The overall goal of the following experiment is to analyze single cells lysates using fluorescence or immunoassays. This is achieved by first creating a microfluidic device and coating the inner chamber with antibodies. Then single cells are immobilized in the traps and are exposed to various reagents to stimulate a cellular response in a second step.
A ring shape valve is quickly closed and lysis buffer is flowed through the device. The ring shaped valve is the opened for a short time and the cells are lysed within the micro chamber, allowing released proteins to bind to the antibodies at a high concentration due to the small volume. Next, the ring shaped valve is opened.
The chamber is washed, and detection reagents are introduced in the closed micro chamber. The increase of fluorescence intensity is monitored over time using a fluorescence microscope, the results demonstrate the ability of this system to quantitatively and reliably detect an analyte of interest released from individual cells. This method can help answer key questions in the field of single A cell analysis.
In particular, it ate systematic investigations on the cellular response to variations in the chemical environment. We can see up or down regulations of intracellular molecules as a result of the chemical stimulus. There are two main advantages of this technique.
The first is that we can expose the immobilized cells to a defined chemical environment that is constant or periodically changing. Secondly, we can reform highly sensitive and specific analysis of the sate without any cross-contamination or loss of analytes due to transport. To begin fabricate the three different SU eight master wafers as described in the accompanying text protocol.
One will be used to create the fluidic layer, one for the control layer, and a third for the micro contact stamp. Then fabricate the microfluidic chips beginning with the preparation of a 10 to one mixture of PDMS and a curing agent. Mix both parts vigorously and degas the solution for 30 minutes or until the mixture is bubble free.
Next, place the wafer within the control layer inside a Petri dish and tape it onto the bottom. Pour approximately 50 grams of PDMS on top to create a four to five millimeter thick layer and place the wafer for at least two hours in an oven at 80 degrees Celsius to assure complete curing of the PDMS. Repeat the same procedure for the micro contact printing wafer, but use approximately 20 grams PDMS instead of 50, resulting in a four to five millimeter thick layer of PDMS.
To prepare the bottom layer spin coat a 40 micron high PDMS layer onto the wafer with the microfluidic layer. Using a rotational speed of 2000 RPM for 60 seconds. Cure the PDMS layer in an oven for one hour at 80 degrees Celsius when all parts have cured.
Remove the control layer from the wafer and cut it into pieces with the razor blade aid. Then punch pressure connection holes using a one millimeter biopsy puncture and cover the channels with tape for storage. Next, remove the PDMS from over the micro contact printing wafer and prepare stamps that are the size of the chamber area.
Store them dust free in a drawer until use. Then place the spin coated wafer and the four to five millimeter thick control layer in a plasma cleaner. Expose the pieces at 18 watts for 45 seconds using oxygen plasma at 0.75 millibar.
After plasma treatment, quickly align both layers under a microscope with a large working distance. Add some spare PDMS around the placed top parts in order to facilitate the removal of the PDMS from the wafer. Then place the wafer in an oven at 80 degrees Celsius for at least one hour.
Once cured, use a scalpel to carefully remove the PDMS from the wafer and to cut out the microchips punch access holes for the fluidic connections with a 1.5 millimeter biopsy puncture. Additionally, create a reservoir in the microchip by cutting the upper part of a 200 microliter pipette tip and using some spare semi cured PDMS. Glue it on top.
Then cure the chip in the oven at 80 degrees Celsius for at least one hour. Begin by cleaning a glass slide and PDMS parts. Clean a glass slide with soap.
Rinse it with distilled water, and then again with ethanol. Dry the slide using a nitrogen stream. Use tape to remove any debris and dust particles from the PDMS.
Then place the PDMS part and the cleaned glass. Slide into the plasma cleaner for 45 seconds at 18 watts to assure a tight bonding, place the chip on a hot plate for five minutes following the plasma treatment After bonding, cut the lower part off of a few 200 microliter pipette tips. Fill them with 10 to 15 microliters of filtered PBS with 4%BSA and place them into the inlets of the device.
Then use cut tips with added 10 to 15 microliters of PBS and put them into the control channels. Next, place the chips into the centrifuge and spin them at 800 times gravity for five minutes to fill the channels with fluid. If air remains in any of the channels, repeat this step until the channels are completely filled.
Incubate the blocking solution for at least 30 minutes at room temperature. Then wash the solution out of the fluid layer with PBS at a flow rate of 10 microliters per minute. Using a syringe pump, the device is now ready for cell experiments to preprint the glass slide for immunoassays incubate PDMS micro contact printing stamps with 0.5%biotinylated BSA in a clean Petri dish.
