RESUMO
The quartz crystal microbalance (QCM) is a surface-sensitive measurement technique to characterize adsorption processes at solid-fluid interfaces. While QCM measurements are routinely applied to study homogeneous thin films, characterizing heterogeneous films of adsorbed particles remains challenging because QCM is sensitive to not only the mass of adsorbed particles but also to that of hydrodynamically coupled fluid. To extract information about adsorbed particles, it is necessary to model these hydrodynamic effects, however, current QCM models are restricted to the limit of either a very low surface coverage or to the extrapolated limit of saturation coverage. Herein, we investigated QCM measurement responses in the intermediate surface coverage regime, by conducting lattice Boltzmann simulations of monodisperse, spherical particles that are attached to an oscillating surface. From the simulations, we relate the overtone-dependent QCM frequency and bandwidth shifts to particle size, interparticle distance, and the relevant hydrodynamic length scale. The corresponding results are in qualitative agreement with experimental QCM data for sub-100 nm, gel-phase liposomes. Furthermore, the data provide a theoretical basis for extracting particle sizes from QCM data in the high surface coverage limit.
RESUMO
Enclosed lipid bilayer structures, referred to as liposomes or lipid vesicles, have a wide range of biological functions, such as cellular signaling and membrane trafficking. The efficiency of cellular uptake of liposomes, a key step in many of these functions, is strongly dependent on the contact area between a liposome and a cell membrane, which is governed by the adhesion force w, the membrane bending energy κ, and the osmotic pressure Δp. Herein, we investigate the relationship between these forces and the physicochemical properties of the solvent, namely, the presence of glucose (a nonionic osmolyte). Using fluorescence microscopy, we measure the diffusivity D of small (â¼50 nm radius), fluorescently labeled liposomes adhering to a supported lipid bilayer or to the freestanding membrane of a giant (â¼10 µm radius) liposome. It is observed that glucose in solution reduces D on the supported membrane, while having negligible effect on D on the freestanding membrane. Using well-known hydrodynamic theory for the diffusivity of membrane inclusions, these observations suggest that glucose enhances the contact area between the small liposomes and the underlying membrane, while not affecting the viscosity of the underlying membrane. In addition, quartz crystal microbalance experiments showed no significant change in the hydrodynamic height of the adsorbed liposomes, upon adding glucose. This observation suggests that instead of osmotic deflation, glucose enhances the contact area via adhesion forces, presumably due to the depletion of the glucose molecules from the intermembrane hydration layer.
Assuntos
Membrana Celular/metabolismo , Glucose/química , Lipossomos/química , Lipossomos/metabolismo , Movimento , Adesividade , Difusão , Bicamadas Lipídicas/metabolismoRESUMO
This paper describes the functionalization of solid supported phospholipid bilayer with decellularized extracellular matrix (dECM) components, toward the development of biomimetic platforms that more closely mimic the cell surface environment. The dECM was obtained through a combination of chemical and enzymatic treatments of mouse adipose tissue that contains collagen, fibronectin, and glycosaminoglycans (GAGs). Using amine coupling chemistry, the dECM components were attached covalently to the surface of a supported lipid bilayer containing phospholipids with reactive carboxylic acid headgroups. The bilayer formation and the kinetics of subsequent dECM conjugation were monitored by quartz crystal microbalance with dissipation (QCM-D). Fluorescence recovery after photobleaching (FRAP) confirmed the fluidity of the membrane after functionalization with dECM. The resulting hybrid biomimetic interface supports the attachment and survival of the human hepatocyte Huh 7.5 and maintains the representative hepatocellular function. Importantly, the platform is suitable for monitoring the lateral organization and clustering of cell-binding ligands and growth factor receptors in the presence of the rich biochemical profile of tissue-derived ECM components.
