Microfluidics, sample delivery and laser illumination Manfred - - PowerPoint PPT Presentation

25.11.2018 1 EMBO practical course BioSAXS 2018

Microfluidics, sample delivery and laser illumination

Manfred Roessle University of Applied Sciences Luebeck

25.11.2018 2 EMBO practical course BioSAXS 2018

Motivation for Microfluidics

Everything goes!

25.11.2018 3 EMBO practical course BioSAXS 2018 A cube of 0.2 mm side length has a volume of 8 nanoliter! A sphere of 0.2 mm diameter has 4 nanoliter! 200 400 600 800 1000 0,2 0,4 0,6 0,8 1

Volume in nanoliter Side lenght in mm

2 4 6 8 10 12 14 16 0,05 0,1 0,15 0,2 0,25

Volume in nanoliter Side lenght in mm

Motivation for Microfluidics

Low sample volume

The volume scales with

L3

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Motivation for Microfluidics

Low Reynolds number

𝑆𝑓 = 𝜍 ∙ 𝑤 ∙ 𝑒 𝜃

Re: Reynolds number ρ: Density of the solution η: Viscosity of the solution d: length scale (e.g. channel width) v: flow velocity

a) Laminar flow (Newtonian Flow) b) Turbulent flow

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.

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Motivation for Microfluidics

Low Reynolds number

𝑆𝑓 = 𝜍 ∙ 𝑤 ∙ 𝑒 𝜃

a) Laminar flow (Newtonian Flow) b) Turbulent flow Example for protein solutions: ρ: ~ 1 g/cm3 η: ~ 1 mPa·s d: ~ 100 µm channel width v: 0.1 m/s flow velocity

Re ~ 10 laminar flow

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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 8 EMBO practical course BioSAXS 2018

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 𝑒

v: Velocity of the flow d: diameter of the tube 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.

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As the surface energy scales with L2 the surface energy dominates over the kinetic energy:

3.5 3.0 2.5 2.0 1.5 1.0 0.5 Ekin/Esurf 1

2 3 4 5 6 7 8

10

2 3 4 5 6 7 8

100

2 3 4 5

diameter of sphere [µm]

surface energy is dominant kinetic energy becomes dominant

Ratio Ekin to Esurf for a droplet velocity v=2.5m/s

Ekin = 4/6 r p r3 v2 Esurf = 4 s p r2

Sphere of radius r

Motivation for Microfluidics

Surface energy dominates

25.11.2018 10 EMBO practical course BioSAXS 2018

Formation of stable droplets, which can sputtered on a surface without splashing!

Motivation for Microfluidics

Surface energy dominates

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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

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Fabrication techniques for microfluidic

Rapid prototyping with 50 µm resolution is possible, even with low- budget 3D printers!

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1 mm 500 µm 10 µm

• Rectangular microchannel cross section
• 1 µm features replicated
• Milled topography replicated

Fabrication techniques for microfluidic

Microfluidic systems contain:

• Valves
• Mixers
• Exact volumes for metering
• T-junctions and distributer
• Flow rate controllers

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X-ray generation

Synchrotron Sources

Shanghai Synchrotron Radiation Facility China European Synchrotron Radiation Facility Grenoble France Advances Photon Source Chicago USA Deutsches Elektronen Synchrotron Hamburg Germany Spring8, Himeij, Japan

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X-ray generation

Synchrotron

Dipole bending magnet (APS)

www.esrf.eu

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Accelerated charges are producing electromagnetic radiation!

X-ray generation

Synchrotron

Synchrotron radiation emitted by a dipole magnet

www.desy.de

e- e-

h = Ei - Ef Ef Ei

“X-ray Bremsstrahlung” Electrons are deflected in the magnetic field of a dipole magnet by the Lorentz- force.

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Undulator

http://www.physics.uwa.edu.au

Most powerful insertion device! A stack of magnetic dipoles 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.

X-ray generation

Synchrotron

High brilliance X-ray beam at ESRF´s ID09!

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PETRA III

Increase of the beam brilliance from X-ray tubes to third generation sychrotron sources to free electron lasers (forth generation)

X-ray generation

Synchrotron radiation Small beams with a lot of photons!!

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205*64 µm2 40*15 rad2 Beam size and divergence at modern SAXS Synchrotron beamlines

High brilliance beamlines for structural biology

• High flux of photons in very small focal X-ray

beam spots!

• Downscaling of the sample container to fit the

beam sizes.

PILATUS 2M P12

X-ray Beamlines

BioSAXS @ EMBL Hamburg

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Eadweard Muybridge (1840 to 1904) British photographer and pioneer in motion pictures

Time resolved Experiments

Access to structural dynamics

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Time resolved Experiments

Access to structural dynamics

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Time resolved Experiments

Access to structural dynamics

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Example: Reaction kinetics of an ATP driven two component protein system. Classical stopped-flow experiment.

quartz capillary mixer

Reactant A Reactant B

• Typical mixing time in the range of several ms
• Suitable for the sub-second time range
• 50µl to 80µl total volume
• on a third generation synchrotron radiation

source about 5 to 10 repetitions necessary

Repetitive measurements High sample consumption Need of a suitable detector system Time resolution ~ 10ms

Time resolved Experiments

Access to structural kinetics

25.11.2018 25 EMBO practical course BioSAXS 2018

Time resolved SAXS

Cooperativity of chaperonin ATPase activity

66 65 64 radius of gyration [Å] 40 30 20 10 time [s] (GroEL+ GroES) + ADP (1mM) (GroEL + GroES) + ATP (0.1mM) GroEL + Buffer (Referenz)

Modulation of the radius of gyration upon the binding of nucleotides. The binding of the nucleotides seems to be highly cooperative.

