2/7/2016 Electrophoresis Techniques Lecture 5/6 Methods in Molecular Biophysics D5 Intermolecular and Surface Forces 14 adahlin@chalmers.se http://www.adahlin.com/ 2016-02-08 Biotechnical Physics 1 Outline Recall the mantra of this course: Biology is complicated, but we can understand it, at least to some extent. One important component is the role of interfaces. A biological sample will contain many different molecules. How can one separate them from one another? (Necessary if we are to study them!) Electrophoresis is one such separation technique, which is based on charge. We will also look closer at gel electrophoresis techniques, which is the standard for separation by size. By using dielectrophoresis, one can even control the position of molecules and direct them to certain regions. (Accumulate molecules and counteract diffusion!) Finally, you will learn what electroosmotic flow is. This relates to the lectures on microfluidics. 2016-02-08 Biotechnical Physics 2 1 2/7/2016 Interfaces are Everywhere Inside the cell you are never more than ~100 nm away from an interface of some kind! transmission electron microscopy image of a cell Rachel Edmonds Animal Cell http://www.thinglink.com/ 2016-02-08 Biotechnical Physics 3 The Electric Double Layer The standard theory for the charged interface is a diffuse Gouy-Chapman layer and a Helmholtz-Stern layer with physically adsorbed ions. Usually you have a bit of both, at least at high potentials. – – + – + – + + – – Helmholtz-Stern model, adsorbed ions. 2015-09-10 + + + – – + – – + – + + + – – – + – – + + – + + – + Gouy-Chapman model, diffuse layer. Soft Matter Physics 4 2 2/7/2016 The Diffuse Layer We want to know the potential ψ and the ion concentration C as a function of distance from the planar surface z. The potential energy change when moving an ion a distance z from the location where the diffusive layer starts (z = 0) is: ψ=0 z + + E z Q z 0 Here ψ0 is the potential at z = 0 and Q is the charge of the ion, which is determined by the valency ν (…, -2, -1, 1, 2, …) by Q = νe. + – – – – – + – – + + – + ψ0 – + + – – + + (The elementary charge is e = 1.602×10-19 C.) 2015-09-10 Soft Matter Physics 5 Poisson-Boltzmann Equation To get ψ(z) at equilibrium, we use Poisson’s equation from electrostatics: eC z 0 2 z 2 Here ε0 = 8.854×10-12 Fm-1 is the permittivity of free space and ε is the relative permittivity of the medium (for a static field). We use Boltzmann statistics for ion concentration: e z C z C0 exp k BT Note that C0 is the concentration in the bulk (not at the surface). We can now combine these into the Poisson-Boltzmann equation with boundary conditions: e z eC0 2 exp z 2 0 k BT z 0 0 z 0 z 0 z Yikes… 2015-09-10 Soft Matter Physics 6 3 2/7/2016 Approximate Solution For low potentials (|ψ| < 80 mV) the equation has a very simple approximate solution: z 0 exp z Clearly, a very important parameter for the solution is κ which is given by: e2 0 k BT 2 i C0i i 1/ 2 bulk solution, bulk properties One refers to κ-1 as the Debye length. It shows how far into a solution a “surface effect” extends! κ-1 changes in ion concentration, potential and all kinds of weird things… For a solution containing only a monovalent salt: 2C e 2 charged interface 1/ 2 0 0 k BT 2015-09-10 Soft Matter Physics 7 Diffuse Layer Exercise Assume we have a water solution with 150 mM NaCl (physiological) at room temperature. Calculate the concentration of Cl- 0.5 nm from a surface with a potential of +200 mV using the Gouy-Chapman model (no adsorbed ions). Comment on the result! 2015-09-10 Soft Matter Physics 8 4 2/7/2016 Diffuse Layer Exercise First calculate the Debye length, for monovalent salt: C0 = 150 mmolL-1 = 150 molm-3 = 150×6.022×1023 m-3 e = 1.602×10-19 C, kB = 1.381×10-23 JK-1, ε0 = 8.854×10-12 Fm-1 Water means ε = 80, room temperature is T = 300 K. 2C0 e 2 0 k BT 1/ 2 1.257... 109 m 1 The potential at z = 0.5 nm is then: z 0.5 nm 0.2 exp 0.5 10 9 0.106... V The sought ion concentration is thus: e z 0.5 nm 9.28... M C 0.15 exp k BT So we get C = 9.3 molL-1, but the maximum solubility of NaCl in water is 6.2 M-1 at room temperature, so the model is not realistic for this surface potential. 