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.

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			<td height="21" style="height:21px;width:344px;">REAGENTS:</td>
			<td style="width:284px;"></td>
			<td style="width:103px;"></td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:344px;">Name of the Reagent</td>
			<td style="width:284px;">Company</td>
			<td style="width:103px;">Catalogue Number</td>
			<td style="width:153px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">SU-8 2015</td>
			<td style="width:284px;">MicroChem Corp. (Netwon, MA)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">1H,1H,2H,2H-Perfluorodecyl-dimethylchloro-silane</td>
			<td style="width:284px;">ABCR (Karlsruhe, Germany)</td>
			<td style="width:103px;">AB103608</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">4-Methylumbelliferyl &#946;-D-N,N&#8242;-diacetylchitobioside hydrate</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">M9763</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Acetone</td>
			<td style="width:284px;">Merck VWR (Darmstadt, Germany)</td>
			<td style="width:103px;">100014</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Avidin</td>
			<td style="width:284px;">AppliChem (Axon Lab AG)</td>
			<td style="width:103px;">A-2568</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">AZ 1518</td>
			<td style="width:284px;">AZ Electronic Materials (Wiesbaden, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">AZ 726 developer</td>
			<td style="width:284px;">AZ Electronic Materials (Wiesbaden, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Bovine serum albumin</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">A-4503</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Bovine serum albumin, biotin</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">A-8549</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Cell dissociation buffer</td>
			<td style="width:284px;">Invitrogen</td>
			<td style="width:103px;">13151-014</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Hexamethyldisilazane (HDMS)</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">40215</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Hydrochloric acid</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:103px;">84422</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Isopropanol</td>
			<td style="width:284px;">Merck VWR (Darmstadt, Germany)</td>
			<td style="width:103px;">109634</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Magnesium chloride hexahydrate</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:103px;">63068</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">MR developer 600</td>
			<td style="width:284px;">Microresist technology GmbH (Berlin, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">PBS</td>
			<td style="width:284px;">Invitrogen</td>
			<td style="width:103px;">10010-031</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">PLL-g-PEG grafted</td>
			<td style="width:284px;">SuSoS, (D&#252;bendorf, Switzerland)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">PLL-g-PEG grafted biotin</td>
			<td style="width:284px;">SuSoS, (D&#252;bendorf, Switzerland)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Potassium chloride</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:103px;">60132</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Protein G, biotin</td>
			<td style="width:284px;">Sigma Fine Chemicals</td>
			<td style="width:103px;">41624</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Silicon wafer</td>
			<td style="width:284px;">Si-Mat (Kaufering, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Sylgard 184 Silicone Elastomer Kit (PDMS)</td>
			<td style="width:284px;">Dow Corning</td>
			<td>39100000</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Tris(hydroxymethyl)-aminomethan</td>
			<td style="width:284px;">Biorad</td>
			<td style="width:103px;">1610716</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Tween 20</td>
			<td style="width:284px;">Biorad</td>
			<td style="width:103px;">1706531</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:344px;">Material Name</td>
			<td style="width:284px;">Company</td>
			<td style="width:103px;">Catalogue Number</td>
			<td style="width:153px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">0.22 &#181;m PES syringe filter</td>
			<td style="width:284px;">TRP</td>
			<td style="width:103px;">99722</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">1/1.5 mm biopsy puncher</td>
			<td style="width:284px;">Miltex, York PA</td>
			<td style="width:103px;">33-31AA/33-31A</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Cell Trics filter 20 &#181;m</td>
			<td style="width:284px;">Partec</td>
			<td style="width:103px;">04-004-2325</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Centrifuge Sigma 3-18K</td>
			<td style="width:284px;">Kuehner</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Hotplate HP 160 III BM</td>
			<td style="width:284px;">Sawatec, Sax, Switzerland</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">MA-6 mask aligner</td>
			<td style="width:284px;">Karl Suess</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Multizoom AZ100M microscope</td>
			<td style="width:284px;">Nikon Corporation</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Photomask</td>
			<td style="width:284px;">Microlitho, Essex, U.K.</td>
			<td style="width:103px;">n.a</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Plasma Cleaner PDC-32G</td>
			<td style="width:284px;">Harrick</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Spin coater Modell WS-400 BZ-6NPP/LITE</td>
			<td style="width:284px;">Laurell</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Spin Modules SM 180 BM</td>
			<td style="width:284px;">Sawatec, Sax, Switzerland</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Step profiler Dektak XT Advanced</td>
			<td style="width:284px;">Bruker</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Syringe pump neMESYS</td>
			<td style="width:284px;">Cetoni</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
	</tbody>
</table>

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&#160;5b) and protein (Figures 5c and&#160;d). For more examples, such as cofactors or small molecules, please refer to Eyer<em...

Watch this Scientific Journal Video about A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells at JoVE.com

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

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 ...

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

Research

JoVE Journal

Bioengineering

14.8K Views.  ETH Zurich, Switzerland. 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. 

Video: A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

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.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

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&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

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

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

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.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

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

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

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.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...