Name: A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells (Video) | JoVE Journal
Uploaded: 2013-10-15T17:00:00.000+00:00
Duration: 15 min 41 s
Description: Learn microfluidic chip fabrication for single cell analysis using fluorescent immunoassays. This method enables precise quantification of intracellular proteins and enzymes with high sensitivity.

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5 minutes

The authors gratefully acknowledge Tom Robinson for proof reading of the manuscript, C. B&auml;rtschi and H. Benz for the construction of the custom-built pressure control system. We would also like to acknowledge the use of the clean room facility FIRST and the Light Microscopy Center (LMC), both at ETH Z&uuml;rich. The work was funded by Merck Serono and the European Research Council (ERC) under the 7th Framework Program (ERC Starting Grant, project no. 203428, n&mu;LIPIDs).

1. SU-8 Master Fabrication
Prepare both master molds for the channels (fluidic and control, for schematics and dimensions see Figure 2) with the following protocol but with different mask patterns. The process is shown in Figure 3a.
<ol>
	<li>Start by heating a 4 inch silicon wafer for 10 min at 180 &#176;C. Load the dehydrated wafer on a spin-coater and use the following protocol for spin-coating SU-8 2015:
	<ol>
		<li>Spin wafer at 100 rpm for 20 sec (open lid of spin-coater, dispense SU-8 during this step, close lid after dispensing).</li>
		<li>Spin wafer at 500 rpm for 10 sec (this will spread the resist over the whole wafer).</li>
		<li>Spin wafer at 1,750 rpm for 30 sec (defines the height of the resist to 20 &#181;m).</li>
	</ol>
	</li>
	<li>Remove resist from the edge of the wafer with acetone (use a swab) as this will otherwise stick to the hotplate in the next step. Transfer the wafer to a hotplate at 95 &#176;C and heat for 4 min. After the softbake, expose the resist through a photomask taped to sodalime glass in a mask aligner with UV light (150 mJ/cm2, measured at 365 nm).</li>
	<li>After the exposure, heat the wafer again at 95 &#176;C for 5 min on a hotplate. Cool down the wafer to RT and then immerse it into a bath of SU-8 developer for 4 min. Shake gently to remove unexposed SU-8. Wash the wafer with clean-room grade isopropanol and blow dry with nitrogen. Check under a microscope if the development was successful (especially the cell traps). If the cell traps are completely developed, the reflection of the silicon wafer should be visible. If necessary, develop the wafer again for a few minutes.</li>
	<li>Bake the wafer in an oven for 2 hr&#160;at 180 &#176;C to remove all residual solvent. Check the height of the channels with a step profiler. If the height differs from the desired height, repeat this protocol starting at step 1.1 with a new wafer and change the spin-speed (step 1.1.3). Finalize the fabrication of the master mold by silanizing the wafer with 1H,1H,2H,2H-Perfluorodecyl-dimethylchloro-silane in a desiccator overnight.</li>
</ol>
2. Master Fabrication for Microcontact Printing
<ol>
	<li>In order to prepare master molds for microcontact printing, dehydrate a silicon wafer for 10 min at 180 &#176;C. Spin-coat 1 ml HDMS onto this wafer at 7,500 rpm for 30 sec (the resist adheres better to a silanized surface). Spin-coat 2 ml of AZ1518 positive resist with this protocol:
	<ol>
		<li>Static dispense of the resist on to the wafer.</li>
		<li>Spin wafer for 5 sec at 500 rpm.</li>
		<li>Spin wafer for 60 sec at 4000 rpm.</li>
	</ol>
	</li>
	<li>After a 50 sec softbake at 100 &#176;C on a hotplate, expose the resist to UV-light (21 mJ/cm2 at 365 nm) through a photomask and on a mask aligner. Develop the resist in a bath of AZ 726 for 75 sec. Rinse the developed wafer with DI-water and blow dry with nitrogen. Remove residual solvent by heating the wafer at 115 &#176;C for 50 sec. Silanize the wafer under vacuum overnight.</li>
