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Microfluidic colocalization set-up for DNA analysis

Detect protein and/or DNA interaction with the help of microfluidics

Introduction to microfluidic colocalization

This user guide will show you how to run microfluidic colocalization studies of single molecule spectroscopy. We will start with the preparation of the flow cell and we will then show you how to apply pressure driven flow control to liquids for DNA detection studies.

DNA Protein interaction colocalization microfluidics elvesys
Image source: Zhao et al., 2017, Biochemistry and Chemical Biology, Structural Biology and Molecular Biophysics https://elifesciences.org/articles/32177#fig1

Schematic depiction of DNA and protein colocalization. Single DNA molecules are attached to the glass surface using streptavidin as intermediate bonding agent. In this article we will describe how to perform microfluidic colocalization using an Elveflow set-up.

Microfluidic colocalization set-up

The imagining chamber consists of a microscope glass slide (1 mm thickness, 25×75 mm size) with etched channels (up to 9 channels on 1 glass slide) covered with a rectangular borosilicate coverslip (0,13 – 0,17 mm thickness, 60 x 22 mm size). The connectors for tubing were bound directly to the microscope glass slide because of its thickness. In this experiment, we analyzed several proteins that we introduced into the flow cell using the ELVEFLOW© MUX distribution valve. Using this set-up a minimal delay in the sequential injection can be achieved.
microfluidic set up for colocalization studies of DNA interaction

Materials for microfluidic colocalization

Hardware:

  1. OB1 flow controller with a channel of 0-2000 mbar
  2. 1x flow sensor MFS2 0-7 μL/min
  3. Kit starter pack luer lock + 1/32 tubings + 1/32 sleeves + custom chip connectors
  4. 1-8 x 15 mL Falcon reservoirs
  5. Microfluidic chip
  6. FRET Microscope for observation

Chemicals:

  1. 20 mM Tris-HCl pH 7.5
  2. 50 mM potassium glutamate
  3. 8 mM MgCl2
  4. 4% glycerol
  5. 2 mM DTT
  6. 0.1% Tween20
  7. 1 mM Trolox
  8. Proteins or DNA molecules (DEPENDING ON EXPERIMENT)

Microfluidic chip design

The bottom part of the chip is made of borosilicate glass, 0.13-0.17 mm. The top glass (cover glass) is a conventional microscope glass slide. The biggest channel length is 40 mm, the smallest 20 mm. Channel depth may differ, here 20-, 30- and 50-micron channels were etched. The chip was produced by glass etching during the study. Channel width was 150 microns. On the top of the microscope glass slide we drilled inlet and outlet holes. Connectors: custom, made as part of the project. Tubing: external diameter 1 mm.

Microfluidic chip design colocalization elvesys

Quick start guide

Glass surface treatment to preassemble the flow cell

  1. Wash the flow cell with bleach and inactivate the bleach with sodium thiosulfate.
  2. Flow a solution of neutravidin, then flow with a casein and/or BSA solution to close gaps on the surface.
  3. For “sticky” proteins, pre-treatment of the bottom glass slide by means of pre-silanization is usually required.

Bonding of DNA to the glass surface

Tips from the expert: Before starting the experiment, proteins and DNA should be labelled and purified using gel filtration. The extended protocol can be requested (classical protein purification).

  1. Connect your OB1 pressure controller to an external pressure supply using pneumatic tubing, and to a computer using the USB cable. For detailed instructions on OB1 pressure driven flow controller setup, please read the OB1 user guide.
  2. Plug the microfluidic reservoir to the OB1 pressure controller outlet. The Elveflow reservoirs connection instructions are covered by a specific guide (see Elveflow microfluidic reservoirs assembly instructions).
  3. For the feedback loop, connect a flow sensor to the OB1. Then, for flow measurement, connect flow sensors between the microfluidic reservoirs and the chip.
  4. Turn on the OB1 by pressing the power switch.
  5. Launch the Elveflow software. The Elveflow Smart Interface’s main features and options are covered in the Smart Interface guide. Please refer to those guides for a detailed description.
  6. Press Add instrument \ choose OB1 \ set as MK3+, set pressure channels if required, give a name to the instrument and press OK to save changes. Your OB1 should now be in the list of recognized devices.
  7. OB1 calibration is required for the first use. Please refer to the OB1 user guide.
  8. Add flow sensor: press Add sensor \ select flow sensor \ analogue or digital \ max flow rate for the sensor, give the sensor a name, select to which device and channel the sensor is connected and press OK to save the changes. For details refer to the Microfluidic flow sensor user guide.
  9. Add MUX distributor: press Add instrument \ choose MUX distribution/injection \ give the instrument a name \ select the number of valves (10 or 12).
  10. Connect the OB1 to the manifold and the manifold to all the reservoirs you need.
  11. Use the supplied 1/32” OD tubing to connect the microfluidic reservoirs to the MUX distributor and one tubing at the outlet of the MUX distributor. Add a dead-end block to the inlets of the MUX-distributor that are not used.

Tips from the expert: Use consecutive MUX inlets for liquid reservoirs and the dead-end. The MUX will switch liquids following the shortest travel distance. Air will enter the system if the MUX passes over an open inlet.

  1. Set pressures (and other parameters if needed) and start pumping liquids into the chip. Wait until all air bubbles escape from the chip and both liquids are flowing.

Tips from the expert: Chip priming: To begin with, run liquids into tubes until the liquid starts to drip for each inlet of the MUX distribution. Only then, connect them to the chip.

  1. Flow continuously at 1µL/min using the flow sensor feedback loop (see Elveflow User Guide Flow sensor Elveflow) the following solutions in this order:

a. 10 µL of PBS
b. 5 µL of bio-BSA 0.1 mg/mL PBS
c. 5 µL of PBS
d. 5 µL of Pluoronics 0.5%
e. 5 µL of Neutravidin 0.2 mg/mL
f. 5 µL of Casein 0.02%
g. 5 µL of 20kb DNA bio (1 µL-0.5 ng) in PCB (19 µL)

Tips from the expert: PBS is Phosphate Buffered Saline and PCB is PBS with BSA (0.4 mg/mL) and casein (0.02%).

  1. Incubate for at least 15 minutes at room temperature without flow.
  2. Restart the continuous flow with the following solutions:

a. 5 µL PCB
b. 5 µL Neutravidin 0.2 mg/mL
c. 5 µL of Pluronics 0.5%


Microfluidic set-up imaging

  1. Different microscope setups can be used, for example the Nikon (Kingston Upon Thames, United Kingdom) Eclipse Ti-E microscope with the ApoTirf 100X/1.49 Oil, 0.13–0.20 WD 0.12 objective.
  2. We used lasers with 150 mW 488 nm, 150 mW 561 nm (both Coherent Sapphire Ely, United Kingdom) and 100 mW 638 nm (Coherent Cube) controlled by an acousto-optic tunable filter (Gooch and Housego, Ilminster, United Kingdom).
  3. Each view field is about 50 x 50 microns, channels have width of 150 microns to observe the molecules in the middle of the channel without any distortion or artifacts.

 

Congratulations – you achieved the binding and monitoring of single molecules on chip! We hope this tutorial is a useful guide in your microfluidic experiments. If you need further details or additional information, do not hesitate to contact our experts!

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 722433 (DNARepairMan).

Want to run a similar experiment? Feel free to contact us at: contact@elveflow.com
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