Assuntos
Materiais Biomiméticos/química , Matriz Extracelular/química , Bicamadas Lipídicas/química , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/química , Fibronectinas/química , Glicosaminoglicanos/química , Hepatócitos/fisiologia , Humanos , Fluidez de Membrana , Camundongos , Fosfatidilcolinas/química , Albumina Sérica/metabolismoRESUMO
Characterizing the deformation of nanoscale, soft-matter particulates at solid-liquid interfaces is a demanding task, and there are limited experimental options to perform quantitative measurements in a nonperturbative manner. Previous attempts, based on the quartz crystal microbalance (QCM) technique, focused on the high surface coverage regime and modeled the adsorbed particles as a homogeneous film, while not considering the coupling between particles and surrounding fluid and hence resulting in an underestimation of the known particle height. In this work, we develop a model for the hydrodynamic coupling between adsorbed particles and surrounding fluid in the limit of a low surface coverage, which can be used to extract shape information from QCM measurement data. We tackle this problem by using hydrodynamic simulations of an ellipsoidal particle on an oscillating surface. From the simulation results, we derived a phenomenological relation between the aspect ratio r of the absorbed particles and the slope and intercept of the line that fits instantaneous, overtone-dependent QCM data on (δ/a, -Δf/n) coordinates where δ is the viscous penetration depth, a is the particle radius, Δf is the QCM frequency shift, and n is the overtone number. The model was applied to QCM measurement data pertaining to the adsorption of 34 nm radius, fluid-phase and gel-phase liposomes onto a titanium oxide-coated surface. The osmotic pressure across the liposomal bilayer was varied to induce shape deformation. By combining these results with a membrane bending model, we determined the membrane bending energy for the gel-phase liposomes, and the results are consistent with literature values. In summary, a phenomenological model is presented and validated in order to show for the first time that QCM experiments can quantitatively measure the deformation of adsorbed particles at low surface coverage.
RESUMO
One challenging aspect of quartz crystal microbalance (QCM) measurements is the characterization of adsorbed particles as the change in resonance frequency (Δf) is proportional not only to the inertia of the adsorbed layer but also to that of the hydrodynamically coupled fluid. Herein, by solving numerically the Navier-Stokes equations, we scrutinize Δf for sparsely deposited, rigid spherical particles that are firmly attached to an oscillating surface. The analysis is shown to be applicable to adsorbed, small unilamellar vesicles (SUVs) of controlled size under experimental conditions in which adhesion-induced vesicle deformation is negligible. The model supports a hydrodynamic explanation for the overtone dependence of Δf, and was fitted to experimental data concerning three monodisperse populations of SUVs with different average sizes ranging between 56 and 114 nm diameter. Using this procedure, we determined the average size of adsorbed vesicles to be within 16% of the size that was measured by dynamic light scattering experiments in bulk solution. In conclusion, this model offers a means to extract the particle size from QCM-D measurement data, with applications to biological and synthetic nanoparticles.
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The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of â¼100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/metabolismo , Hepacivirus/genética , Interações Hidrofóbicas e Hidrofílicas , Fragmentos de Peptídeos/metabolismo , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Fenômenos Biomecânicos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Estrutura Terciária de Proteína , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The multienzyme catalytic phosphorylation of phosphatidylinositol (PI) in a supported lipid membrane platform is demonstrated for the first time. One-step treatment with PI 4-kinase IIIß (PI4Kß) yielded PI 4-phosphate (PI4P), while a multistep enzymatic cascade of PI4Kß followed by PIP 5-kinase produced PI-4,5-bisphosphate (PI(4,5)P2 or PIP2). By employing quartz crystal microbalance with dissipation monitoring, we were able to track membrane association of kinase enzymes for the first time as well as detect PI4P and PI(4,5)P2 generation based on subsequent antibody binding to the supported lipid bilayers. Pharmacologic inhibition of PI4Kß by a small molecule inhibitor was also quantitatively assessed, yielding an EC50 value that agrees well with conventional biochemical readout. Taken together, the development of a PI-containing supported membrane platform coupled with surface-sensitive measurement techniques for kinase studies opens the door to exploring the rich biochemistry and pharmacological targeting of membrane-associated phosphoinositides.
Assuntos
1-Fosfatidilinositol 4-Quinase/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , 1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas de Membrana/antagonistas & inibidores , Fosfatidilinositol 4,5-Difosfato/química , Fosfatos de Fosfatidilinositol/química , Técnicas de Microbalança de Cristal de QuartzoRESUMO
Particle tracking is used to measure the diffusional motion of nanosized (≈100 nm), lipid vesicles that are electrostatically adsorbed onto a solid supported lipid bilayer. It is found that the motion of membrane-adhering vesicles is Brownian and depends inversely on the vesicle size, but is insensitive to the vesicle surface charge. The measured diffusivity agrees well with the Evans-Sackmann model for the diffusion of inclusions in supported, fluidic membranes. The agreement implies that the vesicle motion is coupled to that of a nanoscopic lipid cluster in the upper leaflet, which slides over the lower leaflet. The diffusivity of membrane-adhering vesicles is therefore predominantly governed by the interleaflet friction coefficient, while the diffusivity of single lipids is mainly governed by the membrane viscosity. Combined with fluorescence recovery after photobleaching analysis, the interleaflet friction coefficient and the membrane viscosity are determined by applying the Evans-Sackmann model to the measured diffusivity of membrane adhering vesicles and that of supported membrane lipids. This approach provides an alternative to existing methods for measuring the interleaflet friction coefficient and the membrane viscosity.