2015-09-10 Soft Matter Physics 9 Grahame Equation How can we relate surface potential to charge density σ (C/m2)? The charges inducing the diffusive layer must compensate the net charge of the ions inside it. This gives the Grahame equation: 0 2 2 0 k BT Ci z 0 C0 i i i Remember that we know C if we know ψ! For very low potentials (<25 mV) an approximation is: + 0 0 0 Very important: We are still only considering the diffuse layer! The charge density you get will generally not be that at the actual surface. Soft Matter Physics + – – + – – – – + σ0 – + + – + + – – σs ??? 2015-09-10 + σ=0 – + + 10 5 2/7/2016 Adsorbed Ions The Helmholtz-Stern layer can be thought of as a plate capacitor. The field between two charged plates is E = σ/[εε0] = V/d and thus: s 0 dΓ ion e 0 Here Γion is the surface coverage of adsorbed ions (inverse area). Simple, but the values are very hard to know. The distance d can be approximated with the radius of the adsorbed ion. However, the permittivity will be very different from that of the bulk liquid because the water molecules are highly oriented. d Again very important: Only a part of the surface charges are compensated by ions in the adsorbed layer! ψ0 ψs 2015-09-10 + – + – + – + + – + Soft Matter Physics – + 11 DLVO Theory Colloid stability (double layer repulsion) depends on ionic strength! Includes van der Waals attraction force which scales with separation d as ~1/d2. Simplified interaction energy U for two spheres: U d 2πR 0 0 exp d 2 RZ 12d just proof of principle, only accurate when d << R 15 kinetic barrier 10 van der Waals Example for: R d ψ0 = 20 mV T = 300 K ε = 80 R = 50 nm Z = 10-19 J (Hamaker constant) 2015-09-11 R U/[kBT] 5 0 κ-1 = 100 nm κ-1 = 50 nm κ-1 = 20 nm κ-1 = 1 nm -5 -10 -15 0 Soft Matter Physics 100 200 300 d (nm) 400 500 12 6 2/7/2016 Zeta Potential The zeta potential ζ is defined as the potential at the “no slip” position or the “shear plane” within the electric double layer. This is the distance at which ions and water molecules no longer are “stuck”. When the particle moves, water molecules and ions closer than the point of the zeta potential will move with the particle. + The zeta potential is sometimes assumed to be equal to the potential at which the diffusive layer starts (ζ = ψ0). ζ ψ0 ψs – + – + – – Can be measured for surfaces and for particles! 2015-09-10 + + – – + Soft Matter Physics flow – + – + – – + – + no flow + 13 Electrochemistry Experiments Simplest way to control a surface potential: Set a potential against a reference electrode by running a circuit via a counter electrode. ← e- Pt Ag/AgCl A V – – – electrolyte + + potentiostat + sample 2016-02-08 Biotechnical Physics 14 7 2/7/2016 Electrochemical Detection If the target can induce a current, it can be detected! For glucose, an enzyme (usually glucose oxidase) converts glucose into gluconic acid and hydrogen peroxide. A mediator oxidizes the enzyme. The mediator is then oxidized at the electrode. A steady current is generated as long as there is glucose. The magnitude gives the glucose concentration in the sample. e- glucose oxidase electrochemical mediator glucose gluconic acid, H2O2… e- e- Fe(CN)63- + e- ↔ Fe(CN)64electrode 2016-02-08 ferrocyanide as mediator Biotechnical Physics 15 Assumptions in Electrophoresis • Homogenous electric field (E). • Particle that carries a charge. – – – – – + External field (N/C or V/m) is given by: – V E d + + 2016-02-08 Biotechnical Physics + – + d – – – – + + – + – – – + + + + – + – + • Ions in the surrounding solution. Counterions will on average be closer to the particle and in higher concentration than ions that carry the same charge. – – + – V 16 8 2/7/2016 Electrophoretic Mobility Assume the particle moves at constant velocity. It is reasonable that the steady-state velocity is acquired fast on the nanoscale due to low inertia. We can define the electrophoretic mobility μ as the velocity per field strength: v E Obviously we want to know roughly how large μ is when we design an electrophoresis experiment! This will depend on the forces acting on the charged object. Two forces are obvious: The force from the field and the friction from the liquid. Ffield QE The total charge of the object is Q. The friction force is given by: Fdrag fv For low Reynold’s numbers and spherical objects we have Stokes drag: f 6πR Here η is the dynamic viscosity and R is the particle radius. 