This work was supported by the European Research Council (ERC Starting Grant n&#956;LIPIDS, No. 203428) and ETH Zurich (project number: ETH-49 12-2). The authors of this manuscript would like to thank Bodo Hattendorf for help with the ICP-MS and F. Kurth for cell counting. The authors also would like to thank Christoph B&#228;rtschi and Roland M&#228;der for their support with building the mechanical setup. The clean room facility FIRST at ETH Zurich is acknowledged for support in microfabrication.

1. SU-8 Master Fabrication (Figure 2)
NOTE: Perform the fabrication of the SU-8 master molds in a clean room to prevent defects caused by dust particles. Two wafers are needed for the fabrication, one wafer with microfluidic features and one without.
<ol>
	<li>Prepare the master molds for the microfluidic chip. First apply an adhesion layer to the silicon wafer.
	<ol>
		<li>Dehydrate a silicon wafer for 10&#160;min at 200&#160;&#176;C. Cool the wafer down to RT and load it on to a spin coater and spin coat it with SU-8 2002 with the following protocol.</li>
		<li>Dispense about 3&#160;ml resist onto the wafer.</li>
		<li>Spin the wafer at 500&#160;rpm for 10&#160;sec to spread the resist over the whole wafer.</li>
		<li>Spin the wafer at 2,000&#160;rpm for 30&#160;sec to achieve a resist height of approximately 2 &#181;m.</li>
	</ol>
	</li>
	<li>Remove excess resist from the edge of the wafer with an acetone soaked swab, to prevent sticking of the wafer to the hot plate in the next step. Bake the wafer for 60&#160;sec at 95&#160;&#176;C on a hot plate.</li>
	<li>Expose the whole wafer with ultraviolet light (80&#160;mJ/cm2 at 365&#160;nm). Post-bake the wafer for 120&#160;sec to 95&#160;&#176;C.</li>
	<li>Cool the wafer down and immediately spin coat the wafer again using the following protocol for SU-8 2050:
	<ol>
		<li>Spin the wafer at 100&#160;rpm for 20&#160;sec (dispense about 3&#160;ml SU-8 resist during this step).</li>
		<li>Spin the wafer at 500&#160;rpm for 10&#160;sec to spread the resist over the whole wafer.</li>
		<li>Spin the wafer at 3,250&#160;rpm for 30&#160;sec resulting in a resist thickness of approximately 40&#160;&#181;m.</li>
	</ol>
	</li>
	<li>Again, remove excess resist from the edge of the wafer with an acetone soaked swab and soft bake the wafer on a hot plate for 180&#160;sec at 65&#160;&#176;C and for 360&#160;sec at 95&#160;&#176;C.</li>
	<li>Prepare the photomask by sticking it to a soda-lime glass. See Figure 3 for the mask design. Use a mask aligner to expose the resist with ultraviolet light (160&#160;mJ/cm2, measured at 365 nm) through the prepared mask. Bake the exposed wafer again on a hot plate for 60&#160;sec at 65&#160;&#176;C and for 360&#160;sec at 95&#160;&#176;C.</li>
	<li>After cooling down the wafer to RT, immerse it in a glass Petri dish filled with developer for 5 min to develop the resist. Gently agitate the petri dish to remove unexposed SU-8. Rinse the wafer with isopropanol and blow it dry with a nitrogen gun.</li>
	<li>Examine the wafer under a microscope. In case undeveloped resist remains on the features, develop the wafer again for a few minutes, as described in step 1.7.</li>
	<li>Remove any residual solvent by baking the wafers for 2&#160;hr at 200&#160;&#176;C. Check the height of the features with a step profiler. In case the measured height differs from the desired height begin with this protocol again and adapt the spin speed in step 1.1.4.</li>
	<li>To prevent sticking of the PDMS to the wafer silanize it by placing it in desiccator together with 50&#160;&#181;l of 1H,1H,2H,2H-perfluorodecyltrichlorosilane in a small porcelain dish. Reduce the pressure in the desiccator to 100&#160;mbar and incubate the wafer for 12&#160;hr.
	<ol>
		<li>For the blank PDMS parts silanize another silicon wafer using the method of step 1.10. To save time silanize both wafers at the same time in a single desiccator.</li>
