P16083 Automated Microfluidic Cell Separator

Operator’s Manuel

Rev. 111-28-2015

ContentsSystem Overview:.......................................................................................................................................2

Expected Benefits:......................................................................................................................................2

Terms and Definitions:................................................................................................................................2

Instrument Layout......................................................................................................................................4

Operation:...................................................................................................................................................7

Safety and PPE........................................................................................................................................7

Create the Microchannel:.......................................................................................................................7

Casting and baking the PDMS device.................................................................................................7

PDMS Post Processing.......................................................................................................................10

Sealing the Device to Glass slide.......................................................................................................14

Fabricating Electrodes.......................................................................................................................18

Placing and Sealing the Electrodes and Tubing................................................................................19

Electrode Recycling...............................................................................................................................26

Setting the Voltages:.............................................................................................................................26

Setting the Frequency:..........................................................................................................................26

Setting the Flow Rate:...........................................................................................................................27

Filling the Syringes:...............................................................................................................................27

Loading the Syringe into the Syringe Pump:........................................................................................27

Loading the Microchannel into the Instrument:..................................................................................27

Unloading the Microchannel from the Instrument:.............................................................................28

Unloading the Syringe from the Syringe Pump:...................................................................................28

Digital Microscope Usage:....................................................................................................................28

Full Run:................................................................................................................................................29

Safety:.......................................................................................................................................................29

References:...............................................................................................................................................30

Page | 1

System Overview:Currently, separating a co-culture of cells is a time and labor intensive process. This

instrument will only require the operator to load the sample and set the target signal for the cells. The semi-automation reduces the need for intensive training and reduces the risk of human error. Our instrument will use electric fields to manipulate and sort the cells without the need for expensive reagents or dangerously high shear forces. This project will be used in a teaching lab environment to solidify the understanding of microfluidic techniques to separate cells and particles using dielectrophoresis. The design and prototype must conform to intellectual property and diagnostic laboratory standards so that it may be marketed as a definitive step forwards in cell separation technology.

Expected Benefits:● Provide an inexpensive method of dielectrophoretic cell separation in a standalone

instrument ● Provide a practical teaching tool for microfluidic demonstrations● Contribute to the Biomedical Engineering Department at RIT● Provide a basis for a microfluidic instrument for cell separation and analysis in a medical

setting

Terms and Definitions:Microfluidics – Microfluidics is the science and technology of manipulating and controlling fluids, usually in the range of microliters (10-6) to picoliters (10-12), in networks of channels with lowest dimensions from tens to hundreds micrometers. [1]

Dielectrophoresis – The migration of uncharged particles toward the position of maximum field strength in a nonuniform electric field. [2]

Co-Culture – A cell culture containing growths of two distinct cell types. [3]

Buffer – Fluid used to suspend the co-culture.

Potentiometer – A device with three terminals, two of which are connected to a resistance wire and the third to a brush moving along the wire, so that a variable potential can be tapped off. [4]

Page | 2

Syringe – A small device consisting of a glass, metal, or hard rubber tube, narrowed at its outlet, and fitted with either a piston or a rubber bulb for drawing in a quantity of fluid or for ejecting fluid in a stream, for cleaning wounds, injecting fluids into the body, etc. [5]

Syringe Pump – a small infusion pump (some include infuse and withdraw capability), used to gradually administer small amounts of fluid (with or without medication) to a patient or for use in chemical and biomedical research. [6]

Start Button – Button used to start operation

Emergency Stop – Button used to cut power to all potentially harmful components if a problem occurs.