</ol>
3. Fabrication of the Microfluidic Chip
A two-layer device design is used for these experiments. The two layers are prepared separately and are bonded together, before the chip is finally bonded onto a glass slide (Figure 3b). This step describes the production of the PDMS layers and the PDMS stamp for microcontact printing.
<ol>
	<li>Prepare a 10:1 mixture of PDMS and curing agent. Prepare approximately 80 g PDMS in total (using our designs, this will result in 10 chips). Mix both parts vigorously and degas the PDMS until the mixture is bubble free (~30 min).</li>
	<li>Place the wafer with the control layer inside a Petri dish and tape it onto the bottom. Next, pour &#160;~50 g of PDMS on top and put the wafer for at least 2 hr&#160;in an oven at 80 &#176;C to assure complete curing of the PDMS. Repeat the same procedure for the microcontact printing wafer, for this ~20 g PDMS is needed.</li>
	<li>Spin-coat PDMS on the wafer with the fluidic layer at a rotational speed of 2,000 rpm to form an approximately 40 &#181;m high PDMS layer. Cure the PDMS in an oven for at least 1 hr&#160;at 80 &#176;C.</li>
	<li>When both parts are cured, remove the control layer from the wafer and cut into pieces with a razor blade. Punch pressure connection holes by using a 1 mm biopsy puncher. For storage, it is advisable to put a piece of tape over the channels.</li>
	<li>To bond the two layers, take the spin-coated wafer and the top part and place them in a plasma cleaner23. After plasma treatment, quickly align both parts under a microscope with large working distance. Add some spare PDMS around the placed top parts. This step is not mandatory, however it facilitates the removal of the PDMS from the wafer. Place the wafer in an oven at 80 &#176;C for at least 1 hr.</li>
	<li>Use a scalpel to carefully remove the PDMS from the wafer and to cut the microchips. Punch access holes for the fluidic connections with a 1.5 mm biopsy puncher.</li>
	<li>The PDMS chip is now finished and can be stored for months. Optional: If the chip is used with a reservoir, cut the upper part of a 200 &#181;l pipette tip and use some spare, semi-cured PDMS to glue it on top. Put the chip in the oven again at 80 &#176;C for at least 1 hr.</li>
</ol>
4. Bonding to Glass Slide
To use the device for direct enzymatic assays, the only step required after bonding is the blocking of surfaces. For this protocol, proceed to A. However, to use the microfluidic chip for immunoassays or ELISAs, please refer to protocol B to restrict the binding sites only to the glass slide (i.e. for TIRF microscopy or SPR). If this restriction is not important, refer to A, but use biotinylated conjugates in step 4.3 and continue with step 4.8 in protocol B to create a fully functional surface.
Prior to performing protocol A and B: It is advisable to filter all of the used protein solutions prior to introducing them into the chip. Debris and protein aggregates may otherwise block cell traps, thereby reducing the number of chambers that can be used for analysis. For this purpose, use a nonprotein adsorbing filter unit to filter all solutions before introducing them into the channels.
Protocol A (enzymatic assays)
<ol>
	<li>To finalize the microfluidic device, bond the PDMS parts onto a glass slide. To do this, first clean a glass slide with soap, distilled water and ethanol. Dry the slide using a nitrogen stream. Clean the PDMS part with scotch tape.</li>
