Centre National de la Recherche Scientifique
Name: Christophe Marquette
Title: Permanent researcher, Centre National de la Recherche Scientifique
Professional background: 1999-present, permanent researcher, Laboratoire de Génie Enzymatique et Biomoléculaire, Centre National de la Recherche Scientifique, Lyon, France.
Education: 1999 — doctorat de spécialité, biochemistry, Université Claude Bernard-Lyon, Lyon, France.
Beneath every quality array-based assay there lies superior surface chemistry, and behind all good surface chemistries are teams of researchers scattered around the globe whose objective often boils down to throwing biology at a surface to see what sticks.
Christophe Marquette's lab at France's state-funded Centre National de la Recherche Scientifique in Lyon is devoted solely to developing better surface chemistries for proteins arrayed on artificial substrates.
CNRS’s research is directly linked to R&D groups at bioMérieux and Siemens, two companies that in the long run could profit from Marquette's discoveries.
In its most recent paper, Marquette's group took a swing at tackling one of the central problems with surfaces coated with the poly dimethyl siloxane Sylgard 184. While Sylgard 184 is a popular coating for array surfaces and microfluidic chips because of its low curing temperature and optical transparency, its hydrophobic qualities often interfere with the outcome of biological assays. [Heyries K, et al. Straightforward Protein Immobilization on Sylgard 184 PDMS Microarray Surface. Langmuir. 2007 Apr 10;23(8):4523-7].
To this end, Marquette's group set out to find out what was deactivating proteins immobilized on Sylgard 184, and developed methods for directly immobilizing the proteins without the need for a second immobilizing material, which could have a negative impact on the quality of the assay.
To learn more about the protein array work, BioArray News spoke with Marquette this week.
In your experience why is Sylgard 184 a popular material for use in microarray surfaces? Why are researchers using this?
First, it's very useful for microfluidics devices. So they can do very fast processing of channels and valves. So basically you can do it in any lab just by curing it at 100 degrees Celsius. It's a liquid, so you have to cure it until it solidifies. Then you can make it into a polymer and use it for microfluidic devices. The best part is that it is optically transparent and chemically inert. So all the biologists love this compound because they can do all the optical mirroring they want with it without having problems with interference, et cetera.
Secondly, you can put features on it with a very high length–to-height ratio. You can mold some very small features, and that's what we used for the paper. We molded … proteins. So now that is a very interesting part of this polymer: You can mold very small features.
What are some of the properties that have held back the ability to immobilize features on surfaces comprising Sylgard 184?
There are some problems with its inertness. That's the main topic of our paper because we actually put some proteins in the PMS because it inerts chemically. In the past, you had to modify strongly the surfaces because it’s very basic. With this new concept of transferring the features or proteins directly, you have no modification of PMS to do before immobilizing the proteins.
So, we actually use the molding part instead of trying to make chemistry on the surface. That's the 'new part' of our paper.
If you treat or modify the surface do you think it has an impact on the results of your assay?
Yes. Let's say you want to immobilize different proteins on the surface, and the proteins are different from one another. So if you make the same chemistry to immobilize the proteins, maybe the second protein will be oddly treated and will lose its activity, while the first one will remain active. So the problem is that when you treat the surface trying to immobilize two or three different proteins, if you do the same process, and use the same chemistry for all the proteins, you have a problem with maintaining activity.
Usually when you treat surfaces like PMS with something like plasma or whatever oxidation of the surface to make strong chemistry on the proteins, you lose activity and then you lose something in the assay, so it's always best to keep the proteins as natural as possible. There is definitely an impact of the chemistry of the surface on the assay results.
Why are you assigned to overcome this obstacle? Is this part of your personal research or is the institute involved in this?
Both, actually. The institute is more focused on biomembranes — enzymes and proteins on membranes. There is only a small lab, which is mine, working on artificial membranes. We study the immobilization of proteins on artificial supports and this is used to aid in diagnostic assays and stuff like this. We are working with other artificial surface materials, as well, like gold. So we work with diagnostic companies in France like bioMérieux and companies that want to find good immobilization procedures and new surfaces to immobilize proteins.
They are very interested in surface chemistry. BioMérieux in France and Siemens in Germany, who we also cooperate with, are very interested in immobilizing proteins because they are very interested in the diagnostic area, but they currently use only uni-parameter features and they want to extend their know-how to multi-parameter features. They don’t want to do the research so they work with us. bioMérieux is located near Lyon, so we are pretty tight with them and we are working together.
Maybe you can explain the experiment you used to determine what has been keeping features from being immobilized on surfaces that comprise Sylgard 184.
So what we made basically is immobilized proteins on this particular surface and looked to see if these proteins are still active, because that is the main objective for a diagnostic test on this surface. We looked at the proteins in terms of binding specific antibodies from human sera. So in the paper the proteins are allergens, and we looked at the antibodies against allergens. If you have sera from a patient that is allergic to peanuts, let's say, you can have a positive answer. So we look at the answer from the protein on this surface. This is made by imaging the surface. We used a luminescence interaction between the protein on the surface and the antibody from the sera.
Specifically, about the immobilization what is new is that we took the chance to dry the protein and to cure the proteins at 100 degrees, but the proteins we used kept their activity. This has been the main problem we have overcome — drying and heating the proteins and keeping them active. That is the bottleneck we have gotten through in the lab.
What is the next phase of your work?
Well, our main objective is to show that all of the proteins we test are maintaining their activity, so I think we have done 20 or 25 proteins now, and all of them were done on this surface. We keep trying different proteins because the main objective of the lab is finding a surface that works for all proteins that you can use.
The more interesting part is because you can do something with the materials, we are dealing with making fluidics, making small channels inside PMS, inside which we put the proteins. You can then flow fluid for a test over proteins within the same materials.
That was a driving force from the beginning. Using the Sylgard because it’s used in microfluidics, and trying to put the proteins on the surface. And then in one step you have protein immobilization and then the formation of the microfluidics part. That is the main step for us now.