Assuntos
Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Eletricidade Estática , ViscosidadeRESUMO
Using single-particle tracking, we investigate the interaction of small unilamellar vesicles (SUVs) that are electrostatically tethered to the freestanding membrane of a giant unilamellar vesicle (GUV). We find that the surface mobility of the GUV-riding SUVs is Brownian, insensitive to the bulk viscosity, vesicle size, and vesicle fluidity but strongly altered by the viscosity of the underlying membrane. Analyzing the diffusional behavior of SUVs within the Saffman-Delbrück model for the dynamics of membrane inclusions supports the notion that the mobility of the small vesicles is coupled to that of dynamically induced lipid clusters within the target GUV membrane. The reversible binding also offers a nonperturbative means for measuring the viscosity of biomembranes, which is an important parameter in cell physiology and function.
RESUMO
The recently introduced solvent-assisted lipid bilayer (SALB) formation method allows one to efficiently fabricate planar, lipid bilayers on solid supports and can be used for various applications. It involves the introduction of an aqueous buffer into a mixture of lipid and alcohol, which is incubated on a solid support. The associated phase changes in the ternary bulk system are accompanied by the formation of a lipid bilayer at the solid-liquid interface. While the phase behavior of the ternary bulk system is well understood, the mechanism of bilayer assembly at the solid-liquid interface remains to be elucidated, including whether the adsorption process is limited by diffusion of the lipid in the bulk or by lipid binding kinetics onto the surface. Such factors strongly influence the success of bilayer formation as they pertain to operating conditions, such as lipid concentration, solvent exchange rate and chamber dimensions, and are hence of critical importance for SALB fabrication strategies. Herein, we extend an earlier proposed phenomenological kinetic model of the SALB formation process, based on a volume-averaged treatment of the solvent mixing process. By comparing the model to quartz crystal microbalance with dissipation monitoring (QCM-D) experimental data, we conclude that SALB formation is limited by diffusion of suspended lipid aggregates, with a hydrodynamic radius, that is consistent with aggregate size measurements in the literature. This agreement validates the proposed model to serve as the basis for optimizing conditions for SALB formation.
RESUMO
The adsorption and fusion of small unilamellar lipid vesicles on silica-based substrates such as glass is a common method used to fabricate supported lipid bilayers. Successful bilayer formation depends on a number of experimental conditions as well as on the quality of the vesicle preparation. Inevitably, a small fraction of unruptured vesicles always remains in a supported bilayer, and this kind of defect can have devastating influences on the morphological and electrical properties of the supported bilayer when used as a biosensing platform. In this paper, a simple method is reported to improve the completeness of supported bilayers by adding a vesicle rupturing peptide as a final step in the fabrication process. Peptide treatment reduces the fraction of unruptured vesicles to less than 1%, as determined by epifluorescence microscopy and quartz crystal microbalance-dissipation experiments. This step can easily be incorporated into existing procedures for preparing high-quality supported lipid bilayers.
Assuntos
Bicamadas Lipídicas , Peptídeos/químicaRESUMO
The nanometer-scale discrimination of virus-rupturing peptides is tested using lipid membrane platforms. In combination with single-vesicle analysis of peptide-induced vesicle rupture, a correlation between membrane partitioning and biologically relevant activities is established. Taken together, the findings support that the degree of rupture activity should be balanced by membrane curvature-selectivity for optimal therapeutic properties of antiviral peptides.