2016-02-08 Biotechnical Physics 17 Smoluchowski Approximation If the Debye length is much shorter than the particle size (κ-1 << R), the Smoluchowski equation for the mobility can be used: 0 The model assumes a simple force balance at constant velocity: Ffield = Fdrag The electric double layer theory for a planar surface is used! This is possible if the curvature is low compared with the double layer thickness. Note that zeta potential appears because this is the potential that the external field “senses”, so it will determine the effective charge of the particle. Also, the radius of the particle no longer appears in the equation. The Smoluchowski approximation actually works for particles of arbitrary shape! 2016-02-08 Biotechnical Physics 18 9 2/7/2016 More Forces in Electrophoresis All literature agrees that there is (at least) one additional retardation force from the accumulation of counterions around the particle. These ions want to move in the opposite direction! They will attempt to drag the particle with them, resulting in an additional friction-type force: Ftotal Ffield Fdrag Fretardation κ-1 Taking retardation into account is generally very difficult… However, if R >> κ-1 it seems reasonable to use the Stokes friction coefficient. In this manner, the Smoluchowski approximation “automatically” takes retardation into account. R R does not have to be so large for the approximation to be valid at physiological conditions! 2016-02-08 Biotechnical Physics 19 Electrophoresis Exercise Nanoparticles covered with 0.1 -NH3+ groups per nm2 undergo electrophoresis with a voltage of 20 V applied over a distance of 10 cm. Make a rough estimate how long it will take for the particles to move this distance in 100 mM NaCl (water at room temperature). Can the rate be comparable with Brownian motion? 2016-02-08 Biotechnical Physics 20 10 2/7/2016 Electrophoresis Exercise The field is E = 200 Vm-1. The high ionic strength means a short Debye length, so the Smoluchovski model should work. vE 0 We have σ = 0.1×1.602×10-19×1018 = 0.01602 Cm-2. The inverse Debye length is: 2C0 e 2 0 k BT 1/ 2 1.026... 109 m 1 If there are no ions we can approximate zeta potential as the surface potential: 0.022... V 0 This is quite low, which is a good sign. Smoluchowski gives v = 3.12…×10-5 ms-1 (η = 10-3 Pas). Moving 10 cm takes ~9 h. Even a very small nanoparticle would only diffuse ~1 mm during this time so the electrophoretic mobility dominates. 2016-02-08 Biotechnical Physics 21 Gel Electrophoresis Now we can describe separation based on charge (more specifically ζ potential) by electrophoresis. However, one usually also lets the electrophoresis occur in a gel. The gel is a network of linked polymers. When objects move through the gel they are less likely to be able to pass through if they are larger. Now size will influence mobility directly, not only the total charge. The most common gels are: • Agarose (inhomogenous but large pore size, good for larger molecules). • Polyacrylamide (homogenous but small pore size, good for smaller molecules). 2016-02-08 smaller molecule larger molecule Biotechnical Physics × 22 11 2/7/2016 Denaturation DNA and RNA strands are quite “homogenous” molecules. They have a total charge which is simply proportional to their molecular weight. Mobility is thus simply determined by molecular weight. Proteins are more complicated due to their chemical diversity and complicated structure. It is often preferable to chemically denature proteins that undergo electrophoresis. This means that only their amino acid sequence matters. By using charged surfactants for denaturation, the charge can be controlled. (Negatively charged sodium dodecyl sulphate.) Influence from amino acid type is then minor and electrophoretic separation occurs on the basis of molecular weight. Also, everything moves in the same direction! + – – – + + – – 2016-02-08 – – – – – – – Biotechnical Physics 23 Visualization and Calibration In an electrophoresis experiment, the molecules that undergo separation need to be stained somehow to visualize them in the gel. Despite our fancy models for electrophoretic mobility, it is hard to predict the velocity inside a gel. Usually a size standard is included for calibration. This gives bands that corresponds to the movement of molecules with known molecular weight (and charge). DNA stained with ethidium bromide, UV light 2016-02-08 Biotechnical Physics Wikipedia: Gel Electrophoresis 24 12 2/7/2016 Two Dimensional Electrophoresis A pH gradient can be maintained along the field. In a second run after the initial ordinary separation a protein will stop moving when it comes to a pH region where it has neutral charge. 