Microfluidics, sample delivery and laser illumination Manfred Roessle University of Applied Sciences Luebeck 25.11.2018 EMBO practical course BioSAXS 2018 1
Motivation for Microfluidics Everything goes! 25.11.2018 EMBO practical course BioSAXS 2018 2
Motivation for Microfluidics Low sample volume Volume in nanoliter 1000 800 The volume scales with L 3 600 Volume in nanoliter 400 16 14 200 12 0 10 0 0,2 0,4 0,6 0,8 1 8 Side lenght in mm 6 A cube of 0.2 mm side length has a 4 volume of 8 nanoliter! 2 0 A sphere of 0.2 mm diameter has 4 0 0,05 0,1 0,15 0,2 0,25 Side lenght in mm nanoliter! 25.11.2018 EMBO practical course BioSAXS 2018 3
Motivation for Microfluidics Low Reynolds number 𝑆𝑓 = 𝜍 ∙ 𝑤 ∙ 𝑒 𝜃 Re : Reynolds number ρ : Density of the solution η : Viscosity of the solution a) Laminar flow (Newtonian Flow) d : length scale (e.g. channel width) b) Turbulent flow v : flow velocity The Reynolds number gives a criteria for laminar or turbulent flow. E.g. in a tube, Reynolds number > 1200 are considered to produce turbulent flows. 25.11.2018 EMBO practical course BioSAXS 2018 4
Motivation for Microfluidics Low Reynolds number 𝑆𝑓 = 𝜍 ∙ 𝑤 ∙ 𝑒 𝜃 Example for protein solutions: ρ : ~ 1 g/cm 3 a) Laminar flow (Newtonian Flow) η : ~ 1 mPa·s b) Turbulent flow d : ~ 100 µm channel width v : 0.1 m/s flow velocity Re ~ 10 laminar flow 25.11.2018 EMBO practical course BioSAXS 2018 5
25.11.2018 EMBO practical course BioSAXS 2018 6
Motivation for Microfluidics Hydrodynamic focusing and laminar flow Hydrodynamic focusing produce a laminar flow in a T-junction microchannel. In this very fine and localized flow e.g. small molecules can be mixed in by diffusion. 25.11.2018 EMBO practical course BioSAXS 2018 7
Motivation for Microfluidics Shear stress in microchannels According to Newton mechanics a liquid flow of solution in a tube can be describes as: 𝛿 = 8𝑤 v : Velocity of the flow d : diameter of the tube 𝑒 𝛿 is the shear rate, which leads to shear stress . The forces applied by the shear stress can alter the conformation of the fluid particles, e.g. proteins in solution. Since the diameter of the microfluidic channels d are in the range of several µm, a high shear stress can be applied with low flow rates . 25.11.2018 EMBO practical course BioSAXS 2018 8
Motivation for Microfluidics Surface energy dominates 3.5 Ratio E kin to E surf for a droplet velocity v=2.5m/s 3.0 As the surface energy scales with L 2 the 2.5 surface energy dominates over the kinetic energy: E kin /E surf 2.0 1.5 E surf = 4 s p r 2 Sphere of 1.0 radius r E kin = 4/6 r p r 3 v 2 kinetic energy surface energy is dominant 0.5 becomes dominant 2 3 4 5 6 7 8 2 3 4 5 6 7 8 2 3 4 5 1 10 100 diameter of sphere [µm] 25.11.2018 EMBO practical course BioSAXS 2018 9
Motivation for Microfluidics Surface energy dominates Formation of stable droplets, which can sputtered on a surface without splashing! 25.11.2018 EMBO practical course BioSAXS 2018 10
25.11.2018 EMBO practical course BioSAXS 2018 11
Fabrication techniques for microfluidic Production of a glass or nickel master using microstructuring techniques such as: • Micro milling and other micro machining • Lithographic techniques in silicon or polymers (PMMA) From this master the microfluidic structure can be mass produced by injection molding 25.11.2018 EMBO practical course BioSAXS 2018 12
Fabrication techniques for microfluidic Rapid prototyping with 50 µm resolution is possible, even with low- budget 3D printers! 25.11.2018 EMBO practical course BioSAXS 2018 13
Fabrication techniques for microfluidic • Rectangular microchannel cross section • 1 µm features replicated • Milled topography replicated Microfluidic systems contain: 1 mm • Valves • Mixers • Exact volumes for metering • T-junctions and distributer • Flow rate controllers 500 µm 10 µm 25.11.2018 EMBO practical course BioSAXS 2018 14
X-ray generation Synchrotron Sources Shanghai Synchrotron Radiation Facility China Deutsches Elektronen Synchrotron Hamburg Germany European Synchrotron Radiation Facility Grenoble France Advances Photon Source Chicago USA Spring8, Himeij, Japan 25.11.2018 EMBO practical course BioSAXS 2018 15
X-ray generation Synchrotron Dipole bending magnet (APS) www.esrf.eu 25.11.2018 EMBO practical course BioSAXS 2018 16