CW – Clockwise

CCW – Counterclockwise

PDMS – Polydimethylsiloxane

BOC – Bill of Consumables

BOM – Bill of Materials

PPE – Personal Protective Equipment

awg – American Wire Gauge

°C – degree centigrade/Celsius

Instrument – the fully assembled final product

Device – the microchannel assembly

Page | 3

Instrument Layout

Page | 4

1

2

3 4

5

6 7 8 11

10

14

15

9

13

181716

12

Part #: Description: Part #: Description: Part #: Description:1 Buffer Syringe Pump 8 Voltage Control 2 15 Microchannel Housing2 Co-Culture Syringe

Pump9 Frequency Control 1 16 Cell A Reservoir

3 Buffer Syringe Pump Control

10 Frequency Control 2 17 Cell B Reservoir

4 Co-Culture Syringe Pump Control

11 Flow Rate Control 18 Waste Reservoir

5 Voltage Display 12 Emergency Stop Button

19 Power Entry Module

6 Start Button 13 Flow Rate Display 20 Cooling Fan Vent7 Voltage Control 1 14 Electrode Terminal

Block

Page | 5

19

20

Part #: Description: Part #: Description: Part #: Description:21 Display 24 Menu Control Select 27 Output Amplitude

Adjust Knob22 Menu Control Left 25 Menu Control OK 28 On/Off Switch23 Menu Control Right 26 Main Adjust Knob

Page | 6

28

27262523

2422

21

Operation:Safety and PPE

● Safety:○ Read and understand the hazards of the chemicals being used in all aspects of

manufacturing and operation○ Read and understand the rules of the lab environment being used○ You are held responsible for your safety and the safety of others in the lab○ Processes include the use of an X-acto knife. Use caution throughout all cutting

processes○ Take caution when soldering, soldering iron and other parts will be hot

● PPE○ Nitrile Gloves○ Eye Protection (recommended)

Create the Microchannel:Materials:

● PDMS Microchannel● Microscope Slide● 1cm slip● Copper ● 24 awg Wire ● 100 gram weigh boat

Casting and baking the PDMS device1. Clean a weigh boat with a 70% IPA saturated KIM Wipe and let air dry

Page | 7

2. Mix the PDMS Base and Curing Agent, 10:1 ratio respectively, in the weigh boata. Use 30 grams of the base, 3 grams of the curing agentb. The base can just be poured directly from container

c. Use a serological pipette for the curing agent

Page | 8

3. Mix vigorously with a glass stir rod

4. Place entire weigh boat in vacuum chamber

Page | 9

5. Degas for 10 minutes or until majority of the bubbles/foam have dissipated6. Surround the microchannel mold with aluminum foil, ensuring that the foil is tightly

sealed around the edges of the mold.

7. Pour the entire PDMS contents of the weigh boat on the microchannel mold, the foil should prevent the PDMS from leaking under the mold

8. Using a micropipette tip, move any visible particulates/bubbles away from the channel on the mold

9. Place on the hot plate at 85°C for 40 minutes a. Cover with aluminum foil for entire 40 minutes

10. Set temperature for 135°C for 5 minutesa. Cover with aluminum foil for entire 5 minutes

11. Turn off hotplate

Page | 10

12. Remove foil boat from hot plate and allow to cool to room temperaturePDMS Post Processing

1. Peel the foil off the back of the mask and PDMS

2. Peel the PDMS off of the mask, ensuring PDMS does not rip

3. Punch inlet and outlet holes using the 3mm biopsy puncha. End of each channel should be centered on the punch

Page | 11

4. Punch electrode slits using 1mm biopsy puncha. Follow guide electrode traces on PDMS

Page | 12

5. Cut PDMS into rectangular pieces, maximum of 25mm by 75mm, so that the channel is centered on the cutout

Page | 13

Sealing the Device to Glass slide 1. Wash PDMS microchannels and glass microscope slides by applying 70% IPA and wiping

with gloved fingers. Allow to air dry

Page | 14

2. Once IPA has dried, use tape (Scotch) to clean any dust off of channel and glass slide

Page | 15

3. Wash again with 70% IPA and wiping with gloved fingers4. Place on hot plate at 70°C inside folded foil until IPA has evaporated (note the image

does not have the foil folded over so that contents of foil can be seen. Cover with foil to prevent particles from falling on channels and slides!)

