Hello, my name is Aida Pria and I just completed my PhD from Dr.Katherine KLAS lab here at bu. And today I'm going to demonstrate the fabrication of a thermoplastic microfluidic device using micro hot embossing and thermal bonding. And then I'm going to demonstrate the isolation of nucleic acids using a solid face containing silica particles, which and the extracted nucleic acids can then be used for PCR amplification detection or any other diagnostic module.
Hello, my name is Dominika Kalinski and I'm a graduate student and the biomedical micro devices and Microenvironments Laboratory at Boston University. I will be talking about the formation of a carbon nano tube lysis monolith, and a microfluidic device and the formation of a solid phase extraction monolith for DNA extraction and a microfluidic device. Today we are going to demonstrate an experiment on the production and application of microscale lysis columns and solid face extraction columns within thermoplastic microfluidic channels.
These columns can be used for bacterial cell lysis and isolation of nucleic acids from the cell lysate. The first step in the process is the fabrication of the microfluidic devices. In cyclic polyolefin, we use a polymer, which comes under the trade name xx XX six 90 R has a Gloster edition temperature of 136 degrees Celsius and is UV transparent.
UV transparency is very essential for the light directed chemistry used in the fabrication of the porous monolith within the microfluidic channels. This microfluidic platform is fabricated using micro hot embossing and thermal bonding. At first, I'm going to demonstrate the micro hot embossing process.
So we start with a master mold, which is a nickel cobalt electro formed mold, and it has the negative or the inverse features of what's required on the microfluidic platform. So we put the Zion Nex palettes on top of the features that we require on the on the plastic substrate. The mold is then placed in a hot press, which is heated up to 30 degrees above the GLO edition temperature of the polymer.
This is then covered with another metal plate and then pressed to about anything between two 50 to 500 PSI is good. After five minutes, we can release the pressure and remove the PLAs. We let this cool down a little bit and then the two plates are manually separated and the mold is released from the substrate.
This is how the the substrate looks. After it is released from the mold, we drill 1.5 millimeter holes at the ends of the channel to serve as introduction and collection wells. And this embossed substrate is then thermal bonded with another plain piece of plastic to form and close microfluidic features.
Both the pieces are pasted on the hot plate like this. The temperature is reduced to the glass tion temperature of the polymer, which is 136 Celsius. For the thermal bonding process, again, the the metal plates are placed in the hot press, which is heated to the glass transition temperature that is 136 degrees Celsius and press together at two 50 PSI for two minutes.
At the glass transition temperature, the polymer starts to melt and fuse together, and this is how we get enclosed microfluidic features. After two minutes, the pressure is released. This is how the final microfluidic device looks with the enclosed channels and the access ports for the sample introduction and collection, We're now going to prepare the micro channels for the porous polymer monolith formation.
The first step of this process is a grafting process. We change the surface chemistry of the micro channels and the Xan X six 90 R platform. This is necessary so that we can have covalent attachment of the porous polymer monolith within the microchannel.
This surface modification is a grafting process, which involves a one-to-one ratio of EDA to MMA with a photo initiator benzo pheno. So what I'm doing here is making a one-to-one concentration of MMA and EDA to the solution. I add 3%of benza pheno so that we can cross link this monolith within the channel.
Once the grafting solution has been mixed, we pipe that this into the micro channel and due to capillary action fills the channel. Once the channel is filled, we place this in a UV crosslinker and this will be crosslinked for 10 minutes with an energy level of 200 millijoules per centimeter squared. After the 10 minutes of the UV exposure, the grafting solution and the microchannel are rinsed with methanol, and this is used with vacuum aspiration.
So what I'll do here is place methanol at one end of the channel using a vacuum rinse the channel with methanol. This is to remove any excess solution that was not polymerized. This is a comparison of cyclo, ethanol and cyclin, all with the carbon nanotubes at a concentration of 2.27 molar.
The Cyclin all with carbon nanotubes will be ated for an even dispersion of carbon nanotubes within the solution. After the grafting process, the surface chemistry has been modified and is now ready for a firm attachment of the monolith within the microchannel. Now I'll describe the formation of the carbon nano tube VICIS monolith.
This is composed of genic, solvents and monomers. The four elements involved in this process are al to ecol, butyl methacrylate and EDMA. Additionally, we add a photo initiator so we can form the monolith in sotu using a UV polymerization.
I'll be making a hundred microliter sample. I'll pipette 52.5 microliters of ecol. This needs to be done slowly since ecol is viscous.
After the ecol, we'll add the carbon nanotubes with cyclo anol. This needs to be vortex vigorously for a few minutes to ensure proper dispersion. Again, we need to pipette the cyclo andal and carbon nanotubes slowly because this is Abicus solution.