After half an hour, clean the stamps thoroughly and quickly with distilled water and dry the stamp under a stream of nitrogen. Once dry, quickly place the stamps on a cleaned glass slide to deposit the patterned biotinylated BSA. Do not move the stamp after placing and check that the stamp is in contact with the glass slide.
If not slightly, tap the stamp, but do not press down hard. Then place the stamp together with the top part of the microfluidic chip into the plasma chamber and expose them to oxygen plasma at 18 watts for 45 seconds. After plasma treatment, remove the stamp from the glass slide.
Breathing on the surface will make the pattern visible. Align the micro chambers on top of the printed surface. Once aligned, place the chip on a hot plate at 50 degrees Celsius for 30 minutes to block the remaining surface, introduce a 4%sterile filtered BSA solution in PBS into the chip with a centrifuge as previously shown, incubate the microchip for at least 30 minutes and then wash the solution out of the fluid layer with PBS at a flow rate of 10 microliters per minute.
Using a syringe pump, use the chip directly or stored for up to two weeks at four degrees celsius in a humid box. Next, create a fully functional binding surface by introducing avadon in PBS into the reservoir. Flow the solution to draw the avadon through the fluid channels afterwards.
Rinse the reservoir thoroughly and flush the channels with PBS. Then add protein G to the reservoir and pull it through the channels. Wash out any excess protein G by rinsing the reservoir and the chip with PBS.
Next, add the antibody of interest to the reservoir and flow it through the channels. Then stop the flow to allow binding before washing the channels with PBS fluorescently, labeled analogs can be used to check the quality of the printing process prior to introducing cells into the channels. Harvest adhered cells such as the HE 2 9 3 cells shown here using enzyme free disassociation buffer, and then filter them to reduce the number of cell clusters once filtered, load them into a syringe.
At 300, 000 cells per milliliter, load the cells onto the chip using forward flow on the syringe pump for about three minutes at a rate of 10 to 20 microliters per minute for adhesive cells such as these in order to prevent non-specific attachment of the cells when the traps are filled, close the chambers by pressurizing the top layer with three bars. Then examine the chambers using a microscope. If many cells are found to have non-specifically adhered to the surface inside the chambers, quickly open them and try to wash them away with cell dissociation buffer at a flow rate of 10 to 30 microliters per minute.
For the glucose six phosphate dehydrogenase or G six PDH assay, add two millimolar glucose, six phosphate 0.5 millimolar, and a DP 0.5 units per milliliter DIA arrays and 0.3 millimolar RIN to a lysis buffer and ensure it is well mixed. Once the channels are cleared, draw the G six PDH lysis buffer through the chip at a flow rate of 30 microliters per minute. As the lysis buffer is flowing, quickly open and close the chambers, leaving them open for 500 milliseconds to allow the lysis to enter the chamber immediately after lysis begin to monitor the kinetic reaction.
In this example, a fluorescent product can be monitored over time. Shown here is an array of chambers that were filled with orange food dye and then closed off by pressurizing the control layer above the micro chambers. After chamber closure, green food dye was rinsed through the channels to show the tightness of the chambers.
Each chamber contains a volume of only 625 picoliters of fluid that keeps the concentration of molecules released from the lys cells high enough for analysis. Furthermore, the valves can be opened and closed in a manner to eliminate cross-contamination between the chambers. Within these chambers, it is possible to measure analytes such as the amount of lysosome released from a single PMA activated U 9 37 cell, as it enzymatically catalyzes the production of a fluorescent molecule within the chamber.
The green lines represent the change in fluorescence intensity over time for chambers occupied by one cell. Also shown is the fluorescence intensity for a chamber without cells represented by the black dotted line. For comparison, this assay shows the effect of a toxic molecule on the membrane integrity by measuring an intracellular enzyme G six PDH.
The black line represents the control samples with no toxic influence, whereas the orange line represents cells influenced by the toxin shown. Here is an example of an immunoassay for the intracellular protein. GFP anti GFP antibodies were immobilized on the surface and once cells were lysed, the binding kinetics of the GFP proteins were visualized using turf microscopy.
Time 0.1 marks the introduction of the lysis buffer at time 0.2 GFP is starting to accumulate on the surface, meaning that cell lysis has occurred and GFP has diffused to the surface. Binding of GFP to the antibodies is completed at time 0.3. In this final example, liberated GA DH bound to capture antibodies on the surface and then HRP coupled detection antibodies were added.
Once all non bound detection antibodies were washed away, the detection reagent was introduced and the reaction was over time. Here is a comparison of the amount of formerly intracellular GA DH in animal quantities between U 9 37 and HEC 2 93 cells measured using this method. After watching this video, you should have a good understanding of how to prepare the microfluidic chips, how to modify the surface to enable immunoassays, and finally, how to use the device for single a cell analysis.
This method can be employed to help answer many in triggering questions in the field of single cell biology.
