenomena such as structural rearrangements, cooperative adsorption, overshooting adsorption kinetics, or protein aggregation. In this review recent achievements and new perspectives on protein adsorption processes are comprehensively discussed. The main focus is put on commonly postulated mechanistic aspects and their translation into mathematical concepts and model descriptions. Relevant experimental and computational strategies to practically approach the field of protein adsorption mechanisms and their impact on current successes are outlined.
Assuntos
Proteínas/química , Adsorção , Animais , Humanos , Cinética , Modelos Moleculares , Conformação Proteica , Propriedades de SuperfícieRESUMO
Cooperative effects play a vital role in protein adsorption events on biological interfaces. Despite a number of studies in this field molecular adsorption mechanisms that include cooperativity are still under debate. In this work we use a Monte Carlo-type simulation to explore the microscopic details behind cooperative protein adsorption. The simulation was designed to implement our previously proposed mechanism through which proteins are not necessarily rejected if they approach the surface to an occupied region. Instead, we suggest that proteins can be tracked laterally for a certain distance due to the influence of preadsorbed proteins in order to reach the nearest available binding site. The simulation results were compared with experimental data obtained by using the supercritical angle fluorescence (SAF) microscopy technique. It was found that the tracking distance may be up to 2.5 times the protein's diameter depending on the investigated system. The general validity of this tracking mechanism is supported by a number of linear or upward concave adsorption kinetics reported in the literature which are consistent with our simulation results. Furthermore, the self-organization of proteins adsorbing under cooperative conditions on the surface is shown to necessarily cause density inhomogeneities in the surface distribution of proteins which is also in agreement with experimental observations.
Assuntos
Método de Monte Carlo , Proteínas/química , Adsorção , Cinética , Microscopia de Fluorescência , Propriedades de SuperfícieRESUMO
We present a simple and versatile technique of tailoring functionalized surface structures for protein enrichment and purification applications based on a superhydrophobic silicone nanofilament coating. Using amino and carboxyl group containing silanes, silicone nanofilament templates were chemically modified to mimic anionic and cationic exchange resins. Investigations on the selectivity of the functionalized surfaces toward adsorption of charged model proteins were carried out by means of fluorescence techniques. Due to a high contact area resulting from the nanoroughness of the coating, excellent protein retention characteristics under various conditions were found. The surfaces were shown to be highly stable and reusable over several retention-elution cycles. Especially the full optical transparency and the possibility to use glass substrates as support material open new opportunities for the development of optical biosensors, open geometry microfluidics, or lab-on-a-chip devices.
Assuntos
Nanoestruturas/química , Nanoestruturas/ultraestrutura , Proteínas/isolamento & purificação , Silicones/química , Técnicas Biossensoriais , Materiais Revestidos Biocompatíveis/química , Microscopia Eletrônica de Varredura , Nanotecnologia , Propilaminas , SilanosRESUMO
We investigate nonspecific protein adsorption processes by comparing experimentally measured adsorption kinetics of beta-lactoglobulin with mathematical models. The adsorption and desorption behavior of this protein on a hydrophilic glass surface in citrate buffer (pH 3.0), monitored for a large set of different bulk concentrations (0.5x10(-8) M-1.5x10(-6) M) using a supercritical angle fluorescence (SAF) biosensor, is reported. Increasing adsorption rates and overshootings in the beginning of the adsorption are observed as well as a transition to an almost irreversibly bound state of the protein in the long term. Furthermore, rinsing experiments prove that adsorbed proteins abruptly change their desorption behavior from irreversible to reversible when a critical surface coverage theta(crit) is reached. Based on all experimental observations, a mathematical model composed of three adsorbed states differing in their surface affinity is proposed. Terms to account for lateral interactions between surface-bound proteins are included, which yield an excellent fit of the measured kinetics. For the first time, several phenomena that have been discussed in theoretical studies are confirmed by comparing experimental data with a single model.
Assuntos
Técnicas Biossensoriais/métodos , Lactoglobulinas/química , Adsorção , Soluções Tampão , Corantes Fluorescentes/química , Vidro/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Químicos , Propriedades de SuperfícieRESUMO
To achieve a better understanding of the nonspecific adsorption process of proteins on solid surfaces, the mechanism of this interaction was investigated by a model system comprising the structurally flexible ("soft") protein goat anti-rabbit immunoglobulin G and a set of chemically defined surfaces. The thermodynamic properties of both protein and surfaces were derived from contact angle measurements by applying the Lifshitz-van der Waals acid-base approach, and the Gibbs free enthalpy of interaction was calculated. The protein shows two conformational states, one hydrophobic and the other hydrophilic. The interaction energy indicates that the hydrophobic conformation favorably adsorbs onto the surfaces. With real-time binding kinetics, measured by a supercritical angle fluorescence biosensor, we show that during the nonspecific adsorption the protein performs a reorientation in its three-dimensional amino acid structure from a hydrophilic to a hydrophobic molecular structure. Unlike the rates of adsorption and desorption, the transition rate is independent of the type of surface and only influenced by the structural reorganization of the protein.