Team:BIOTEC Dresden/Methods Vesicles

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=== Vesicles - Methods ===
=== Vesicles - Methods ===
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Microfluidics chambers
 
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In order to make vesicles we use a microfluidic chamber incorporated with pumping system, controlled by the software. The experiment is observed by means of Biozero fluorescent microscope.
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==== Setup of the microfluidic system ====
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Microfluidic chamber
 
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The chamber consist of two parts: glass coverslide and a PDMS layer with the microstructure printed on it. Glass does not require any special preparation but plasma oven treatment for 30 seconds at 15 mBars.  
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The microfluidic system consists of a flow chamber made of Polydimethylsiloxane (PDMS) and a pump system that controls the flow rates of the various liquids into the chamber. Droplets are created within a defined space in the chamber and are propagated along a grid that allows containment and imaging. Two types of chambers have been used, differing in the geometry of the space where droplets were produced. One featured a T-junction, and the other one a V-junction (Fig1).
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PDMS (Polydimethyloxane) is an organic, silicon based polymer. [SiO(CH3)2]  is the monomer (Fig. 1).  PDMS is chemically inert, transparent, stable at room temperature, non-toxic and non-flammable and thus can be used as a material for the chamber.
 
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Fig1: Different geometries of the intersection between aqueous and oil phase in flow chambers
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{|
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| [[Image:tchamber2.jpg|thumb|400|alt=t shaped junction|T shaped junction]]
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| [[Image:Vchamber.jpg|thumb|400|alt=v shaped junction|V shaped junction]]
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|}
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In order to prepare PDMS layer one needs a silicon wafer with litographically etched microstructures of required configuration and PDMS mixed with silicon elastomer curing agent in proportion 10:1. For two wafers that we have (Fig.2) we used 12g of PDMS and 1.2g of curing agent. Those two substances have to be well-mixed, degassed and placed on the clean wafer. Polymerization takes about 30 minutes at 150C. After the heating drops of PDMS 2-3 mm high should be placed on top of the structure in the places where the inlets are supposed to be. The polymerized PDMS should be carefully removed from the wafer and the holes for the inlets should be made. There are two ways how one can do those holes: by means of 0.8 mm needle or using a laser cutter. Both methods have advantages and disadvantages. Using needle requires a lot of training and most of samples get cracks around the holes, which means that the chamber will leak. Laser cutting is more accurate but it's difficult to place the PDMS precisely.
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[[Image:Silicon wafer.jpg|200px|thumb|right|Silicon wafer we used for microfluidics chamber preparation]]
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Production of flow chambers:
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* mix PDMS and curing agent in 10:1 ratio
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* degas and pour on wafer with etched microstructures
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* polymerize on heat plate at 150 ºC for 30 min
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* add unpolymerized PDMS mixture to points on microstructure where microtube inlets are to be pierced
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* polymerize on heat plate at 150 ºC for 30 minutes
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* remove polymerized PDMS from wafer, cut to fit onto glass cover slide (24 x 60 mm), and use clean needles    (0.8 mm) or laser cutter (Trotec Speedy 100TM) to pierce tube inlets
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* ionize PDMS and glass slide in plasma chamber for 30 sec to make it reactive
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* align PDMS on glass slide and seal
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* seal irreversibly by heating on plate at 60ºC for 6 hours
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When PDMS layer with microstructure printed on it is ready one cuts out the needed size for a chamber and treats it in plasma oven for 30 seconds (together with cover glass). This treatment makes the surface hydrophilic. After that polymer layer is placed onto the glass and the chamber is baked at 60C for 6 hours.
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This video shows the laser cutting of the microtube inlets:
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In order to check if the chamber is working one tests in under the light microscope with mineral oil. 1 mm tube connected with the syringe with oil is inserted in lower inlet and oil is to fill the whole chamber. When the camber is washed with oil one should check every upper inlet in the similar way. This allows to check if the chamber is not leaking at the inlets and if it is working properly. Also, this makes the surface more hydrophobic which facilitates vesicles formation.
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The next step is to connect the chamber with the pumping system.
 
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Pumping system
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The pumping system (ceDOSYS SP-4) allows to control of syringes filled with aqueous material and surfactant treated with mineral oil, respectively. The syringes access the chamber via the tubing inlets. Two inlets are used to pump in material in the aqueous phase; the remaining one is used for the oil phase. The flow rates of the syringes are controlled via a ceDOSYS user interface software.
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Our setup allows simultaneous control of pumping rate of 4 syringes driven by the software.
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Control via pump system:
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* two syringes are loaded with 1ml each of material in the aqueous phase; during the first trial, distilled water
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Measurement
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*      another is filled with a 1ml solution of 0.5% span 80 in oil
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* use flow rate on ceDOSYS interface to flood the chamber first with oil phase
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The chamber should be placed on the microscope sample holder, connected with the syringes (inlet 1 – oil, inlet 2 water and DNA plasmid, 3 – water and an expression kit). The microscope is focused on the spot where channels bringing oil and water meet. In order to make the vesicles one should play around with the flow rates. Usually the ratio of oil/water flow rate is about 1/0.75. The expression of GFP in the vesicles is proved by fluorescent microscopy.
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* gradually introduce aqueous phase and modify rates of both phases until the shear stress breaks the aqueous phase into droplets at the T- or V- junctions in the respective chambers
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Latest revision as of 03:50, 22 October 2009

Vesicles - Methods

Setup of the microfluidic system

The microfluidic system consists of a flow chamber made of Polydimethylsiloxane (PDMS) and a pump system that controls the flow rates of the various liquids into the chamber. Droplets are created within a defined space in the chamber and are propagated along a grid that allows containment and imaging. Two types of chambers have been used, differing in the geometry of the space where droplets were produced. One featured a T-junction, and the other one a V-junction (Fig1).


Fig1: Different geometries of the intersection between aqueous and oil phase in flow chambers

t shaped junction
T shaped junction
v shaped junction
V shaped junction
Silicon wafer we used for microfluidics chamber preparation

Production of flow chambers:

  • mix PDMS and curing agent in 10:1 ratio
  • degas and pour on wafer with etched microstructures
  • polymerize on heat plate at 150 ºC for 30 min
  • add unpolymerized PDMS mixture to points on microstructure where microtube inlets are to be pierced
  • polymerize on heat plate at 150 ºC for 30 minutes
  • remove polymerized PDMS from wafer, cut to fit onto glass cover slide (24 x 60 mm), and use clean needles (0.8 mm) or laser cutter (Trotec Speedy 100TM) to pierce tube inlets
  • ionize PDMS and glass slide in plasma chamber for 30 sec to make it reactive
  • align PDMS on glass slide and seal
  • seal irreversibly by heating on plate at 60ºC for 6 hours

This video shows the laser cutting of the microtube inlets:



The pumping system (ceDOSYS SP-4) allows to control of syringes filled with aqueous material and surfactant treated with mineral oil, respectively. The syringes access the chamber via the tubing inlets. Two inlets are used to pump in material in the aqueous phase; the remaining one is used for the oil phase. The flow rates of the syringes are controlled via a ceDOSYS user interface software.

Control via pump system:

  • two syringes are loaded with 1ml each of material in the aqueous phase; during the first trial, distilled water
  • another is filled with a 1ml solution of 0.5% span 80 in oil
  • use flow rate on ceDOSYS interface to flood the chamber first with oil phase
  • gradually introduce aqueous phase and modify rates of both phases until the shear stress breaks the aqueous phase into droplets at the T- or V- junctions in the respective chambers
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