Then we add 15 microliters of butyl meth Aly. And finally, we add 10 microliters of EDMA. We'll vortex this briefly, add our photo initiator and then sonicate this.
The photo initiator that we add is DM PAP to the lysis monolith. This is added at a 1.13 concentration to the entire volume of this a hundred microliter solution. After it is added, because this is a photo initiator, we want to cover our license monolith solution to ensure no exposure while we sonicate this.
This will beated for 30 minutes at a 50%amplitude with a 50%duty cycle. This 50%duty cycle is to avoid overheating the solution. It is extremely important to take the solution after sonication and quickly work.
This is because we want to ensure an even dispersion of the carbon nanotubes in the solution because we will be pipetting the solution into the microchannel. Here we have the lysis solution. Just after sonification and working very quickly, we pipette the volume into the micro channel using capillary action, it flows into the channel.
We place this into the crosslinker for 0.9 minutes at an energy level of 120 Millies per centimeter square. After this 0.9 minutes, we flip the device. So this flipping is essentially to allow for more even polymerization and for a more even distribution of carbon nanotubes in the channel.
Once the monolith has been polymerized and rinse with methanol, the monomer solution, the monolith inside will no longer be clear, but instead, a vivid white, there will be tiny specks of black, which will be the carbon nanotubes in the monolith. The solid phase extraction monolith is made in a similar manner to the carbon nanotube, lysis, monolith. This is the bacteria and liquid culture, and here we have a sample of our media.
Once we have diluted the sample, we'll pipette this into a vet and centrifuge it. We then decant the media being sure not to disturb the bacteria pellet at the side of the einor tube. Then the bacteria are resuspended in a solution with gudo, dium, thio, SDS, and protease K.The gu diem thiocyanate that is pipetted is in the same volume that the bacteria was suspended in.
Here, the bacteria was suspended in one mil, so I will resuspend the bacteria in one mil of gguumm thiocyanate to the gu thiocyanate. I'll add proteinase K.I'm adding 45 microliters and RNAs. The last solution that I'm adding is SDS.
After this, the solution is vortex. After vortexing, we'll pipette the bacteria sample into a disposable syringe. Here we're using a three mil Excel disposable syringe.
This has a lure fitting, which connects to peak tubing, and we will connect to our device via nano ports. So I will pipette the solution into the disposable syringe. We want to aspirate to make sure that there's no air in the system.
You want to do this until you get one drop or two coming out of the other end. Now that we have our solution ready for connection to our device, we'll place our disposable syringe on our KDS syringe pump and we'll connect the end of our fitting to our license device, which has nano ports that we're eped on each end. This allows for a firm connection to the device.
These are screw fittings, and usually we like to tape the device down to make sure it does not flip. Here we will set the flow rate for four or five mils per hour. This is a displacement pump, which will force the solution through the syringe, through the tubing into this connection through the channel.
And we'll collect the sample through this exit nano port. We can collect the pollutant using a pipette, and this pollutant will be run through the next phase, the solid phase extraction process to isolate the bacterial DNA For the isolation of the nucleic acids. The solid phase containing the silica particles is conditioned with guad theo signate containing lysis buffer.
This is done by filling a syringe with the lysis buffer and attaching it to the syringe pump, and the buffer is flowed through the channel at a flow rate of 300 microliters per hour. This is followed by the loading of the sample. The sample here is the bacterial cell lysate that we obtained from the previous lysis step.
Again, the load, this sample is loaded for three minutes at a flow rate of 300 microliters per hour. After this, the channel is washed with 70%at ethanol, again at a flow rate of 300 microliters per hour. This wash step takes out all the absorb protein and contaminants, and the waste is collected from the collection.
Well using a pipette after the wash step, which removes all the contaminants. The DNA that was bound to the silica is then eluted in water. So this is a syringe containing really pour water.
It is attached to the to the chip and, and again, the water is flowed at a flow rate of 300 microliters per hour, and we collect the sample at the other end. This is done for three minutes, and we collect 15 microliter of ient, which is just pure DNA in water, and is PCR ready for the fabrication of the thermoplastic device. The biggest challenge is the thermal bonding aspect because the bonding is done at a very high temperature, at or near the glass transition temperature of the polymer with the application of some pressure and slight variation of the pressure or the temperature can completely obliterate the channel or create a poor seal between the two plastic parts.
So maintaining the optimum temperature and pressure is very important and it can be a little challenging. The key elements that are challenging in this process, we're obtaining an even distribution of carbon nanotubes in the monomer solution that is in situ UV polymerized. This is also a similar challenge to an even dispersion of the silica particles in the porous polymer monolith for solid phase extraction.