	</ol>
	</li>
</ol>
2. LADE Chip Fabrication
NOTE: The LADE chip is made out of two PDMS pieces that are bonded together by adhesive bonding24. The first part contains the microfluidic features. The other part is flat and used to seal the channels. Bonded together, they form the round shape necessary to interface the chip with the droplet transport system. Here, the fabrication of the two parts and their bonding is described. All process steps are shown in Figure 4.
<ol>
	<li>Prepare 44&#160;g of PDMS by mixing 40&#160;g of prepolymer with 4&#160;g of the PDMS curing agent (this will result in up to 6 chips). Degas the PDMS in a desiccator until it is bubble free (this will take about 20&#160;min).</li>
	<li>Replica molding of the structured halves.
	<ol>
		<li>Place the casting form on top of the wafer and snap it into place using the guiding structures around the design (see Figure 5). Skip the snapping into place for the flat PDMS halve.</li>
		<li>Pour approximately 3 to 4&#160;g of the degased PDMS in the casting form and place it for 6&#160;min on a hot plate at 150&#160;&#176;C. Cool down the cured PDMS in the casting form and carefully lift the casting form wafer using a spatula.</li>
		<li>In order to prevent any contamination of the microfluidic channels cover the side of the chip that was previously in contact with the wafer with tape. Carefully cut the tape along the edge of the PDMS part to remove excess PDMS.</li>
	</ol>
	</li>
	<li>To fabricate the flat PDMS halves repeat the above-mentioned steps 2.2.1 to 2.2.3 with the blank silanized wafer.</li>
	<li>Peel of the tape and punch fluid connection holes into the structured halves with a biopsy puncher. Protect the structures with tape during storage.</li>
	<li>Bond the PDMS parts together by adhesive bonding using the PDMS curing agent24.
	<ol>
		<li>Take an untreated silicon wafer and spin coat it with PDMS curing agent for 30&#160;sec at 6,000&#160;rpm. Take the wafer out of the spin coater.</li>
		<li>Remove the tape from the structured halves and place them with the structures facing downwards onto the wafer. Gently push on top of the PDMS to remove air bubbles.</li>
		<li>Remove the tape from the blank PDMS halves. Take a structured halve from the wafer and manually align it on top of the flat PDMS halve. Gently squeeze the part together to remove air bubbles and let the assembled chip cure for 24&#160;hr at RT. Do not push the parts together with force as this can cause the channels to collapse.</li>
	</ol>
	</li>
	<li>Cut the tip of the chip along the indicator line orthogonal to the nozzle channel with a utility knife to open the outlet nozzle. Use an alignment device to ensure a straight cut, which is necessary for a straight liquid ejection. Inspect the chip under a microscope for defects in the microfluidic channels and dust particles. Put a tape over the inlet holes to protect the chips during storage.</li>
	<li>Connect a Woulff bottle with tubing to a dry nitrogen source and to all inlets of the LADE chip. Deposit 50&#160;&#181;l of 1H,1H,2H,2H-perfluorodecyltrichlorosilane at the bottom of the Woulff bottle and close it.</li>
	<li>Silanize the microfluidic channels by flushing all channels for 20&#160;min with the nitrogen stream carrying 1H,1H,2H,2H-perfluorodecyltrichlorosilane at a flow rate of approximately 1&#160;ml/sec. The chips are ready for experiments and can be stored for at least several weeks at RT.</li>
</ol>
3. Preparations for Measurement / Droplet Transport System
NOTE: Build the whole droplet transport system on top of an optical table, since it is necessary to construct a stable supporting structure for the setup. A scheme of the whole droplet transportation system is depicted in Figure 6.
<ol>
