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Bedside Dx in the Future for MIT’s Protein Screening Method?


Daniel Pregibon
PhD candidate
Massachusetts Institute of Technology
Name: Daniel Pregibon
Position: PhD candidate, chemical engineering, Massachusetts Institute of Technology, 2003 to present.
Background: BS, chemical engineering, Case Western University, 2003

Daniel Pregibon, a PhD candidate at MIT, Patrick Boyle, a professor of chemical engineering at MIT, and Mehmet Toner, a professor at Harvard Medical School, have developed a method to screen for biomolecules, including proteins, DNA, and RNA.
Their device and method is based on particles equipped with a bar-coded ID and one or more probe regions that fluoresce when they detect specific targets. They say that their work is applicable for disease monitoring, drug discovery, and genetic profiling.
A paper on their work will appear March 9 in the online edition of Science. Pregibon is the lead author of the paper.
Below is an edited version of a conversation ProteoMonitor had with Pregibon this week.
Describe the work that you did and what you found.
We’ve developed a method to test for multiple analytes in a single sample, and the way we’re doing this is we’re using multifunctional porous particles, so the way we make these particles is we do it in a microfluidic device, we flow two monomer streams, or more monomer streams down this device. And then we can UV polymerize particles out of those streams.
And now particles have multiple distinct regions. One region has a bar code written on there and that kind of identifies what specific targets that certain particle is looking for. And then in the other sections of the particle, we can actually have a molecular probe that specifically binds to a certain target. We can have that probe loaded in that region. So what we have when we’re done, we can create a library of particles each of which has a different bar code and is looking for a different target in a solution.
So what we do, we can mix up this library of particles, we can put them in a sample, and then have the specific targets bind to those particle that have their corresponding probes. Then we can actually detect this interaction via fluorescence. What we do now, we put these particles in a scanning device, and we can scan each particle, read off the bar code, and then determine if that corresponding target has bound or not.
This is kind of our method for doing the multiplexed detection.
Are these targets determined ahead of time? Do you decide what you’re looking for specifically, or is it more open-ended?
As a proof of principle, we were looking for specific DNA oligomers, so DNA with specific sequences. And so what we do is made particles where the probe is a DNA sequence and the target is actually the complementary strand. That was our proof of principle. We’re very confident that we can apply the same kind of technology to actually distinguish proteins, to detect RNA and that sort of thing, cytokines, and other various biomolecules.
These particles you describe, how do they work?
The way we make the particles, we use monomer streams. It’s a liquid monomer. We mix that with a photo-initiator, photo-sensitive species that will initiate a reaction in the monomer. And we can also load a given monomer stream with a fluorescent dye or a biomolecule.
We mixed up two monomer solutions, one which had a fluorescent dye and then one which had a specific DNA sequence. Then we flowed these two streams side by side. What we do is we have a microscope set up where we have a mask. It’s similar to a transparency mask or something you would use on an overhead projector. And we can shine a quick burst of ultraviolet light through that mask. What this does is set off a chemical reaction in the monomer stream to actually solidify the liquid wherever it’s hit with the UV light.
By doing this we can make very defined shapes that have two functionalities — one that’s fluorescent and one that’s loaded with this DNA probe. That’s how we make the particles. We make this is a batch process. We make a bunch of them and then collect them and rinse them. They’re immediately ready for use in a bioassay.
That’s different from other technologies because we’re actually making the particles. We’re encoding these particles with the barcode, and we’re functionalizing them with a DNA probe, all in a single step. That’s one of the things that makes our technology cost-effective, because the synthesis is so efficient.
How did you go about developing this method?
We actually developed a new process last year in our lab called continuous-flow lithography, and this was an enabling technology that allowed us a new way to make microparticles of various morphologies and various chemical entities. We based this technology off that technology. There were various certain things that were beneficial from that technology like the morphology. We had very, very precise control over the morphology of particles we make.
We could also make particles with multiple chemistries, so we exploited these two things for this multiplexing technique.
You said that you think this will be useable for protein detection and analysis. Will you be using conducting further studies using proteins?
We will be a lot of further studies. We’re determining exactly what we want to do now.
Will there need to be more work done on protein biomarkers before your method is fully functional for protein targets?
We don’t want to make much of a comment on that. We can say that other people have used similar polymers with proteins.
What about quantification? Will there need to be more work done on quantitative protein analysis or proteomics before your method can be adapted for protein work?
We do plan on doing a lot more work with proteins and quantification. We haven’t started on any of that yet.
What about the high dynamic range of protein found in fluids such as blood? How will your method be able to work around that?
We’re not sure. We haven’t looked too far into that yet. We’re still working on that.
Is the goal to develop this into a point-of-care, point-of-service diagnostic, or is this something that will be used in a laboratory setting?
We want to take it to the bedside eventually. Right now, we’ve only done proof-of-principle experiments in the lab. The way we envision this thing is we can make a device that can be placed at a bedside for a rapid and very cheap diagnostic.
How will you do that?
We’re still developing new ways of doing that and ways of making the device cheaper and pulling it away from the microscope. But I don’t want to make any further comment on that.
Are there any specific diseases that you’re developing this for?
Not right now. We’re using this as a very general platform technology, similar to the microarray, or what people at Luminex would do. We’re keeping it a very broad platform right now.
Why should protein researchers and proteomics researchers care about your technology?
I think our technology is going to be beneficial because we’re hoping to do very rapid, very inexpensive diagnostics, and this should be enabling for things like monitoring progression of diseases or doing very rapid, very cheap diagnostics at the bedside.

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