	<li>Put the PDMS part and the cleaned glass slide into the plasma cleaner for 45 sec at 18 W. To assure a tight bonding, put the chip on a hot plate (100 &#176;C) for 5 min.</li>
	<li>After bonding, fill the chip with fluids. An easy way to achieve this is to use centrifugal force. Cut the lower part of 200 &#181;l pipette tip. For the fluidic channels, fill them with the blocking solution (bovine serum albumin (4% w/v) or poly-L-lysine) grafted polyethylene glycol (0.05% w/v) and place the tips into the inlets. Fill the control layer inlets with either water or with an isosmotic solution, e.g. PBS. Place the chip into the centrifuge (800 x g) for 5 min. The channels should be now filled with fluid completely. If not, repeat this step.</li>
	<li>Incubate the blocking solution for at least 30 min&#160;at room temperature. Wash the solution out of the fluid layer with PBS at a flow rate of 10 &#181;l/min using a syringe pump. The device is now ready for cell experiments.</li>
</ol>
Protocol B (immunoassays)
For a complete overview of the surface modification from step 4.7 onwards, please refer to Table 1.
<ol start="5">
	<li>Incubate PDMS stamps for microcontact printing with a bBSA/BSA solution (e.g. 1-100). After 30 min, clean the stamps thoroughly with distilled water and dry the stamp under a stream of nitrogen. Quickly place the stamps on a cleaned glass slide (Figure 4).</li>
	<li>Expose the glass slide with the stamp together with the top part of the microfluidic chip to oxygen plasma for 45 sec at maximum power (18 W) in a plasma cleaner. After plasma treatment, remove the stamp and align the printed surface underneath the microchambers of the device. Place the chip for 30 min on a hot plate at 50 &#176;C.</li>
	<li>To block the remaining surface, introduce sterile-filtered BSA solution (4% (w/v) in PBS) into the chip with a centrifuge (see step 4.2). Incubate for 1 hr and wash the solution out of the fluid layer with PBS (see step 4.3). The chip can then be used directly or stored for up to 2 weeks at 4 &#176;C in a humid box.</li>
	<li>For a fully functional binding surface, introduce avidin (0.025% (w/v) in PBS) into the reservoir. Use a flow rate of 5 &#181;l/min for 30 min&#160;to withdraw the avidin solution through the fluid channels. Afterwards, flush PBS for 10 min. Rinse the reservoir thoroughly.</li>
	<li>Afterwards, add Protein G (0.0025% (w/v) in PBS) and flush for another 30 min. Wash with PBS for another 10 min&#160;afterwards. Next, add the antibody of interest (0.0001% (w/v) in PBS) for 10 min. After 10 min, stop the flow for 15 min&#160;to allow binding. Next, wash with PBS for 10 min.</li>
</ol>
5. Cell Experiments
The protocol is written in general terms because of the variety of possible assays. As an example the reagents needed for the G6PDH toxicity assay are given. The initial cell trapping efficiency of the device is around 2.5%, i.e.&#160;5 out of 200 cells will be trapped. The final occupancy of the cell traps strongly depends on the used cell line, the protocol to suspend the cell line and the time the cells are flushed through the channel. For cells naturally growing in suspension (such as U937), single cells can be found in about 75% of the chambers after a few minutes. The other chambers are either not filled, or occupied by two or more cells. In general, the single-cell occupancy is smaller for adherent cell lines. When using the rather mild suspending protocol presented here, the percentage decreases to about 30% (HEK, MLT cells). The single-cell occupancy can be improved by trypsin treatment of the cells, but this may also alter the results of the experiments.
