A Microfluidic Chip for ICPMS Sample Introduction

The overall goal of this procedure is to generate and quantitatively analyze the elemental composition of mono dispersed micro droplets containing single cells or aqueous solutions by I-C-P-M-S. This is accomplished by first fabricating a microfluidic chip using replica molding with PDMS. The second step is to connect syringe pumps to the microfluidic chip.

The pumps will supply the aqueous sample and the highly volatile and admissible or organic carrier phase PFH. When the syringe pump is turned on, the microfluidic chip generates mono dispersed droplets of the aqueous sample solution or a cell suspension by flow, focusing with PFH and ejects these droplets with additional PFH from the chip as a liquid jet. The final step is to insert the chip into a transport assembly consisting of a cartridge heater that evaporates the droplets and a membrane de salvator that removes the solvents prior to the ionization and detection of the droplet content by an I-C-P-M-S.

Ultimately, the recorded signals and a calibration measurement with a standard solution are used to determine the elemental composition of the sample solution or the cells in the suspension. The main advantages of our microfluidic technique over existing methods are the adjustable droplet size, the low sample consumption, and the low risk of nologging when working with cell suspensions or concentrated salt solutions. Furthermore, the low cost disposable microfluidic chip avoids cleaning time and eliminates cross-contamination.

Demonstrating the procedure will be all vin sc, a PhD student from Professor GTAs Laboratory and Pascal Fabrica, a PhD student from my laboratory. To begin the MICROFABRICATION process, measure 40 grams of PDMS and four grams of PDMS curing agent in a large plastic dish. Mix both parts evenly with a spatula and place the dish inside a vacuum desiccate for 20 to 30 minutes to degas all bubbles from the mixture.

While degassing. Place a casting mold on top of a micro structured silicon wafer and use the guiding structure around the design to snap it into place on a flat silicon wafer. Place another casting mold on top freely.

Pour approximately three to four grams of the Degas PDMS mixture onto both casting molds. Transfer both silicon wafers onto a hot plate. Cure the PDMS at 150 degrees Celsius for six minutes, and then cool.

The solidified PDMS structures on the bench at room temperature with a metal spatula. Carefully separate the solid PDMS blocks from the molds. Protect the bonding surfaces of both blocks from particle and fingerprint contamination.

By applying a layer of clear scotch tape, the PDMS pieces can now be handled and trimmed to size with scissors. After trimming, remove the tape from the micro structured PDMS block and use a biopsy hole punch to create the microfluidic inlet ports. This completes the fabrication process for the micro structured half of the device, and it is now ready to be bonded and assembled with its flat counterpart.

To prepare the services for PDMS to PDMS bonding, begin by spin coating a layer of PDMS curing agent onto a blank silicon wafer at 6, 000 RPM for 30 seconds while the curing agent is still wet, bring the bonding interface of the micro structured PDMS block into contact with the wafer and gently apply pressure on top of the PDMS to form a seamless seal without bubbles. In a separate step, remove the protective tape from the flat counterpart. Then slowly separate the micro structured PDMS from the silicon wafer.

Carefully align the two PDMS pieces and bring the two bonding interfaces into contact. Gently squeeze the two blocks to remove any interfacial bubbles and cure the assembled PDMS chip for 24 hours At room temperature using a utility knife and an alignment device, make a cut along the indicator line orthogonal to the outlet nozzle. Inspect the chip under a microscope for trimming defects and particle contamination adjacent to the exposed outlet nozzle.

After assessing the fabrication quality, the internal surfaces of the PDMS microchannel are ready to be siloized. In a chemical hood connect one end of a wolf bottle to a dry nitrogen source and connect the other end of the bottle to the inlets of the PDMS chip. Using multiple tubes connected to a central manifold, then pipette 50 microliters of the seline into the wolf bottle and seal the top lid.

