Biomolecular Diffusion in Nanofluidics

Imagine a small integrated biomedical analysis laboratory, connected to your home computer, which would be capable of diagnosing illnesses, a lack of vitamins, or the over-presence of substances from samples of blood, urine or saliva. This hypothetic system would be able to give a diagnosis within minutes, finally advising the user about the optimal targeted medicines to take or the right specialist to consult for fast recovery. Of course this system will not be ready in the near future, but this thesis aims to bring some new elements to this exciting project by investigating the diffusion of proteins in well-defined nanometer-sized confined areas. Understanding molecular dynamics in nanoconfinement volumes is fundamental for designing the appropriate lab-on-a-chip devices able to transport, pre-concentrate, separate and sense biomolecules. However, a multitude of phenomena occurring at the nanoscale are still to be discovered and currently, there is a lack of accurate theoretical models to predict the transport of proteins in nanofluidics. Based on measurements performed in 50 nm high 1D nanochannels, where the surface-to-volume ratio is extremely high, protein-surface interactions were initially investigated. Using electrical measurements, the adsorption and desorption kinetics of highly concentrated bovine serum albumin proteins was characterized in different scenarios. Ionic strength conditions were identified, where the electrical conductance is dominated by volume effects due to the adsorbed or bound proteins, leading to potential applications of rapid immunology on-chip. Other situations, where the protein charges were directly influencing the nanochannel conductance, were also highlighted, giving a better understanding of how the adsorbed proteins counterions modify the surface charges. Furthermore, the transport of single proteins diffusing through nanochannels was analyzed using fluorescence correlation spectroscopy. Direct measurements inside nanochannels has allowed the identification of different regimes of interacting proteins, depending on the thickness of the electrical double layer (constituted of immobile ions which equilibrate the surface charges). Taking into account the steric exclusion due to the small channel size, the reversible surface adsorption, and the exclusion-enrichment effect due to the charge of the proteins and ionic strength of the solution, novel theoretical models describing the hindered diffusion of proteins were elaborated. Conditions where the diffusion of proteins through the nanochannels were of the same magnitude as in the bulk were both predicted and experimentally verified. Finally, a novel method is presented to measure the apparent diffusion coefficients of fluorescently-labeled molecules directly inside a nanofluidic system. This technique, based on steady-state dispersion of proteins in a transversal nanoslit, demonstrates that under specific ionic conditions, the apparent diffusion coefficient of wheat germ agglutinin proteins is four orders of magnitude lower than its free diffusion value. Based on this system, the binding affinity of two different proteins was directly measured, demonstrating the potential of this method to be used as a biosensor for quantifying rapid protein complex formation. This thesis mainly deals with fundamental studies related to surface physics and physical chemistry applied to life sciences. The work points out novel, important, experimentally-verified complements to define solid theoretical models, in order to go forward with the design of complex nanofluidic systems applied to biomedical and biological applications.