Depending on the target molecule, adsorption or absorption to PDMS can influence the results. Adsorption can be reduced by blocking the surfaces with BSA or PLL-g-PEG. Absorption is unlikely for most hydrophilic biomolecules, but it should be checked for (small) lipophilic cell components.
<ol>
	<li>Prepare the cells according to the specific protocol (e.g. drug addition). For suspension cells, add cells directly to the chip. For adhesive cell lines, use enzyme-free dissociation buffer to produce a cell suspension. Filter the cells before introducing them to the chip to reduce the amount of cell clusters.</li>
	<li>Load the cells onto the chip using a syringe pump and forward flow, rather than to withdraw the cell suspension. Use a flow rate of 5 &#181;l/min for suspension cell lines&#160;and higher flow rates of 10-20 &#181;l/min for adhesive cells since to reduce nonspecific attachment of the cells onto the channel walls.</li>
	<li>When the traps are filled, close the chambers. If many cells adhere nonspecifically to the surface, try to wash them away with cell dissociation buffer at a flow rate of 10-30 &#181;l/min.</li>
</ol>
Protocol A (enzymatic assays)
<ol start="4">
	<li>Withdraw lysis buffer through the chip. For this example, this buffer is a hypoosmolar buffer with added Tween 20 (10 mM Tris-HCl, 10 mM KCl, 1.5 mM MgCl2, 1% (v/v) Tween 20). For lysis, quickly open and close chambers (i.e. 500 msec for flow rates of 10-50 &#181;l/min 7,21). For the G6PDH assay, add 2 mM glucose-6-phosphate, 0.5 mM NADP, 0.5 U/ml diaphorase and 0.3 mM resazurin to the lysis buffer. Start monitoring the kinetic reaction immediately after lysis.</li>
</ol>
Note: Choose any buffer which is suitable for the assay, however bare in mind that strong detergents (like Triton, SDS) lyse cells immediately and will lead to a loss of analyte during chamber opening.
Protocol B (immunoassays)
For a short description of the ELISA cell experiments (flow rates, chamber status, etc.), please refer to Table 1.
<ol start="5">
	<li>For ELISA, add the required detection reagent (e.g. secondary antibody, labeled antigen) directly to the lysis buffer. The herein added Tween 20 reduces the nonspecific adsorption. Lyse the cells according to step 5.4. Incubate the mixture for 10-15 minutes to allow binding (incubation time depending on the used antigen and antibody).</li>
	<li>Antibody enzyme conjugates that adsorbed nonspecifically to the channel walls can lead to a conversion of the detection reagent already inside the reservoir and channels. To reduce issues from this adsorption, flush a permanent inhibitor of the used detection enzyme through the chip during the incubation time (chambers closed, before adding the detection reagent). For HRP, use 20 mM HCl in water to permanently inactivate the nonspecifically adsorbed enzymes.</li>
	<li>After incubation time, introduce the detection reagent (e.g. Amplex Red and hydrogen peroxide for HRP) to the chip. Open the chambers shortly again to wash the nonbound secondary antibody away and to add the detection reagent. Start monitoring the kinetic reaction immediately.</li>
	<li>Calibration: Immobilize the antibody against the target of interest. Close chambers. Prepare different concentrations of analyte in buffer or in cell lysate off-chip. Apply a concentration to the chip and quickly exchange the volume inside the chambers (500 msec opening time). This opening time is sufficiently short to ensure that no additional accumulation occurs from analyte flushing, and only a specific number of molecules from the volume of the microchamber are detected. From the known concentration of the antigen inside the solution and the volume of the chamber (625 pl), calculate the amount inside the chamber. Afterwards, continue with step 5.5 and introduce the detection moiety.</li>
</ol>