Page | 16

5. Remove from hot plate and use Scotch Tape/equivalent to clean off any dust or water spots

6. Corona treat the PDMS channel cutout and the microscope slide for a minimum of 30 seconds each

Page | 17

7. Adhere the PDMS channel cutout to the microscope slide by applying pressure by hand

8. Place device on hot plate at 70°C for 5 minutes

Page | 18

Fabricating Electrodes1. Cut electrodes to 5mmX10mm from copper stock (found in BOM and BOC)

a. Recommend using bench shear found in Machine Shop (GLE-2430)2. Solder wire, 15-20 cm in length, to the copper pieces

a. Follow standard soldering practices for soldering wire to a flat plate3. Solder and wire must be only on half the copper pieces lengthwise

Placing and Sealing the Electrodes and Tubing 1. Cast 17 grams of PDMS onto aluminum casting block using the 10:1 base to curing

agent ratio. Aluminum foil should be used to surround the aluminum casting block and prevent PDMS from leaking

2. Remove sheet of PDMS from the aluminum casting block, ensuring the sheet does not rip

Page | 19

3. Cut out squares, approximately 1 cm square, of the PDMS slip

4. Wash PDMS microchannels and 1cm slip by applying 70% IPA and wiping with gloved fingers

5. Once IPA has dried, use tape to clean any dust off of channel6. Corona treat the PDMS channel cutout and 1cm slip

Page | 20

7. Adhere the 1 cm square to the PDMS channel cutout directly over the electrode by applying pressure by hand

Page | 21

8. Cut electrode slits in the 1 cm slip using an X-acto knife flowing the pattern in the PDMS microchannel

Page | 22

9. Insert the fabricated electrodes with the soldered wire coming out of the PDMS channel assembly

Page | 23

10. Ensure the electrode are flush with the glass microscope slide11. Insert ⅛” tubes into all the Input/Output holes. Note that the image shows blue tubing

as a connector. This blue tubing can be replaced with the tubing connectors provided

12. Make 10 grams of liquid PDMS per channel (10:1 base to curing agent)13. Surround the microchannel assembly in foil

Page | 24

14. Pour all the liquid PDMS on the microchannel assembly

15. Bake in oven at 80°C for 8 hoursa. Between markers 4 and 5 on the oven thermometer gauge

Page | 25

Electrode Recycling1. Turn off the instrument2. Disconnect wires from the electrode terminal block3. Slide an X-acto knife between each side of the copper electrodes and PDMS to break the

seal4. Using needle nose pliers, remove the copper electrodes from the PDMS microchannel

assembly5. Clean using 70% IPA6. Dry electrodes7. Inspect electrodes and solder joint for any corrosion or damage

Setting the Voltages:

Page | 26

1. Turn on the instrument, if not already ona. Part #: 19

2. Locate the knob for controlling the voltage for the desired electrodea. Part #: 7 or 8

3. Rotate the knob to change the voltage (CW to increase the voltage and CCW to decrease the voltage)

4. Stop rotating the knob when the correct voltage is reacheda. Part #: 12

Setting the Frequency:1. Turn on the instrument, if not already on

a. Part #: 192. Locate the knob for controlling the frequency for the correct electrode

a. Frequency Control Part #: 9 or 10 using Main Adjust Knob Part #: 263. Rotate the knob dial to change the frequency (CW to increase the frequency and CCW to

decrease the frequency)4. Stop rotating the knob when the correct frequency is reached

a. Frequency Control Part #: 9 or 10 using Main Adjust Knob Part #: 21

Setting the Flow Rate:1. Turn on the instrument, it not already on

a. Part #: 192. Locate the knob for controlling the frequency for the flow control

a. Part #: 113. Rotate the knob dial to change the flow rate (CW to increase the flow and CCW to

decrease the flow)4. Stop rotating the dial when the correct flow rate is reached

a. Part #: 13

Filling the Syringes:1. Locate two syringes, suspended co-culture in buffer, and plain buffer2. Screw in barb to luer lock fitting into the syringe3. Connect 15 cm of tubing to the barbed fitting4. Aspirate 6-8 mL of plain buffer in a syringe5. Aspirate 4-5 mL of the co-culture in another syringe