Assuntos
Antivirais/síntese química , Desenho de Fármacos , Membranas Artificiais , Peptídeos/síntese química , Lipossomas Unilamelares/química , Antivirais/química , Microscopia de Fluorescência , Peptídeos/químicaRESUMO
As a simple and efficient technique, the solvent-assisted lipid bilayer (SALB) formation method offers a versatile approach to fabricating a planar lipid bilayer on solid supports. Corresponding mechanistic aspects and the role of various governing parameters remain, however, to be better understood. Herein, we first scrutinized the effect of lipid concentration (0.01 to 5 mg/mL) and solvent type (isopropanol, n-propanol, or ethanol) on SALB formation on silicon oxide in order to identify optimal conditions for this process. The obtained fluid-phase lipid layers on silicon oxide were investigated by using the quartz crystal microbalance with dissipation monitoring, epifluorescence microscopy, and atomic force microscopy. The experimental results indicate that, in alcohol, lipid attachment to the substrate is reversible and reaches equilibrium in accordance with the bulk lipid concentration. During the solvent-exchange step, the water fraction increases and the deposited lipids are converted into planar bilayer fragments, along with the concurrent adsorption and rupture of micelles within an optimal lipid concentration range. In addition, fluid-phase lipid bilayers were successfully formed on other substrates (e.g., chrome, indium tin oxide, and titanium oxide) that are largely intractable to conventional methods (e.g., vesicle fusion). Moreover, gel-phase lipid bilayers were fabricated as well. Depending on the phase state of the lipid bilayer during fabrication, the corresponding adlayer mass varied by approximately 20% between the fluid- and gel-phase states in a manner which is consistent with the molecular packing of lipids in the two arrangements. Taken together, our findings help to explain the mechanistic details of SALB formation, optimize the corresponding procedure, and demonstrate the general utility for fabricating gel- and fluid-phase planar lipid bilayers.
Assuntos
Membrana Celular/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Solventes/química , Adsorção , Fosfatidilcolinas/químicaRESUMO
In this study, we employed the solvent-assisted lipid bilayer (SALB) formation method to fabricate charged membranes on solid supports. The SALB formation method exploits a ternary mixture of lipid-alcohol-aqueous buffer to deposit lamellar phase structures on solid supports upon gradual increase of the buffer fraction. Using the quartz crystal microbalance with dissipation (QCM-D) technique, we investigated the formation of negatively and positively charged membranes via the SALB formation method and directly compared with the vesicle fusion method on two different oxide films. Bilayers containing an increasing fraction of negatively charged DOPS lipid molecules were successfully formed on both SiO2 and Al2O3 substrates using the SALB formation method at physiological pH (7.5). In contrast, the vesicle fusion method did not support bilayer formation on Al2O3 and those containing more than 10% DOPS ruptured on SiO2 only under acidic conditions (pH 5). Characterization of the fraction of negatively charge DOPS by in situ annexin 5A binding assay revealed that the fraction of DOPS lipid molecules in the bilayers formed on Al2O3 is significantly higher than that formed on SiO2. This suggests that the SALB self-assembly of charged membranes is predominantly governed by the electrostatic interaction. Furthermore, our findings indicate that when multicomponent lipid mixtures are used, the relative fraction of lipids in the bilayer may differ from the fraction of lipids in the precursor mixture.
Assuntos
Óxido de Alumínio/química , Bicamadas Lipídicas/síntese química , Dióxido de Silício/química , Solventes/química , Bicamadas Lipídicas/químicaRESUMO
The solvent-assisted lipid bilayer (SALB) method offers a general strategy to fabricate supported lipid bilayers on solid surfaces. In this method, lipids dissolved in alcohol are deposited on the target substrate in parallel with their aggregation during exchange with aqueous buffer solution which promotes spontaneous bilayer formation. Herein, a combination of experimental and theoretical approaches is employed in order to understand the key aspects of the SALB formation process. Epifluorescence microscopy experiments are conducted in order to measure the spatiotemporal dynamics of bilayer formation on a glass substrate in a microfluidic channel. Corresponding snapshots of bilayer formation at different stages are rationalized by a numerical simulation of solvent displacement inside the channel. Comparing simulation with experiment indicates that in close proximity to the side walls of the present setup, the bilayer formation is confined to a relatively thin region behind the moving solvent displacement front.
Assuntos
Bicamadas Lipídicas/química , Solventes/química , Recuperação de Fluorescência Após Fotodegradação , Corantes Fluorescentes/química , Microscopia de Fluorescência , Fosfatidilcolinas/químicaRESUMO
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
Visualization of phase coexistence in the ß region of cholesterol-phospholipid mixtures consisting of high cholesterol concentrations has proved elusive in lipid bilayers. Here, using the solvent-assisted lipid bilayer approach to prepare supported membranes with high cholesterol fractions close to the cholesterol solubility limit, we report the observation of coexisting liquid phases using fluorescence microscopy. At â¼63 mol % cholesterol, supported membranes consisting of mixtures of DOPC and cholesterol exhibit large-area striping reminiscent of the stripe superstructures that characterize the proximity of the second critical point in the miscibility phase diagram. The properties of the two phases are consistent with condensed complex-rich and cholesterol-rich liquids. Both phases exhibit long-range lateral mobility, and diffusion through a given phase is favored over hopping across the phase boundary, producing an "archipelago effect" and a complex percolation path.