2D electrophoresis makes separation much more efficient since two proteins are unlikely to have both similar mass and isoelectric point. Invented in 1975 and still heavily used today in proteomics! high pH protein spots first electrophoresis gel second electrophoresis 2016-02-08 low pH Biotechnical Physics 25 Background: Isoelectric Point Proteins have amino acids that can be basic or acidic. The isoelectric point of a protein is the pH at which it carries no net charge. The electrophoretic mobility is then zero! + + + + + + + low pH basic side chains protonated positive charge – – + + – pH = isoelectric point no net charge – – – – – – high pH acidic side chains deprotonated negative charge Wikipedia: Amino Acid 2016-02-08 Biotechnical Physics 26 13 2/7/2016 Dielectrophoresis In an inhomogenous field, an additional electrophoretic force FDEP appears. The force acts along the gradient of the field (zero gradient means homogenous field). Field gradients appear at any type of pointy electrode geometry! ∂E/∂z = 0 + + + – – – + – – – + ∂E/∂z > 0 + 2016-02-08 Biotechnical Physics 27 The Dielectrophoretic Force An object does NOT have to be charged to feel the force. The force depends on the polarizability of the object. net force Since the field is not uniform, one pole will experience a greater electric field and thus a higher force! For a spherical object, the time averaged force is (for induced dipole): FDEP 2 E 2πR 3 m 0 Re 2 p p m m – – – – + + + + Here ε* represents complex relative permittivity: i 0 Here σ is the conductivity of the surrounding medium and ω is the angular frequency of the field. 2016-02-08 Biotechnical Physics 28 14 2/7/2016 DEP Applications It is possible to use DC fields and still get a DEP force, so why use AC fields? First, periodically reversing the field allows elimination of ordinary electrophoretic motion due to inherent charge. One can also determine electrical properties of particles by varying ω. The main applications of DEP is to separate cells and particles like lipid vesicles. Macromolecules like DNA or proteins can also be manipulated. electrode↓ field→ DC AC Planar Ordinary electrophoretic mobility only. No net movement from electrophoresis, no dielectrophoretic force either. Structured Both electrophoretic and dielectrophoretic mobility. No net movement due to electrophoresis, but movement due to dielectrophoresis. 2016-02-08 Biotechnical Physics 29 Electroosmotic Flow Remember the counterions that leads to an additional force hindering the movement of an object in electrophoresis. These ions are dragging liquid with them! This principle can be used to generate flow in narrow channels that have charged walls. There must be an excess of counterions inside the channel. The flow will follow the field lines (positive to negative) if the walls are negative and vice versa. – – – – – + – – + – – – – + – + – + If κ-1 << channel width, the velocity can be approximated as: E v 0 4π + + – – + – – + – + – + – – – + – Here ζ is the zeta potential of the channel surface! 2016-02-08 Biotechnical Physics 30 15 2/7/2016 Capillary Electrophoresis The electroosmotic flow gives a constant flow profile (plug flow)! This is in contrast to the parabolic velocity profile from pressure driven flow. Naturally, a charged object placed inside a capillary channel will experience the electroosmotic velocity together with the ordinary electrophoretic force. The electroosmotic flow is always strongest. (Not obvious why…) In capillary electrophoresis one takes advantage of the flow. All objects go through the channel in the same direction with the flow, but those that carry charge will either go slower or faster depending on the charge. (A type of chromatography!) – 2016-02-08 + 0 Biotechnical Physics 31 Nanopipettes Very small openings at the end of a tip which can be moved with high precision. A nice mixture of everything: • Electrophoretic mobility. • Dielectrophoretic trapping. • Electroosmotic flow. Can be used for delivery or trapping at the tip! L.M. Ying Biochemical Society Transactions 2009 2016-02-08 Biotechnical Physics 32 16 2/7/2016 Reflections & Questions ? 2016-02-08 Biotechnical Physics 33 17