	<li>Install a custom cyclonic poly(methyl methacrylate) (PMMA) adapter with an 50&#160;cm attached stainless steel tube vertically. Attach the adapter to a helium source with a mass flow controller. Attach a (high-speed) camera and a lamp to the adapter on the opposite sites for droplet visualization.</li>
	<li>Place a cartridge heater in the middle of the steel tube and use poly(vinyl chloride) (PVC) tubing and a Legris tube connector to connect the end of the steel tube with the inlet of the membrane desolvator.</li>
	<li>Connect the outlet of the desolvator with another PVC tubing to a laminar flow adapter, which is directly connected to the ICPMS inlet. Connect the laminar flow adapter to an argon source with a mass flow controller and later on use it to admix Argon to achieve a stable operation condition.</li>
	<li>Align the adapter as well as the steel tube vertical with a spirit level. If the alignment is not accurate, it may lead to significant losses of droplets. Insert a plug into the adapter to prevent gases leaking out during the system warm up time.</li>
	<li>Start all above-mentioned gas flows and devices using the settings from Table 1. Allow the system to warm up for 15 min. The cartridge heater needs 2 hr to stabilize the temperature, switch it on in advance.</li>
	<li>Place the syringe pumps on a rack at the height of cyclonic helium adapter. Keep the distance between the syringe pumps and the adapter as short as possible.</li>
</ol>
4. Measurements
NOTE: The following protocol is written in general terms because of the variety of solutions and suspensions that can be used. However, cell suspensions should be diluted to a concentration of &#60; 1 x 107 cells/ml, when single cell analysis is performed, to ensure that the majority of the droplets carry only one cell. For measurements with cells place the syringe pumps at an angle so that the outlet of the syringes point downwards and install the tubing in such a way that they point downwards.
<ol>
	<li>Attach tubing to the syringes. Load two 5&#160;ml syringes with perfluorohexane and one 1&#160;ml syringe with a sample solution or suspension. Remove all bubbles trapped in the syringes and tubing.</li>
	<li>Install the syringes in a syringe pump and connect them to the inlets of the chip. Start the syringe pumps using the start settings from Table 1 (or higher flow rates). Give the flows 3 to 5 min to stabilize.
	<ol>
		<li>Remove excess liquid from the tip of the chip with a tissue. The liquids should now eject from the chip in a straight jet. If a straight ejection cannot be achieved by wiping with a tissue replace the chip and start over with this step.&#160;</li>
	</ol>
	</li>
	<li>Remove the plug from the adapter and carefully insert the chip into the adapter. Lubricate the chip with FC-40 if necessary. A chip can be used for at least 2 hr of experiments.</li>
	<li>Change the flow rate to be within the recommended measurement settings from Table 1. Lower flow rate of the PFH not only saves PFH but also reduces signal background, caused by isobaric interferences.</li>
	<li>Give the system 2-5&#160;min to stabilize (depending on the chosen flow rates). Optimize the ICPMS for the highest signal intensity of the analytes of interest.
	<ol>
		<li>Successively adjust the flows of all the gases on the mass flow controllers (see the recommended ranges in Table 1) until the maximum signal intensity of the analytes of interest is achieved. Tune the plasma power and the focusing lens voltages (according to the manufacturer&#39;s recommended ranges) on the ICPMS in the same way.</li>
	</ol>
	</li>
	<li>Set the ICPMS to a dwell time of 10&#160;msec (applied for the very ICPMS used but can be adjusted with other instruments to ensure a time-resolved acquisition). Start recording the signal of a particular m/z using manufacturer&#8217;s protocol.</li>