Loading the Syringe into the Syringe Pump:1. Locate the filled syringes

a. 1 with plain buffer b. 1 with the co-culture

2. Place the syringe with the plain buffer into Buffer Syringe Pump

Page | 27

a. Part #: 13. Place the syringe with the co-culture in Co-Culture Syringe Pump

a. Part #: 24. Use the syringe pump controls to move the plunger controller so that the plunger can fit

in both the slots in the syringe holder and the plunger controller (Push the toggle switch towards the syringe pump to move the plunger controller forward, pull the toggle switch away from the syringe pump to move the plunger controller backwards)

a. Part #: 3 and 45. Slide the syringe lock plate over the syringe

Loading the Microchannel into the Instrument:1. Ensure instrument is turned off

a. Part #: 192. Locate a sealed Microchannel Assembly for use in this instrument3. Place glass slide down in the channel with the “Y” towards the syringe pump and the

three pronged side towards the edge of the instrumenta. Part #: 15

4. Press the tubing into each access holea. 6 cm for each of the 2 inlet holesb. 15 cm for each of the 3 outlet holes

5. Press the barb-to-barb fittings into the inlet tubing6. Attach the microchannel tubing to the syringe tubing through the barb-to-barb fittings7. Feed the outlet tubing into the reservoirs

a. Part #: 16, 17, and 188. Attach the electrode wires to the electrode terminal block

a. Part #: 149. Adjust the tubing and wires so that the glass lies flat on the microchannel mount

Unloading the Microchannel from the Instrument:1. Ensure the instrument is turned off

a. Part #: 192. Carefully remove the tubing of the microchannel from the barb-barb fittings3. Remove the electrode wires from the electrode terminal block

a. Part #: 144. Lift the microchannel out of the housing5. Clean the microchannel as stated in the operation below

Unloading the Syringe from the Syringe Pump:1. Follow procedure for Unloading Microchannel from the Instrument if the syringe is

connected to the microchannel

Page | 28

2. Slide the syringe lock plate off the syringea. Image

3. Remove the syringe from the syringe pumpa. Image

4. Carefully pull the barb-to-barb fitting from the 15 cm tubinga. The barb-to-barb fitting can be autoclaved and reused

5. Remove the 15 cm of tubing from the luer lock to barb fitting6. Unscrew the luer lock tip from the syringe7. Dispose of material accordingly

Digital Microscope Usage:1. Ensure proper connection to a computer and software is working2. Position the microchannel glass slide so that the channel is visible3. Use the focus dial in order to digitally focus on the channel4. Tighten the hand screws to ensure the microchannel glass slide does not move5. Follow software instructions for video and picture capture

Full Run:1. Ensure the instrument is laying on a flat surface2. Follow steps for filling the syringes3. Follow the steps for load the microchannel into the instrument4. Follow the steps for load the syringes into the syringe pumps5. Follow the steps for setting the voltages and frequencies for the electrodes6. Start the run cycle by pressing the start button7. Observe the run to ensure no problems occur

a. If a problem arises, press the emergency switch8. Wait for the run to conclude9. Follow the steps for unloading the microchannel from the instrument10. Follow the steps for unloading the syringes from the syringe pumps11. Follow the steps listed below for cleaning

Safety:Warning

● This instrument handles biological cells, treat the instrument appropriately● This instrument has exposed electrodes at high voltage, do not touch micro-channel

when instrument is running● There is an electric field in contact with fluid during operation, DO NOT interact with the

microchannel, fluid, or electrodes while the instrument is on

Page | 29

● Do not open exterior shell of device - electronics inside may contain charge even when device is unplugged.

● Read and understand the hazards of the chemicals being used in all aspects of manufacturing and operation

● Read and understand the rules of the lab environment being used● You are held responsible for your safety and the safety of others in the lab● Processes include the use of an x-acto knife and biopsy punches. Use caution

throughout all cutting processes

Warning

Page | 30

References:1) http://www.fluigent.com/what-is-microfluidics/2) "Definition of Dielectrophoresis in English:." Dielectrophoresis: Definition of

Dielectrophoresis in Oxford Dictionary (American English) (US). N.p., n.d. Web. 28 Nov. 2015.

3) "Coculture." - Biology-Online Dictionary. N.p., n.d. Web. 28 Nov. 2015.4) http://www.thefreedictionary.com/potentiometer5) http://dictionary.reference.com/browse/syringe6) https://en.wikipedia.org/wiki/Syringe_driver

Page | 31