• M. Roessle et. al. J.Appl. Cryst.

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• L. Pollack PNAS 1999

Akyama, PNAS 2002

• fast mixing times ~10µs to ~100µs
• continuous flow method but small sample

consumption!

• micromachining or lithographic technology

Akiyama et al. PNAS 2002

Time resolved SAXS

Fast mixing in continuous flow

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Mixing of the droplets by collision is very fast tmix ~ 10µs Following the reaction by scanning the flow after the mixing with the X-ray microbeam. Example: droplet volume: 65pl droplet frequency: 1000Hz exposure time : 10s time points : 100 65 µl Volume

Reagent B Reagent A High brilliance X-ray beam t0 treag

Online sample preparation

Micro reactors for fast structural kinetics

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• A. Martel et. al. Biomicrofluidics 2008

Online sample preparation

Micro reactors ESRF microfocus beamline ID 13 Sample environment depends on scientific question e.g. silk fiber maturation under shear forces

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Time resolved SAXS/WAXS

Access to structural dynamics

Kinetics: Every individual is behaving individual. Estimation on the reaction velocity and the

• rder of the reaction

Dynamics: All individuals are behaving in the same way. Analysis on the reaction intermediates and intermediate structures.

25.11.2018 30 EMBO practical course BioSAXS 2018 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 nucleotide, but the protection group can be cleaved from the nucleotide by a strong, fast light flash.

Caged ATP

Photosensitive protection group

Reactive ATP

Time resolved SAXS/WAXS

Caged compounds

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ATP-dependent dimerization of soluble nucleotide-binding domains (NBDs) from a bacterial lipid flippase, MsbA.

Time resolved SAXS/WAXS

Caged compounds

Photocage-initiated time-resolved solution X-ray scattering investigation of protein dimerization

• I. Josts, S. Niebling, Y. Gao, M. Levantino, H. Tidowa, & D. Monteiroa; IUCrJ (2018). 5, 667–672

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Cho, H. S.; Schotte, F.; et.al: Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-Ray Scattering. J. Am. Chem. Soc. 2016, 138 (28), 8815–8823.

Time resolved SAXS

Investigation of Structural Dynamics

Different reaction intermediates of the photoactive yellow protein PYP upon its photo cycle. The different states has be initiated by illumination with a laser (wavelength 390nm & 473nm). Combination of different methods including Laue time resolved crystallography.

25.11.2018 33 EMBO practical course BioSAXS 2018 Dolev Rimmerman; et.al: Direct Observation of Insulin Association Dynamics with Time- Resolved X-ray Scattering; J. Phys. Chem. Lett. 2017, 8, 4413−4418

Time resolved SAXS

Investigation of Structural Dynamics

Insulin dimerization upon temperature changes

25.11.2018 34 EMBO practical course BioSAXS 2018

Time resolved SAXS/WAXS

Problem of light absorption For triggering reactions in a water- protein solution, the light has to be transmitted completely into the sample cell! For light outside the visible range, the sample cell has to be very small!

25.11.2018 35 EMBO practical course BioSAXS 2018

Time Resolved SAXS/WAXS

Sample environment for pump probe experiments

Micro fabricated sample flow cell:

• Fitting the trigger-pump pulse to the absorption of the sample
• Path length for the X-ray beam as large as possible
• Coupling of the pump optics to the microstructure
• Experts in micro structuring needed!

X-rays

Standard capillary:

• Large absorption of the triggering light pulse in the cell
• Large part of the sample may not be illuminated by the trigger

pulse nor the X-rays

X-rays

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Microfluidic THz cell

Sample environment for pump probe experiments

Main parts are 3d printed from polyethylen.

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Time Resolved SAXS/WAXS

Where does the SAXS signal coming from? How can small changes introduce large conformational changes?

Cho, H. S.; Schotte, F.; et.al: Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time- Resolved Small- and Wide-Angle X-Ray

• Scattering. J. Am. Chem. Soc. 2016, 138

(28), 8815–8823.

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Time resolved SAXS/WAXS

Access to structural dynamics-Motions and time scale

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Time Resolved SAXS/WAXS

Where does the SAXS signal coming from?

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Time resolved SAXS/WAXS

Protein Dynamics induced by Tera-Hertz radiation

With pump-probe experiments these vibrations can be exited by THz radiation and probed with an intensive X-ray beam.

X-Ray THz radiation

• B. P. Born, “Terahertz Spectroscopy of Proteins”, Diss.; RU Bochum 2010

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P12 beamline Hamburg

Time resolved SAXS/WAXS

THz setup at P12 BioSAXS beamline

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x-ray THz

Time resolved SAXS/WAXS

THz setup at P12 BioSAXS beamline

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Time resolved SAXS/WAXS

Conformational changes introduced by THz radiation Rg/on = 2.95 nm Rg/off = 3.30 nm

Rg change because of conformational changes triggered by THz radiation?

25.11.2018 44 EMBO practical course BioSAXS 2018 Hamburg BioSAXS group Dmitri Svergun Martin Schroer Clement Blanchet Universität zu Lübeck Young-Hwa Song Christian Hübner Projects: PureSAXS TTSAS RöntgenFlex RÅC Project TTSAS: THz Excitations Sia Schewa Till Zickmantel

Acknowledgment

University of Gothenburg Gergely Katonas group CoSAXS team: Tomas Plivelic Ann Terry ESS: Hanna Wacklin Andrew Jackson