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The authors declare that they have no competing financial interests.

Microfluidics technology has opened new and fascinating possibilities for single-cell analysis. In particular, the possibility to trap and immobilize cells individually by microfluidic tools has allowed systematic short and long-term studies on single-cell properties and response. Additionally, encapsulation of cells in high frequency microdroplets, generated on a microchip, has enabled single cell secretion studies, which cannot be performed with conventional cytometry devices. The microdroplet approach, however, has so...

Many studies in the past have revealed cell-to-cell differences within a large cell population1-3, in particular signaling processes4,&#160;or the amounts of intracellular biomolecules such as proteins5,6, metabolites, and cofactors7,8. These heterogeneities are considered to be fundamentally important for cell adaptation and evolution9, but also play a key role in the emergence and treatment of diseases such as cancer10-13. Therefore, studies on the single-cell level are of high interest in biological and pharmacological research, particularly if these studies reveal the different cell responses after treatment with bioactive chemical substances.
In recent years, many analytical platforms have been developed that facilitate the analysis of single living cells or the chemical composition of the cell content. While fluorescent activated cell sorting (FACS) is the gold standard for very high-throughput analysis of single living cells, the method cannot be employed for the quantification of intracellular or secreted compounds. The emergence of microfluidic platforms has promised novel analytical strategies for positioning, treatment and observation of single cells. A milestone in microfluidics was reached with the integration of flexible PDMS valves realized by Quake and coworkers14,15. These valves are useful since they can isolate regions on chip, e.g. separate two cultures 16. Furthermore, they are especially applicable for single cell analysis and therefore help to reduce analyte dilution problems. The power of this approach for single-cell analysis has been recently demonstrated by Hansen and coworkers, who analyzed the gene expression from hundreds of single cells in parallel17.
When targeting proteins and metabolites, the analysis is very difficult due to the lack of suitable amplification methods, the large number of different compounds present, and their variations in chemical nature. Furthermore, most intracellular biomolecules are expected to be present in low copy numbers in the order of a few ten thousands18, hence the analytical method used must have a high sensitivity. More powerful assays such as immunoassays and enzyme-linked immunoassays (ELISA) are difficult to integrate into microfluidic devices since they require several washing and incubation steps as well as surface immobilization.
Due to these challenges, it is not surprising that only a few examples have been reported where proteins or metabolites were quantified on the single-cell level. For example, studies on the secretion of fluorescent compounds have been reported19,20. Recently, the implementation with ELISA was presented for the analysis of secreted (nonfluorescent) proteins from a cell culture (THP-1 cells)21 and single (immune) cells10. Targeting intracellular proteins, Shi et al. developed a microfluidic device that facilitated the identification of intracellular proteins for the analysis of signaling pathways in tumor cells by means of an immunoassay11. However, only relative amounts of proteins were determined and no enzymatic amplification was used to increase the signal for low abundance proteins.
Recently, we were able to combine a single-cell trapping microdevice with fluorescence assays8 and immunoassays22 (Figure 1). Cells are passively trapped in microsized hurdle structures, which allow supply and (rapid) exchange of medium and other chemical agents without any movement of the cells. A ring-shaped valve around each trap enables isolation of the cell in a very small volume (&#8220;the microchamber&#8221;). This valve is actuated immediately after introducing a cell-lysing (hypoosmolar) buffer, hence preventing intracellular molecules or secreted molecules to diffuse away. Most importantly, due to the small size of the volume (625 pl) large dilution of the molecules is avoided. Furthermore, since lysis and analysis are performed in at the same position in the chip, there is no loss of analytes due to transportation. The chip design described here comprises 8 alternating rows of either 7 or 8 microchambers, totaling 60 microchambers. The chambers are actuated in rows, so that cross-contamination along a line is precluded.
The platform can be used in combination with fluorescence assays as well as immunological assays (Figure 1d). For the latter, we established protocols for immobilization of the antibodies, which are compatible with the chip production and assembly process. Hence the platform opens the way for sensitive, reliable and quantifiable assays at the single cell level. Up to now, we have used the device for the analysis of intracellular and secreted enzymes (relative quantification by enzymatic assays), intracellular cofactors, proteins and small molecules (absolute quantification by endpoint assays or ELISA). In the following, we describe the process of chip fabrication by means of multilayer soft lithography and the protocols for patterning of the antibodies by means of microcontact printing and surface chemistry. Additionally, some examples of chip use and operations are given.

