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 li...

<ol>
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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>
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		<tr height="22">
			<td height="22" style="height:22px;"></td>
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			<td></td>
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		</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>
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		</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>
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		</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>
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			<td style="width:103px;">04-004-2325</td>
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			<td style="width:284px;">Sawatec, Sax, Switzerland</td>
			<td style="width:103px;">n.a.</td>
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			<td height="22" style="height:22px;width:344px;">MA-6 mask aligner</td>
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</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 vistas.  ETH Zurich, Switzerland.  En este artículo se presenta un chip de microfluidos para el análisis de células individuales. Permite la cuantificación de las proteínas intracelulares, enzimas, cofactores, y segundos mensajeros por medio de ensayos fluorescentes o inmunoensayos. 