微流控一次性诊断的应用

My name is Catherine CloudBridge and I'm a professor in manufacturing, engineering and biomedical engineering at Boston University. And I'm the director of the Biomedical Micro Devices and Microenvironments Laboratory. We work primarily on making disposable diagnostics for applications in resource limited settings.

So we use microfluidics in order to make those devices. We're interested in actually two fields of study. Primarily we're interested in looking at how biomolecules and cells interact with plastics polymer surfaces.

That's our major umbrella. That work is embodied in microfluidic applications for disposable diagnostics. So in the laboratory, what we make are microfluidic devices.

In thermoplastic materials we don't use, typically use PDMS, which is the rubbery material that most people use to make microfluidics in the laboratory. We make our devices out of thermoplastics. We're interested in using those thermoplastic devices to do molecular biology at the microscale in such a way that that molecular biology could be done in the field far away from a centralized laboratory.

How that works out in, in the lab is we we're making little plastic cards and inside of those little plastic cards are solid phase extraction columns or cell lysis columns or mixing columns. We also do polymerase chain reaction on the chip as a detection technique. All of these things are integrated into a small device that eventually, in the long term outlook of the research would end up being an integrated device that would be able to be handheld and carried out into the field or into settings where a large scale laboratory with all the attending center fusions and heating incubators and so forth would not be available.

So we'd like to be able to ideally start with a human sample like blood urine stool or a nasopharyngeal swab, for example, which which would be mucus. And then at the output of the chip have an answer whether or not yes or no, someone's infected with a particular microorganism. And the way that we do that is by looking for the DNA of that particular or RNA, depending on the microorganism of that particular microorganism in the patient's sample.

So the technical challenges that arrive are largely in the sample preparation side of that equation. So when you're starting with blood or stool or urine, you have to get to from a situation where you have a messy human sample with a lot of potentially particulate in it or inhibitors to PCR or other amplification techniques. And you wanna end up the end with a very clean eluded sample of nucleic acids that you can then use to amplify or look for a particular DNA or RNA that will tell you whether a a microorganism that you're looking for was there.

So in order to clean up those samples, you have to do a few things. First, you have to filter the sample, which depending on the sample, can involve course filtration or a quick spin step before the sample goes on the chip. So we always try to start with samples as they come from the clinician.

So with urine and stool and nasopharyngeal swabs, our goal is to just have the clinician do their job like they normally would and take the sample like they normally would. And then we are take that sample and put it onto the chips. So then if any more filtration needs to happen at that step, that filtration would happen in the chip.

The second step involves, because we're doing molecular assays, we have to lyse the cells and the microorganisms that are in that sample. So we then perform a lysis step, which usually involves forcing the sample through a filter that has a small pore size. Sometimes that filter has also in it nanoparticles like carbon nanotubes, which have sharp edges, which can be used to rip apart some of the organisms that have more robust cell walls like C difficile, which is a gram-positive bacteria that we work with, which is very difficult to lys.

So we have to have more robust lysis columns. But typically we're doing a combined chemical and mechanical lysis inside of the chip. After that, our goal is to take the lysate and then extract the purified nucleic acids.

So we pass the lysis over, we pass the lysate over a solid phase extraction column that's resonant in the microfluidic chip. And that solid phase extraction column is made out of plastic and it's made using photo initiated chemistry inside of the microfluidic channel after it's already fabricated. And it's also impregnated with usually silica particles because we want to bind and release nucleic acids.

In some cases, we've incorporated oligo DT beads if we want to bind and release mRNA specifically, for example, or beads that have particular antibodies on them, if we wanna bind and release particular proteins. And we can make extraction columns that do all of those things. But today we'll show you how to do make the sase extraction column with the silica beads.

So after we've done that, pass the lysate over the silica column, it works just like a kyogen kit would work in the macro scale laboratory. We then wash to get rid of all the other things that we don't want, all the carbohydrates and the lipids and other stuff in the cell lysate and in the rest of the human sample that we don't want. And then the very end, we're alluding with water.

And what's nice about these microscale solid phase extraction columns is that we can elute with a very small amount of water, usually on the order of one to five microliters. And we're getting back almost all of the nucleic acids that we put in. We have about a 70 to 90%efficiency rate and the first 10 microliters that we get back off of that channel.

So if we wash through twice with two to 10 microliters, we get back almost all the nucleic acids that we put there. So it's not only a clean sample that's PCR able, it's also a sample that's much more highly concentrated than you would be able to get with a standard bench top kit. So the end goal of all of these sample preparation techniques is to make things that are compatible with some of the other work that people have done.

Very nice work in the field showing that you can do PCR amplification on a chip. You can do the reverse transcriptase reaction on a chip if you're dealing with an RNA virus or if you wanna do a gene expression experiment. Our sample preparation techniques can be directly coupled with PCR on a chip, so you wouldn't have to do any of that cleanup and sample preparation at the bench.

And, and, and also the concentration step that you would need to do in order to get the tiny volumes necessary to do multiplexed PCR on a small chip. So the experiments that we'll show you today are showing how we assemble the microfluidic chip, which is made out of thermoplastic and not PDMS. So it requires a little bit different fabrication process than is typically used, how those chips are sealed.

And then how we do the light initiated chemistry inside of the chip to make the polymer monoliths that are used for both cell lysis and for solid phase extraction. And at the end of that process, which right now are modular processes in the lab, but they can and have been integrated, give you at the end a highly concentrated sample of nucleic acids, RNA and DNA in water, that can then go downstream to a PCR module or another amplification module where identification of a microorganism or an infection can occur. So we actually have a grant that we're working on right now where we've got, we're beginning to collect human samples from Boston Medical Center from patients that may or may not be infected with Influenza A.So since it's flu season, those are the samples that we're collecting.

And then in the laboratory, we're doing the gold standard assays for looking at whether or not someone's infected with Influenza A, which is basically a culture assay, which takes several days to carry out. And side by side with that, we're putting these samples through our, right now, the modules of our chip. So we put them through the lysis column, we put them through the solid phase extraction column, and then we perform the PCR to see how well we do in comparison with the gold standard methods.

So that's the stage we're at right now. If those experiments pan out, I think over the next, you know, four to six years maybe we'll be in a situation where we have an integrated chip and a complete assay for influenza A and also we're working on clostridium difficile and stool samples. And we're, we're, we're also very close to having not only the device integrated, but also the, the assay integrated.

So both the assay development and the chip design have to happen hand in hand. So for a particular application, getting to the bedside, it's, it's really a target oriented process. You have to know, you know, the sample that you're starting with and the microorganism that you're looking for in order to optimize the chip for a bedside use.

So I, I don't think we're that far away from making that happen in a prototype manner. Those are our end goals, so I hope we get there.