Turn on the nitrogen carrier stream and flush the micro channels with a saline containing vapor for 20 minutes at a flow rate of approximately one milliliter per second. Finally, disconnect the inlet tubes from the wolf bottle. The coated PDMS chips are ready for mass spectrometry experiments for single cell mass spectrometry measurements.

First, dilute the input sample with an appropriate buffer such that the effective cellular concentration is less than 10 million cells per milliliter In preparation for mass spectrometry, begin by attaching tubing to the sample syringes. Load two five milliliter syringes with the PFH and load the input sample in a separate one milliliter syringe. Remove all bubbles trapped within the syringes and tubing and load the two syringes onto a syringe pump.

Make sure the transport tube is aligned properly and all gas tubings are connected. Next, connect the syringe tubings to the inlet ports of the PDMS chip and activate the syringe pump for three to five minutes. During this flow stabilization process, remove any excess liquid from the outlet port with a tissue.

If the fluid exiting, the chip does not form a straight jet. After five minutes, try to wipe away any potential obstructions at the chip outlet with a clean tissue. If unsuccessful, discard the existing chip and replace the connection with a new chip.

Remove the plug from the cyclonic sprayer adapter and carefully insert the flow stabilized PDMS chip into the adapter. Change the flow rate according to the recommended settings, and restabilize the flow for two to five minutes. At the same time, adjust the flow rates for all gases until the maximum signal intensity of the analyte of interest is achieved.

Also, tune the plasma power and the focusing lens voltage on the mass spectrometer to further enhance the signal intensity upon signal optimization. Set the mass spectrometer to a dwell time of 10 milliseconds and begin data acquisition. After measurements, transfer the raw data to a data analysis program.

In a typical data analysis routine, all measurements are first separated into data bins with each bin representing the number of counts per 10 milliseconds of dwell time, then a histogram plot is generated for all bins and fitted with the Gaussian distribution function. The mean and spread of individual fits represent both the mean signal intensity and standard deviation at a particular mass to charge ratio. To begin the calibration process, first, measure a standard solution at the same flow rate as your sample.

Using the PDMS chip for droplet generation, set up two five milliliter syringes for PFH and one one milliliter syringe for the standard solution. Then using a Petri dish as a microscope specimen holder, place the PDMS chip in the Petri dish and bring the chip into focus under low magnification mirroring previous protocol steps. Stabilize the droplet generation within the PDMS microchannel for three to five minutes with a high speed camera.

Start recording images of aqueous droplet on chip at the secondary junction to obtain the average droplet diameter from the recordings. Use an image analysis software with a dedicated droplet morph, optometry and velo symmetry suite. Assuming each droplet can be modeled as a spherical object, the average droplet volume can be estimated by using the average droplet diameter obtained by the software.

Next, divide the known analyte concentration of the sample by the average droplet volume to obtain the mass of analyte contained in one droplet. Then convert the resulting mass into the number of atoms. Finally, calculate the overall detection efficiency by dividing the number of measured ions per droplet by the previous result.

This detection efficiency can now be used as a calibration quantity to ascertain the number of atoms in an unknown sample. Using a microfluidic droplet generator, single cells can be encapsulated within mono dispersed aqueous droplets at an average diameter of less than 50 microns, and analyzed by I-C-P-M-S. The organic PFH sheath surrounding the aqueous droplet can easily be removed prior to the sample reaching the I-C-P-M-S detector.

When measuring solutions, the high droplet mono dispersity can be observed as low variances among groups of signals coming from either a single double or a triple droplet counts within a given dwell time. After proper calibration, the iron content from an ensemble of single bovine red blood cell, each suspended in single droplets can be ascertained. After watching this video, you should have a good understanding of how to prepare and use these microfluidic chips for quantitative elemental analysis of very small sample volumes or single particles and cells.

The fabrication of these chips requires less than 15 minutes, hands on time per chip if it's performed properly well. Additionally, the easy to modify chips enables the integration of further microfluidic modules for advanced sample pretreatment and simultaneous introduction of samples and standards to improve efficiency and samples throughput.