X-ray generation Synchrotron Electrons are deflected in the magnetic field of a dipole magnet by the Lorentz- “X - ray Bremsstrahlung” h = E i - E f force. E i Accelerated charges are producing electromagnetic e - radiation! e - E f Synchrotron radiation emitted by a dipole magnet www.desy.de 25.11.2018 EMBO practical course BioSAXS 2018 17
X-ray generation Synchrotron Most powerful insertion device! A stack of magnetic dipoles Undulator generate a high flux of photons in a very small source size. The specific arrangement of the dipoles with distance d=n* produces a discrete spectrum with coherent properties. http://www.physics.uwa.edu.au High brilliance X-ray beam at ESRF´s ID09! 25.11.2018 EMBO practical course BioSAXS 2018 18
X-ray generation Synchrotron radiation Increase of the beam PETRA III brilliance from X-ray tubes to third generation sychrotron Small beams sources to free electron lasers with a lot of (forth generation) photons!! 25.11.2018 EMBO practical course BioSAXS 2018 19
X-ray Beamlines BioSAXS @ EMBL Hamburg Beam size and divergence at modern SAXS Synchrotron beamlines PILATUS 2M P12 205*64 µm 2 High brilliance beamlines for structural biology 40*15 rad 2 • High flux of photons in very small focal X-ray beam spots! • Downscaling of the sample container to fit the beam sizes. 25.11.2018 EMBO practical course BioSAXS 2018 20
Time resolved Experiments Access to structural dynamics Eadweard Muybridge (1840 to 1904) British photographer and pioneer in motion pictures 25.11.2018 EMBO practical course BioSAXS 2018 21
Time resolved Experiments Access to structural dynamics 25.11.2018 EMBO practical course BioSAXS 2018 22
Time resolved Experiments Access to structural dynamics 25.11.2018 EMBO practical course BioSAXS 2018 23
Time resolved Experiments Access to structural kinetics Example: Reaction kinetics of an ATP driven two component protein system. Classical stopped-flow experiment. • Typical mixing time in the range of several ms • Suitable for the sub-second time range • 50µl to 80µl total volume Reactant B Reactant A • on a third generation synchrotron radiation source about 5 to 10 repetitions necessary Repetitive measurements quartz capillary mixer High sample consumption Need of a suitable detector system Time resolution ~ 10ms 25.11.2018 EMBO practical course BioSAXS 2018 24
Time resolved SAXS Cooperativity of chaperonin ATPase activity 66 radius of gyration [Å] 65 (GroEL+ GroES) + ADP (1mM) (GroEL + GroES) + ATP (0.1mM) GroEL + Buffer (Referenz) 64 Modulation of the radius of gyration upon the binding of nucleotides. The binding of 10 20 30 40 time [s] the nucleotides seems to be highly M. Roessle et. al. J.Appl. Cryst. cooperative. 25.11.2018 EMBO practical course BioSAXS 2018 25
Time resolved SAXS Fast mixing in continuous flow • fast mixing times ~10µs to ~100µs • continuous flow method but small sample consumption! • micromachining or lithographic technology Akyama, PNAS 2002 L. Pollack PNAS 1999 Akiyama et al. PNAS 2002 25.11.2018 EMBO practical course BioSAXS 2018 26
Online sample preparation Micro reactors for fast structural kinetics Mixing of the droplets by Reagent A Reagent B collision is very fast t mix ~ 10µs Following the reaction by scanning the flow after the mixing with the X-ray microbeam. t 0 High brilliance X-ray beam Example: droplet volume: 65pl droplet frequency: 1000Hz exposure time : 10s t reag time points : 100 65 µl Volume 25.11.2018 EMBO practical course BioSAXS 2018 27
Online sample preparation Micro reactors ESRF microfocus beamline ID 13 Sample environment depends on scientific question e.g. silk fiber maturation under shear forces A. Martel et. al. Biomicrofluidics 2008 25.11.2018 EMBO practical course BioSAXS 2018 28
Time resolved SAXS/WAXS Access to structural dynamics Kinetics: Every individual is behaving individual. Dynamics: All individuals are behaving in the same way. Estimation on the reaction velocity and the order of the reaction Analysis on the reaction intermediates and intermediate structures. 25.11.2018 EMBO practical course BioSAXS 2018 29
Time resolved SAXS/WAXS Caged compounds Caged ATP Possible reaction triggering by flash photolysis of so called caged components such as caged nucleotides (caged ADP, caged ATP etc.) . A photosensitive protection group inhibits the normal hydrolysis process of the Photosensitive protection group nucleotide, but the protection group can be cleaved from the nucleotide by a strong, fast light flash. Reactive ATP 25.11.2018 EMBO practical course BioSAXS 2018 30
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