	<li>After the measurement, transfer the raw data to data analysis program for evaluation. Bin the data, given in counts per 10 msec, with a built-in-function, and plot resulting bin center values against counts. Fit each peak in the plotted frequency distribution histogram with a Gauss function. The mean and sigma of the fit represent the mean signal intensity and its standard deviation, respectively.</li>
</ol>
5. Calibration Concept
<ol>
	<li>Measure a single or multi-element standard solution containing the element or elements of interest at the same flow rates as the sample.</li>
	<li>Place a LADE chip in a petri dish on a microscope. For a better image quality use a non-round chip. Fabricate this chip as described in step 2, but using a simple rectangular casting form instead of the partially round shaped one.</li>
	<li>Follow the steps 4.1 to 4.2.1 to start the droplet generation. Set the flow rates to the flow rates used in step 5.1.</li>
	<li>Record images of the aqueous droplets with a high-speed camera attached to a microscope (20X objective). Use an image analysis software like the droplet morphometry and velocimetry software by Basu25 to obtain the average droplet diameter from the recordings.</li>
	<li>Use average droplet diameter to calculate the droplet volume, assuming the droplet is a spherical object.
	<ol>
		<li>From this volume and the known concentration of an analyte in the droplet calculate the number of corresponding atoms. Divide the number of measured ions per droplet by the number of atoms to obtain the detection efficiency. Use this detection efficiency to calculate the number of atoms in an unknown sample. 
		NOTE: Since the variations between individual chips are small22, it is not necessary to repeat the measurement of the droplet size for every chip or solution if the flow rates remain the same. A list of the droplet sizes and frequencies according to the specific flow rates is published by Verboket et al.22.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pillar-induced+droplet+merging+in+microfluidic+circuits.">Pillar-induced droplet merging in microfluidic circuits.</a> Lab Chip. 8 (11), 1837-1841 (2008).</li><li>Song, H., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond+kinetics+on+a+microfluidic+chip+using+nanoliters+of+reagents.">Millisecond kinetics on a microfluidic chip using nanoliters of reagents.</a> J. Am. Chem. Soc. 125 (47), 14613-14619 (2003).</li><li>Song, H., Chen, D. L., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactions+in+droplets+in+microflulidic+channels.">Reactions in droplets in microflulidic channels.</a> Angew. Chem., Int. Ed. 45 (44), 7336-7356 (2006).</li><li>Lombardi, D., Dittrich, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+microfluidics+with+magnetic+beads:+a+new+tool+to+investigate+drug-protein+interactions.">Droplet microfluidics with magnetic beads: a new tool to investigate drug-protein interactions.</a> Anal. Bioanal. Chem. 399 (1), 347-352 (2011).</li><li>Edgar, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+chemically+separated+components+into+droplets.">Compartmentalization of chemically separated components into droplets.</a> Angew. Chem., Int. Ed. 48 (15), 2719-2722 (2009).</li><li>Edgar, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis+separation+in+the+presence+of+an+immiscible+boundary+for+droplet+analysis.">Capillary electrophoresis separation in the presence of an immiscible boundary for droplet analysis.</a> Anal. Chem. 78 (19), 6948-6954 (2006).</li><li>Baret, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+droplet+sorting+(FADS):+efficient+microfluidic+cell+sorting+based+on+enzymatic+activity.">Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity.</a> Lab Chip. 9 (13), 1850-1858 (2009).</li><li>Agresti, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh-throughput+screening+in+drop-based+microfluidics+for+directed+evolution.">Ultrahigh-throughput screening in drop-based microfluidics for directed evolution.</a> Proc. Natl. Acad. Sci. U. S. A. 107 (9), 4004-4009 (2010).</li></ol>