Assuntos
Colesterol/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Substâncias Macromoleculares/química , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Planar lipid bilayers on solid supports mimic the fundamental structure of biological membranes and can be investigated using a wide range of surface-sensitive techniques. Despite these advantages, planar bilayer fabrication is challenging, and there are no simple universal methods to form such bilayers on diverse material substrates. One of the novel methods recently proposed and proven to form a planar bilayer on silicon dioxide involves lipid deposition in organic solvent and solvent exchange to influence the phase of adsorbed lipids. To scrutinize the specifics of this solvent-assisted lipid bilayer (SALB) formation method and clarify the limits of its applicability, we have developed a simplified, continuous solvent-exchange version to form planar bilayers on silicon dioxide, gold, and alkanethiol-coated gold (in the latter case, a lipid monolayer is formed to yield a hybrid bilayer) and varied the type of organic solvent and rate of solvent exchange. By tracking the SALB formation process with simultaneous quartz crystal microbalance-dissipation (QCM-D) and ellipsometry, it was determined that the acoustic, optical, and hydration masses along with the acoustic and optical thicknesses, measured at the end of the process, are comparable to those observed by employing conventional fabrication methods (e.g., vesicle fusion). As shown by QCM-D measurements, the obtained planar bilayers are highly resistant to protein adsorption, and several, but not all, water-miscible organic solvents could be successfully used in the SALB procedure, with isopropanol yielding particularly high-quality bilayers. In addition, fluorescence recovery after photobleaching (FRAP) measurements demonstrated that the coefficient of lateral lipid diffusion in the fabricated bilayers corresponds to that measured earlier in the planar bilayers formed by vesicle fusion. With increasing rate of solvent exchange, it was also observed that the bilayer became incomplete and a phenomenological model was developed in order to explain this feature. The results obtained allowed us to clarify and discriminate likely steps of the SALB formation process as well as determine the corresponding influence of organic solvent type and flow conditions on these steps. Taken together, the findings demonstrate that the SALB formation method can be adapted to a continuous solvent-exchange procedure that is technically minimal, quick, and efficient to form planar bilayers on solid supports.
Assuntos
Ouro/química , Bicamadas Lipídicas , Dióxido de Silício/química , Solventes/químicaRESUMO
This paper describes the application of a solvent-exchange method to prepare supported membranes containing high fractions of cholesterol (up to â¼57 mol %) in an apparent equilibrium. The method exploits the phenomenon of reverse-phase evaporation, in which the deposition of lipids in alcohol (e.g., isopropanol) is followed by the slow removal of the organic solvent from the water-alcohol mixture. This in turn induces a series of lyotropic phase transitions successively producing inverse-micelles, monomers, micelles, and vesicles in equilibrium with supported bilayers at the contacting solid surface. By using the standard cholesterol depletion by methyl-ß-cyclodextrin treatment, a quartz crystal microbalance with dissipation monitoring assay confirms that the cholesterol concentration in the supported membranes is comparable to that in the surrounding bulk phase. A quantitative characterization of the biophysical properties of the resultant bilayer, including lateral diffusion constants and phase separation, using epifluorescence microscopy and atomic force microscopy establishes the formation of laterally contiguous supported lipid bilayers, which break into a characteristic domain-pattern of coexisting phases in a cholesterol concentration-dependent manner. With increasing cholesterol fraction in the supported bilayer, the size of the domains increases, ultimately yielding two-dimensional cholesterol bilayer domains near the solubility limit. A unique feature of the approach is that it enables preparation of supported membranes containing limiting concentrations of cholesterol near the solubility limit under equilibrium conditions, which cannot be obtained using conventional techniques (i.e., vesicle fusion).
Assuntos
2-Propanol/química , Colesterol/síntese química , beta-Ciclodextrinas/química , Colesterol/química , Tamanho da Partícula , Solventes/química , Propriedades de SuperfícieRESUMO
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.