Towards the Glass Transition in Vibrated Granular Matter

Alessandro Luigi Sellerio

Granular materials are large sets of macroscopic particles that interact solely via contact forces. The static behavior depends on the contact network and on the surface friction forces between grains; when they are set in motion (typically by vibrations) their dynamics is dominated by inelastic collisions. For these reasons granular media show an extremely rich phenomenology, ranging from fluid-like properties (if strongly vibrated), to "jamming", glassy, behavior (if weakly vibrated), to aging and hysteretical phenomena observed when they become trapped in frozen, amorphous states. The objective of this work is to study these states and transitions, and to characterize the analogies found between the dynamic behavior of vibrated granular media and the glass transition observed in thermal glass-formers. These analogies justify the interest in granular materials, because granular media can be seen as simplified model systems useful in the study of out of equilibrium thermodynamics, and, in general, to the larger framework known as "complexity". The granular materials considered here are composed of spheric, polished glass spheres. Since the surface state plays an important role in the grain-grain interaction, some measurements were also performed with acid etched beads, having different surface roughness. The samples are vertically vibrated to achieve vibrofluidization. Different kinds of vibration are used, to highlight different properties of the system. We first consider the transition between the fluid and the subcooled glassy phase, using different experimental techniques. The most important one is a torsion oscillator, that interacts with the granular media via immersed probes. The torsion oscillator can be used in forced mode. A torque is applied on the probe, and we measure the mechanical response function (complex susceptibility). In general, a relaxation is found and it is interpreted as the signature of the irreversible energy loss (damping) in granular collisions. This relaxation has an intrinsic time scale, and systematic analysis of it shows that a clear parallel can be traced to the behavior of "strong" glasses. In particular, it is found that (i) the relaxation time is a function of a unified control parameter, proportional to the square root of the average vibration, and phenomenologically equivalent to an effective temperature; (ii) the functional form with which the relaxation times approach the final "frozen" state has an Arrhenius, or Vögel-Fulcher-Tamman (VFT) behavior. The same torsion oscillator is employed in free mode. In this case, no external torque is applied, and the probe moves adapting its position under the effect of the continuous rearrangements in the sample. The system is studied by computing the power spectral density of the (angular position) time series. The resulting spectra represent a "configurational noise" as the system randomly hops from one configuration to the following. This allows to define, using a completely different approach, the same intrinsic time scale observed in forced mode measurements. The comparison of the two techniques allows to obtain a more complete and detailed picture of the dynamics in the jamming region. From this comparison, it was inferred that the system is also influenced by an effective vibration frequency, and that the relaxation time has indeed a non-Arrhenius behavior as a function of a control parameter defined as as = √ Γ/ωs. A model was developed combining rheological observations to a statistic approach describing extremal phenomena. This model justifies the appearance of both the control parameter and the VFT evolution of the relaxation. Furthermore, the model is predictive and exposes the effect of a few other rheologic properties of granular system. The effect of surface roughness are considered, showing that the static and dynamic surface friction coefficients are well described by the model. A second relevant part of this work is devoted to an explicit verification that macroscopic probes act as Brownian objects. This fact is often used to interpret experimental data (also in the present work) and to propose theoretical model. However, no explicit evidence has ever been discussed. This is hard to do, using a constrained system such as the torsion oscillator, because the restoring coefficient influences the dynamics of diffusion. To overcome the problem we built a different apparatus, called "Brownian motor", where the probes are mounted on ball bearings, so that they are free to turn without constraint. The properties of the time series of the position of the free turning probe and of the torsionally constrained oscillator can finally be analyzed and compared with simple simulations. The data show an overall diffusion-like behavior, that is influenced by the presence of constraints. Using fractal analysis we estimate the diffusion, or Hurst exponent. This allows to verify that a "macroscopic" object (the probe) immersed in the "microscopic" granular medium indeed behaves as a Brownian object, and that its dynamics can be studied in detail, showing that it undergoes anomalous diffusion. This work is concluded with a discussion on a few possible developments. The most promising idea is a novel approach to the study of the geometrical properties of the contact network of granular assemblies, that is responsible for many of the properties of the granular sample. By using Magnetic Resonance Imaging, the static 3-D structure of granular media can be reconstructed with unprecedented accuracy, resolution and ease of reproducibility. From the spatial information we can extract all the properties of static granular media: the compaction factor, the grain-grain correlation function, the free volume and other observables. Systematic studies could allow experimental confirmations of the many theoretical models that have been proposed in the last years and that still lack a thorough comparison with experiments. This idea does not conclude the perspectives of this work, that are vast and intriguing. A few promising subjects are reviewed more into detail in the corresponding Perspective section. To name a few we cite: measurements of induced aging in non-vibrated samples, the Brownian motor, stick and slip phenomena and their comparison with earthquakes.

EPFL2012

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed.

EPFL2006

Strong Coupling between Nanofluidic Transport and Interfacial Chemistry: How Defect Reactivity Controls Liquid-Solid Friction through Hydrogen Bonding

Gabriele Tocci

Defects are inevitably present in nanofluidic systems, yet the role they play in nanofluidic transport remains poorly understood. Here, we report ab initio molecular dynamics (AIMD) simulations of the friction of liquid water on defective graphene and boron nitride sheets. We show that water dissociates at certain defects and that these "reactive" defects lead to much larger friction than the "nonreactive" defects at which water molecules remain intact. Furthermore, we find that friction is extremely sensitive to the chemical structure of reactive defects and to the number of hydrogen bonds they can partake in with the liquid. Finally, we discuss how the insight obtained from AIMD can be used to quantify the influence of defects on friction in nanofluidic devices for water treatment and sustainable energy harvesting. Overall, we provide new insight into the role of interfacial chemistry on nanofluidic transport in real, defective systems.