The authors declare that they have no competing financial interests.

Although the fabrication of the chips is very reliable there are some critical points during the fabrication that require special attention. First, cleanliness during the assembly is highly important to prevent contamination of the chip by dust. The dust can block the channels and prevent a stable droplet generation. Second, it is especially important that the tip is cut orthogonal to the nozzle channel. The angle of the cut strongly influences the ejection angle. If the liquid is ejected at an angle it can cause a loss ...

Elemental analysis of liquid samples by inductively coupled plasma mass spectrometry (ICPMS) is commonly carried out using nebulizers in combination with spray chambers as introduction system1. In this sample introduction system the sample is sprayed by a nebulizer to generate a polydisperse aerosol. A downstream spray chamber is used to filter out large droplets. This method is associated with high sample consumption (&#62;0.3 ml min-1)2 and an incomplete sample transport. Thus, it becomes impractical for applications where only microliter sample volumes are available, as in biological, forensic, toxicological and clinical studies3. To reduce the sample consumption, nebulizers with smaller nozzle dimensions were developed3. However, the reduced nozzle size increases the risk of clogging when samples of undigested biological fluids or concentrated salt solutions have to be analyzed3.
A different approach for sample introduction was proposed by Olesik et al.4. The authors injected a liquid into ICPMS in the form of monodisperse discrete microdroplets, which were produced by a piezo-electrically driven micropump. Even though this very system did not find wide application, it initiated the further development of the concept of discrete droplet introduction in ICPMS. Today, piezo-electrically driven dispensing systems, which can generate droplets in size of 30, 50, 70 and 100 &#181;m and at frequencies of 100-2,000 Hz, can be purchased. The droplets can be transported into ICPMS with close to 100% efficiency5. These microdroplet dispensers have been applied for quantitatively measuring single nanoparticles5,6 as well as characterizing individual biological cells7. A similar system based on thermal inkjet technology8 was tested for analysis of biological samples9. Although the available single droplet introduction systems are very efficient, can be used for small sample volumes and are promising for the analysis of nanoparticles and cells, they have several limitations. For a fixed nozzle size, the droplet size can be varied only slightly (unless custom settings are used10). Changes of the physical properties of the liquid (pH, salt content) can alter the droplet characteristics (size, injection speed). Also, these devices are rather expensive, prone to clogging and are difficult to clean.
Another method to generate droplets is known in the field of droplet microfluidics11. In recent years droplet microfluidics has gained interest for (bio-)chemical reactions12-15 and for single cell studies16,17. Additionally, this technique was applied for introducing samples in electrospray ionization mass spectrometry18,19 and for preparing samples in matrix-assisted laser desorption/ionization mass spectrometry20,21.
Recently, we introduced a microfluidic based system for sample introduction in ICPMS22. The key component of our introduction system is the liquid assisted droplet ejection (LADE) chip. This chip consists completely of poly(dimethylsiloxane) (PDMS). In the first channel junction flow focusing is used to generate monodisperse droplets of an aqueous sample solution (Figure 1). For this purpose the highly volatile (boiling point of 58-60&#160;&#176;C23) and immiscible carrier phase perfluorohexane (PFH) is used (Figure 1). These PFH properties enable a stable droplet generation and fast removal of the carrier phase. Changes in the properties of the sample liquid influence this generation method less, compared to other droplet generators. The droplet size is adjustable over a wide range by changing the flow rates of the aqueous phase and the PFH. In a downstream secondary junction, more PFH is added to increase the flow speed to at least 1 m&#160;sec-1. At this speed the liquid can be ejected from the chip in stable and straight jet (Figure 1) without droplet destruction (Figure 1 inset). This double-junction design allows controlling the jet stability independent of droplet generation. The droplets are transported to the ICPMS with a customized transport system. This system comprises a falling tube and a membrane desolvator to remove the PFH. The dried residues of the aqueous droplets are subsequently ionized in the plasma of the ICPMS and a mass detector measures the ions. The front part of the chip is barrel-shaped to ensure a tight connection with the droplet transport system. The ejection of the aqueous sample as droplets in PFH is beneficial, because contact with the nozzle is avoided. This considerably lowers the risk of nozzle clogging, which can be a problem when working with cell suspensions or concentrated salt solutions. The LADE chips, fabricated by PDMS soft lithography, are cheap (material cost approximately $2 per chip), disposable and easy to modify. In combination with the fabrication that requires only a small amount of manual work each experiment can be performed with a new chip. Therefore, a laborious cleaning is not needed and cross contamination is minimized.
Here, the fabrication of the LADE chip by soft lithography and its application for ICPMS are described. Examples of measurements with an aqueous solution and a cell suspension are presented.

