In microscale thermophoresis (MST), the directed movement of fluorescent molecules in a local temperature gradient is measured. The underlying effect is called thermophoresis
3.5 Binding Probed by Microscale Thermophoresis 31 and was first mentioned by Carl Ludwig in 1856 [71]. MST is a technology that allows the evaluation of the interaction of molecules such as binding or unbinding by studying the changes in the characteristic motion of the sample molecules [72, 73].
3.5.1 Basics of Microscale Thermophoresis
In a temperature gradient∇Tin a liquid, molecules with thermophoretic mobilityDT move with pacev= DT· ∇T. Differences in the local concentrationcare induced by the thermophoretic motion, which causes diffusion of the molecules along the gradient. The resulting total molecular flow is described by [71]:
j =jD+jD
T =−∇c·D−c· ∇T·DT . (3.21) Dis the diffusive mobility. In equilibrium, thermophoresis and ordinary mass diffusion (j= 0) compensate each other:
dc
c =−DT
D ·dT. (3.22)
From this relation the equilibrium distribution of the concentration can be obtained:
c(x)
c0 =exp
−DT
D ·(T(x)−T(x0))
. (3.23)
c0 describes the concentration at site x0. The Soret coefficient ST, a measure for the intensity of thermodiffusion in the stationary state, is defined as
ST = DT
D . (3.24)
The distribution of the concentration caused by thermophoretic motion can be interpreted as a local change in the Gibbs free enthalpyGusing a Boltzmann distribution for small, quasi-continuous temperature steps [74] :
c(T1)
c(T2) =exp
−G(T1)−G(T2) kBT
. (3.25)
From the last two equations, we deduce an expression that relates the Soret coefficient to the overall entropyH:
−H = ∆G
∆T =ST·kBT. (3.26)
Hdescribes the local negative entropy of the solvent-solute system for individual parti-cles at constant pressure. For biophysical experiments, this solvent is ordinarily water or watery solutions. In this case, the entropy of the particles is determined by the entropy of ionic shielding and the entropy of hydration, described by
ST = A
kBT −shydr+ βσ
2 eff
4εε0T ·λDH
!
(3.27) with the hydration entropyshydrper surface area A, the effective surface charge density σeff, the dielectric constant of waterε, the vacuum permittivityε0and the Debye screening lengthλDH. λDHdepends on the concentration of salt of the solution. The relative change of the solvent’s dielectric constantε(T)andλDHwith temperature is characterized byβ.
3.5.2 Analysis of Binding
The Soret coefficientST depends on several parameters: size, conformation, charge and solvation entropy of a particle. Interactions such as binding may alter some or all of the named characteristics. The impact on at least one of these parameters is used in MST to put the interaction of molecules on a quantitative level. The change in concentration in the stationary state with the temperature change∆T =T(x)−T(x0)at the respective positionxis determined byST [75]:
chot=ccold·exp(−ST·∆T) . (3.28) In practice, the temperature gradient inside the sample chamber is generated by a low-powered infrared laser [76]. Usually, the sample chamber is a capillary. Fluo-rescent labelling of the molecules under observation allows the visualization of the thermophoretic motion as a change in the fluorescence intensity detected (see Figure 3.4). The ratioFhot/Fcold =Fnormof the averaged fluorescence intensity during the times when the laser is switched on (Fhot) and off (Fcold), respectively, is used to quantify the thermophoretic behavior. Normally, the amount of the unlabeled interaction partner is titrated against a fixed concentration of the fluorescently labeled partner. This unlabeled partner acts as receptor. If the thermophoretic depletion is plotted against the concentra-tion of receptors, we obtain a binding isotherm that can be analyzed using the formalism presented in the previous section (Equation 3.7 or 3.8, respectively).
