A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

The overall goal of this procedure is to perform automated small volume particle separations using a microfluidic photic device. This method can enable key procedures in the biomedical engineering field, including robust processing and separation of samples for bio detection and clinical research applications. The main advantages of this technique are modularity, precision, robustness, and automation, making it adaptable and flexible not only for retic separation, but wide variety of flow through microfluidic separation devices.

We first recognized the need for this capability to routinely and reliably run separations on sub-millimeter biological sample volumes without significant dilution or loss of analyte. The integration of sample metering and automated routing from source to collection files together with standard syringe pump operations is critical for working successfully at this scale. To begin design the acoustic retic device and the layout for the two photo masks as described in section one of the accompanying text protocol.

Then pattern the backside fluid ports onto a double side polished 1 0 0 silicon wafer. Using standard positive resist photolithography Etch this geometry using deep reactive ion etching to a depth of 350 to 400 micrometers. Next, turn the wafer over and pattern the front side fluid channel geometry.

Again, using standard positive resist photolithography, then mount the patterned wafer to a second blank silicon carrier wafer. Using photo resist now etch the exposed channels to a depth of 200 micrometers. Using deep react ion etching, then soak the device wafer in a resist stripping solution to det it from the blank silicon wafer.

Next, clean the device wafer and a featureless 0.5 millimeter thick boro silicate glass wafer using piranha solution. Once clean seal the fluid channels ionically bonding the glass and silicon wafer using the conditions shown here. Finally, cut the individual chips apart with a diamond blade on a dicing saw from a two component low viscosity epoxy kit.

Weigh out the recommended ratio of both components and mix them thoroughly. Then place the lead Zirkin eight titanate or PCT piezo ceramic in a suitable jig and use a pipette to spread approximately 10 microliters of the epoxy mixture evenly across it to create a thin even layer. Next, place the chip into the jig and align the epoxy side of the pizo ceramic with the microfluidic chip.

It may be necessary to tack the chip down with tape to hold it in place while aligning. Clamp the assembly in a vice taking care not to crack any of the components and cure the temperature and duration that are recommended by the epoxy manufacturer. After the epoxy has cured, use a fine tip soldering iron to attach fine gauge wire leads to each side of the pizo ceramic.

If necessary, use the appropriate flux to prepare the pizo surface. Take care to make the briefest possible contact with the pizo To avoid thermally depolarizing it. Attach the chip to a fluidic breadboard using clamping fixtures.

Also, attach a cooling fan to the breadboard to regulate temperature during acoustic experiments. Next, screw in the chip to world connectors. Mount the breadboard onto the microscope stage and join the world to chip connectors to additional tubing at the inlets and outlets.

Using standard quarter 28 threaded unions. For one 16th inch tubing, connect the microfluidic chip to the syringe pump, computer controlled multiport selection valves, PC interfaced flow meters and tubing as shown here and described in figure four of the accompanying text protocol. Prior to running the separation, fill the cleaning reagent reservoirs and prime their corresponding fluid lines.

Also set valves three and four at the chip outlets to initially flow to the waste reservoirs. Next, turn on the cooling fan. Connect the pizo ceramic leads to the power amplifier and set the function generator at the resonant frequency for the acoustic chip that is being used.

Adjust the voltage set point on the function generator so that the RF amplifier outputs between 12 and 25 volts peak to peak as appropriate for the desired separation. Then connect the appropriate collection vials and the buffer input vial to the system. Use the sample buffer suitable for the cells or particles to be separated or as required by the analysis assay to be used after separation.

Next, load the control software and use it to control the valves, flow sensors and pump to carry out the fully automatic prime and separation routine. Immediately before starting the separation procedure, vortex the sample vial briefly to resuspend any particles that may have settled. Alternatively, pipette the sample up and down 10 times to mix biological samples that are more susceptible to damage or clumping by vortexing.

Then attach the sample vial to the input line and initiate the prime and separation routine without delay. Start by using the software to prime the air inlet of valves one and two to ensure that the tubing contains no fluid. Simultaneously prime the tubing connecting the buffer input vial to valve two and the sample input vial to valve one.

Have the syringe pump withdraw approximately 15 microliters from both vials to completely fill the tubing, connecting them to the valves. Finally, switch valves one and two to waste to expel any excess fluid or air that has entered the loading coil. Next, set up the program to load the sample coil by first withdrawing 25 microliters of air at 50 microliters per minute.

Then loading 250 microliters of sample at 200 microliters per minute, followed by 35 microliters of leading buffer at 200 microliters per minute, and finally, another 25 microliters of air at 50 microliters per minute. Then set the syringe pumps to infuse the loaded fluid plugs at 100 microliters per minute and monitor the flow rates at the small and large particle outlets using flow sensors. The appropriate flow ratio can be determined as described in the accompanying text protocol.

When the flow sensors detect a spike in flow rate indicating passage of the first air gap, switch the corresponding output valve from the waste vial to a sample collection vial. Ensure that the flow is stable as the sample transits the chip and is separated by observing flow sensor readings. As the sample passes through the chip, the separated fractions are collected at the outlet valves.

At the end of the sample plug, observe the flow sensor, detect the second air gap. At this point, switch the output valves back to waste. After the full volume is dispensed, stop the syringe pump infusion and terminate the automation routine.

After the separation experiment is complete, disconnect the small and large particle sample collection vials and store them appropriately for subsequent analysis. Secure the sample pickup tube in empty vials to collect excess cleaning solutions that will be flushed through the tubes. Then start the automated system cleaning routine as described in the accompanying text protocol.

During this process, the program will control the valves and syringe pump to sequentially load the holding coil with cleaning reagents and flush them through the system. Once the automated cleaning and decontamination process completes discard the excess flushing solutions and the vials in which they collected by following appropriate procedures for handling biological or chemical waste. Shown here is a representative frequency scan from a well coupled pizo and microfluidic device.

When the coupling between the pizo and microfluidic device is good particles focus tightly at the resonant frequency, resulting in a clear peak in the fluorescent intensity and migration to the expected focusing position. In contrast, where there is poor coupling particles will not focus well and the device is unsuitable when high quality focusing or fast flow are critical for the required application. Each line on this graph represents separation results using various polystyrene particle sizes and pizo driving voltages.

In general, higher voltages are required to extract smaller particles. Dry voltages cannot be indefinitely increased, however, due to greater heat dissipation and increasing effects of acoustic streaming. To demonstrate the utility of this platform for biological particle separation, human raji cells with an average diameter of eight to 10 micrometers were spiked with dengue virus having an approximate diameter of 50 nanometers and then separated using the acoustic microfluidic device.

Once a system like this is assembled and programmed, separations can be routinely carried out in approximately five minutes. Around three samples per hour can be processed with complete cleaning and decontamination between samples. Following this procedure, other methods can be performed such as cell counting, western blotting or next generation sequencing in order to confirm the purity of the separation and elucidate biological significance.

After watching this video, you should have a good understanding of how to fabricate a typical microfluidic separation device. Use world to chip connections to interface with hardware and run an automated separation procedure in a precise and robust fashion. Don't forget that working with infectious materials can be extremely hazardous and must only be carried out by properly trained personnel using the appropriate institutionally prescribed equipment and procedures for biological safety.