<table border="0" cellpadding="0" cellspacing="0" width="919">
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			<td height="21" style="height:21px;width:269px;">Name of Reagent/ Equipment</td>
			<td style="width:320px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
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		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Silicon wafer 100 mm</td>
			<td align="left" style="width:320px;">Si-Mat (Kaufering, Germany)</td>
			<td style="width:147px;">n. a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">SU-8 2002</td>
			<td align="left" style="width:320px;">Microchem Corp. (Massachusetts, U.S.A.)</td>
			<td style="width:147px;">n.a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">SU-8 2050</td>
			<td align="left" style="width:320px;">Microchem Corp. (Massachusetts, U.S.A.)</td>
			<td style="width:147px;">n.a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Acetone</td>
			<td align="left" style="width:320px;">Merk VWR (Darmstadt, Germany)</td>
			<td style="width:147px;">100014</td>
			<td></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:269px;">MR-developer 600</td>
			<td align="left" style="width:320px;">Microresist Technology GmbH (Berlin, Germany)</td>
			<td style="width:147px;">n. a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Isopropanol</td>
			<td align="left" style="width:320px;">Merk VWR (Darmstadt, Germany)</td>
			<td style="width:147px;">109634</td>
			<td></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:269px;">1H,1H,2H,2H-perfluorodecyltrichlorosilane</td>
			<td align="left" style="width:320px;">ABCR-Chemicals (Karlsruhe, Germany)</td>
			<td style="width:147px;">AB111155</td>
			<td></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:269px;">Sylgard 184 silicone elastomer kit (PDMS)</td>
			<td align="left" style="width:320px;">Dow Corning (Michigan, U.S.A.)</td>
			<td style="width:147px;">39100000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Perfluorohexane 99%</td>
			<td align="left" style="width:320px;">Sigma-Aldrich (Missouri, U.S.A.)</td>
			<td style="width:147px;">281042</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">FC-40</td>
			<td align="left" style="width:320px;">ABCR-Chemicals (Karlsruhe, Germany)</td>
			<td style="width:147px;">AB103511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Phosphate-buffered saline&#160;</td>
			<td align="left">Life Technologies (Paisley, U.K.)&#160;</td>
			<td style="width:147px;">10010-015</td>
			<td></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:269px;">Red blood cells in phosphate-buffered saline</td>
			<td align="left">Rockland Immunochemicals Inc. (Pennsylvania, U.S.A.)&#160;</td>
			<td style="width:147px;">R400-0100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:269px;">Single-element standard solutions Na, Fe</td>
			<td align="left" style="width:320px;">Inorganic Ventures (Virginia, U.S.A.)</td>
			<td style="width:147px;">n. a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Multielement standard solution&#160;</td>
			<td align="left" style="width:320px;">Merck Millipore (Massachusetts, U.S.A.)</td>
			<td align="left" style="width:147px;">IV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Nitric acid</td>
			<td align="left" style="width:320px;">Sub-boiled</td>
			<td style="width:147px;">n. a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Ultrahigh-purity water</td>
			<td align="left" style="width:320px;">Merck Millipore (Massachusetts, U.S.A.)</td>
			<td style="width:147px;">n. a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;"></td>
			<td style="width:320px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name of Equipment</td>
			<td style="width:320px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Hot plate HP 160 III BM</td>
			<td align="left" style="width:320px;">Sawatec (Sax, Switzerland)</td>
			<td style="width:147px;">n. a.</td>
			<td align="left">used for wafer preparation</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Spin modules SM 180 BM</td>
			<td align="left" style="width:320px;">Sawatec (Sax, Switzerland)</td>
			<td style="width:147px;">n. a.</td>
			<td align="left">used for wafer preparation</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">MA-6 mask aligner</td>
			<td align="left" style="width:320px;">Su&#776;ss MicroTec (Garching, Germany)</td>