3.5 Binding Probed by Microscale Thermophoresis 33
a) b) IR on IR off
Fcold Fhot
time
fluorescence
capillary
-IR beam
-fluorescence -excitation -objective
-Figure 3.4: MST. a) Thermophoresis in a sample solution containing biomolecules is induced by a focused infrared laser. The directed motion of the molecules in the established temperature gradient is detected via the local change in fluorescence. The fluorescent particles are excited using an LED. The thermophoretic motion causes a concentration gradient inside the capillary. b) Fluorescence time trace. Before the infrared laser is switched on, the cold fluorescence Fcold is measured. After starting heating, a steep decrease in fluorescence is detected. This phase is followed by the chracteristic thermophoretic motion, from whichFhotis gained. After turning the laser off, the fluorescent molecules start to diffuse back.
Chapter 4
Proteins and Plasma
In this chapter the background of the used biomaterial is presented. The binding of various proteins to different NPs is discussed in this dissertation (Chapter 5 and 6).
Knowledge of the protein properties and its biological function is essential for experi-ments and interpretation of results. Additionally, experiexperi-ments are performed in complex media such as plasma or serum. Biofluids are scattering media, which are crowded by macromolecules and hence strongly disturb FCS experiments by distortion of the confo-cal volume. The distinction between plasma and serum is explained. The dependence of the viscosity on the temperature and the composition of the medium is demonstrated for mixtures of phosphate buffered saline (PBS) and FBS. The impact of these solutions on FCS measurements is discussed.
4.1 Biomolecular Corona
When bare NPs are exposed to biological fluids, proteins and other biomolecules adsorb to them. The resulting layer(s) of biomaterial is called “protein corona” or “biomolecular corona” (Figure 4.1 a) [8–11]. The composition of the corona gives the NP its biological identity and therefore determines the fate of the NP inside a living system in dependence on its composition [77]. However, the corona is not static. It evolves with time and environmental changes when traveling through different compartments of the body.
Certain proteins will be replaced with fresh ones from the new local environment, resulting in a change of the biological identity. The particle may then show characteristics of both the original milieu and the new one [9, 78]. Understanding of the evolution of the corona is mandatory regarding the general safety of NPs, but also for potential
a) b)
Figure 4.1:a) Inside an organism, NPs will almost invariably be surrounded by a mixture of biomolecules. These biomolecules adsorb fast and strongly onto the NP’s surface. Due to very strong adsorption, the biomolecules might never come off again. This implies that the identity of the NP is effectively hidden by the covering biomolecules on its surface. The corona is divided into two sections. The hard corona (inner circle, dark colors) and the soft corona (outer circle, light colors). The hard corona is often considered to be irreversibly bound due to its slow exchange, while the loosely associated molecules of the soft corona exhibit dynamic exchange with its surroundings. b) NP with hard corona after removal of the surrounding protein solution. Removal can be performed by centrifugation as in publication P1 in Chapter 5. This way, the hard corona becomes analytically accessible.
therapeutic applications as drug carriers. Biomolecules within organisms, such as lipids, sugars or proteins, adsorb strongly to the NP’s surface. In the beginning, abundant proteins bind that are slowly replaced by proteins with a higher affinity. The original NP and its physico-chemical properties are thereby effectively masked. These biophysical properties may differ significantly for the coated and the uncoated NP. Thus, the object whose eventual hazards need to be investigated, is not the original bare NP, but rather the complex consisting of NP and the involved biomolecules from the organism. The biomolecular corona itself can be divided into two parts: the hard and the soft corona (Figure 4.1 a). The molecules of the hard corona are in direct contact with the bare NP surface and remain there for a relevant time [79]. In comparison, the molecules of the soft corona are only loosely associated and exhibit a dynamic exchange with its environment. While the soft corona can be almost completely replaced, the hard corona is robust and long-lived in the presence of added full plasma. Depending on the time-scale of the experiment, the hard corona is also termed irreversible [21, 80].
Studies of the biomolecular corona often use techniques to separate the hard corona-NP complexes from solutions (Figure 4.1 b). Methods such as centrifugation, size exclusion
4.2 Plasma Proteins 37