<table><tbody><tr><td>&lt;strong&gt;REAGENTS:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Name of the Reagent</td><td>Company</td><td>Catalogue Number</td><td>Comments (optional)</td></tr><tr><td>SU-8 2015</td><td>MicroChem Corp. (Netwon, MA)</td><td>n.a.</td><td></td></tr><tr><td>1H,1H,2H,2H-Perfluorodecyl-dimethylchloro-silane</td><td>ABCR (Karlsruhe, Germany)</td><td>AB103608</td><td></td></tr><tr><td>4-Methylumbelliferyl &amp;beta;-D-N,N&amp;prime;-diacetylchitobioside hydrate</td><td>Sigma Aldrich</td><td>M9763</td><td></td></tr><tr><td>Acetone</td><td>Merck VWR (Darmstadt, Germany)</td><td>100014</td><td></td></tr><tr><td>Avidin</td><td>AppliChem (Axon Lab AG)</td><td>A-2568</td><td></td></tr><tr><td>AZ 1518</td><td>AZ Electronic Materials (Wiesbaden, Germany)</td><td>n.a.</td><td></td></tr><tr><td>AZ 726 developer</td><td>AZ Electronic Materials (Wiesbaden, Germany)</td><td>n.a.</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A-4503</td><td></td></tr><tr><td>Bovine serum albumin, biotin</td><td>Sigma Aldrich</td><td>A-8549</td><td></td></tr><tr><td>Cell dissociation buffer</td><td>Invitrogen</td><td>13151-014</td><td></td></tr><tr><td>Hexamethyldisilazane (HDMS)</td><td>Sigma Aldrich</td><td>40215</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fluka</td><td>84422</td><td></td></tr><tr><td>Isopropanol</td><td>Merck VWR (Darmstadt, Germany)</td><td>109634</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Fluka</td><td>63068</td><td></td></tr><tr><td>MR developer 600</td><td>Microresist technology GmbH (Berlin, Germany)</td><td>n.a.</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>10010-031</td><td></td></tr><tr><td>PLL-g-PEG grafted</td><td>SuSoS, (D&amp;uuml;bendorf, Switzerland)</td><td>n.a.</td><td></td></tr><tr><td>PLL-g-PEG grafted biotin</td><td>SuSoS, (D&amp;uuml;bendorf, Switzerland)</td><td>n.a.</td><td></td></tr><tr><td>Potassium chloride</td><td>Fluka</td><td>60132</td><td></td></tr><tr><td>Protein G, biotin</td><td>Sigma Fine Chemicals</td><td>41624</td><td></td></tr><tr><td>Silicon wafer</td><td>Si-Mat (Kaufering, Germany)</td><td>n.a.</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit (PDMS)</td><td>Dow Corning</td><td>39100000</td><td></td></tr><tr><td>Tris(hydroxymethyl)-aminomethan</td><td>Biorad</td><td>1610716</td><td></td></tr><tr><td>Tween 20</td><td>Biorad</td><td>1706531</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Material Name</td><td>Company</td><td>Catalogue Number</td><td>Comments (optional)</td></tr><tr><td>0.22 &amp;micro;m PES syringe filter</td><td>TRP</td><td>99722</td><td></td></tr><tr><td>1/1.5 mm biopsy puncher</td><td>Miltex, York PA</td><td>33-31AA/33-31A</td><td></td></tr><tr><td>Cell Trics filter 20 &amp;micro;m</td><td>Partec</td><td>04-004-2325</td><td></td></tr><tr><td>Centrifuge Sigma 3-18K</td><td>Kuehner</td><td>n.a.</td><td></td></tr><tr><td>Hotplate HP 160 III BM</td><td>Sawatec, Sax, Switzerland</td><td>n.a.</td><td></td></tr><tr><td>MA-6 mask aligner</td><td>Karl Suess</td><td>n.a.</td><td></td></tr><tr><td>Multizoom AZ100M microscope</td><td>Nikon Corporation</td><td>n.a.</td><td></td></tr><tr><td>Photomask</td><td>Microlitho, Essex, U.K.</td><td>n.a</td><td></td></tr><tr><td>Plasma Cleaner PDC-32G</td><td>Harrick</td><td>n.a.</td><td></td></tr><tr><td>Spin coater Modell WS-400 BZ-6NPP/LITE</td><td>Laurell</td><td>n.a.</td><td></td></tr><tr><td>Spin Modules SM 180 BM</td><td>Sawatec, Sax, Switzerland</td><td>n.a.</td><td></td></tr><tr><td>Step profiler Dektak XT Advanced</td><td>Bruker</td><td>n.a.</td><td></td></tr><tr><td>Syringe pump neMESYS</td><td>Cetoni</td><td>n.a.</td><td></td></tr></tbody></table>

a microfluidic chip for the versatile chemical analysis of single cells

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

Our platform is able to analyze a variety of intracellular as well as secreted molecules present in or produced by single cells. Here, we would like to present different example studies to underline the variety of possible assays. We will give an example for a secreted enzyme (Figure 5a) as well as an intracellular enzyme (Figure 5b) and protein (Figures 5c and d). For more examples, such as cofactors or small molecules, please refer to Eyer et al.</...

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

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.

a-microfluidic-chip-for-versatile-chemical-analysis-single

Research

JoVE Journal

Bioengineering

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Fluorescence detection methods for microfluidic droplet platforms

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40−60 µm at frequencies from 90 to 7,000 Hz.

A Microfluidic Chip for ICPMS Sample Introduction

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Counting Proteins in Single Cells with Addressable Droplet Microarrays

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

The protocol describes an advanced microfluidic platform to quantitatively measure cytokine secretion dynamics of individual human peripheral blood mononuclear cells. The platform measures up to three cytokines in parallel (IL-6, TNFα, and IL-1β) for each individual cell stimulated with lipopolysaccharide as an example.

Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells

Learn microfluidic chip fabrication for single cell analysis using fluorescent immunoassays. This method enables precise quantification of intracellular proteins and enzymes with high sensitivity.

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

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