			<td style="width:147px;">n. a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">High resolution film photomask</td>
			<td align="left" style="width:320px;">Microlitho (Essex, U.K.)</td>
			<td style="width:147px;">n. a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Step profiler Dektak XT advanced</td>
			<td align="left" style="width:320px;">Bruker&#160; (Massachusetts, U.S.A.)</td>
			<td style="width:147px;">n. a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Hot plate MR 3002</td>
			<td align="left" style="width:320px;">Heidolph (Schwabach, Germany)</td>
			<td style="width:147px;">n. a.</td>
			<td align="left">used for replica molding&#160;</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">1.5 mm biopsy puncher</td>
			<td align="left" style="width:320px;">Miltex (Pennsylvania, U.S.A.)</td>
			<td style="width:147px;">33-31AA/33-31A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Spin coater&#160; WS-400 BZ-6NPP/LITE</td>
			<td align="left" style="width:320px;">Laurell (Pennsylvania, U.S.A.)</td>
			<td style="width:147px;">n. a.</td>
			<td align="left">used for adhesive bonding</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Syringe pump neMESYS</td>
			<td align="left" style="width:320px;">Cetoni (Korbussen, Germany)</td>
			<td style="width:147px;">n. a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">1 mL syringe&#160;</td>
			<td align="left">Codan (Lensahn, Germany)&#160;</td>
			<td style="width:147px;">62.1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">5 mL syringe&#160;</td>
			<td align="left">B. Braun (Melsungen, Germany)&#160;</td>
			<td style="width:147px;">4606051V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">PTFE tubing&#160;</td>
			<td align="left">PKM SA (Lyss, Switzerland)&#160;</td>
			<td style="width:147px;">PTFE-AWG-TFT20.N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Quadrupole-based ICPMS ELAN6000</td>
			<td align="left">PerkinElmer (Massachusetts, U.S.A.)&#160;</td>
			<td>n. a.&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Membrane desolvator CETAC6000AT+</td>
			<td align="left">CETAC Technologies (Nebraska, U.S.A.)&#160;</td>
			<td style="width:147px;">n. a.</td>
			<td align="left">only the desolvator unit is used</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">High speed camera Miro M110</td>
			<td align="left">Vision Research (New Jersey, U.S.A.)</td>
			<td style="width:147px;">n. a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Data analysis program Origin pro</td>
			<td align="left" style="width:320px;">OriginLab Corp. (Massachusetts, U.S.A.)</td>
			<td align="left" style="width:147px;">version 8.6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:269px;">Microscope</td>
			<td align="left" style="width:320px;">Olympus (Tokyo, Japan)</td>
			<td align="left" style="width:147px;">IX71</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The presented system can be employed to measure small volumes of solutions or suspensions containing cells or nanoparticles. Examples of a measurement of a standard solution and characterization of single cells are shown here. More examples can be found in Verboket et al.22.
Typically the signal of a single droplet of a solution is a very short event. It usually lasts for a few 100 &#181;sec26. With the ICPMS used here (dwell time 10 msec) short signals like thes...

Watch this Scientific Journal Video about A Microfluidic Chip for ICPMS Sample Introduction at JoVE.com

A Microfluidic Chip for ICPMS Sample Introduction

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 ...

a-microfluidic-chip-for-icpms-sample-introduction

Research

JoVE Journal

Bioengineering

11.1K Views.  ETH Zurich.  نقدم نظام إدخال العينة قطيرة منفصلة عن بالحث البلازما الطيفي (ICPMS). لأنه يقوم على رقاقة ميكروفلويديك رخيصة ويمكن التخلص منها الذي يولد قطرات monodisperse غاية في نطاق حجم 40-60 ميكرومتر على ترددات من 90 إلى 7،000 هرتز. 

Video: A Microfluidic Chip for ICPMS Sample Introduction

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 ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

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.

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 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 ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...