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Biochip Designed to Measure Calcium Flux in Plant Cells May Find Use in Ion Channel Drug Discovery


Marshall Porterfield
Associate professor, agricultural and biological engineering
Purdue University
Name: Marshall Porterfield
Position: Associate professor, agricultural and biological engineering; and co-director, Physiological Sensing Facility, Purdue University
Background: Assistant professor, biological sciences and electrical and computer engineering, University of Missouri-Rolla; postdoc, Woods Hole Marine Biology Lab; PhD, Louisiana State University

A biochip developed by researchers at Purdue University to help them measure how calcium ion currents in plant cells change in response to changes in gravity may be used in ion channel-based drug discovery, according to research published in the November issue of Sensors and Actuators.
Marshall Porterfield, an associate professor of agricultural and biological engineering at Purdue and lead author on the study, believes the MEMS-based lab-on-a-chip could eventually replace patch-clamping as the go-to method for cellular electrophysiology.
Porterfield took a few moments recently to discuss the technology with Cell-Based Assay News.
Tell me about the development of this biochip.
For years I’ve been using microelectrodes to scan and measure these calcium currents that enter in the bottom of the cell and come out the top. That calcium current basically points like a compass, and tells us which way is up and which way is down. That’s important in this instance in the development of this plant system so that it can orient itself to the environment.
But these mechanisms associated with cell electrophysiology and calcium current and cell polarity are the exact same mechanisms that are active in early embryo development, cellular development, neuronal polarity – every cell that develops and has any kind of polarity or directionality to its development uses these same types of mechanisms. It’s a fundamental physiological process.
When did you start considering applying this to ion channel drug discovery?
I knew early on that if it would work for our experimental system then eventually it would provide an advanced throughput format for doing any kind of electrophysiological experimentation, including pharmacological drug discovery. We already have plans in place now – we did calcium originally, but we’re looking at developing different specificity in terms of the membranes that we apply to the chip for sodium, potassium, chloride, and other ions of interest that would be targets for electrophysiological studies.
Can you explain the membrane component of these chips? Are these not whole-cell studies?
It’s a selective polymer membrane that we apply to the electrode surface to give it ion specificity. Then, the cells are actually integrated into that chip with the polymer membrane. That membrane is an ion-selective sensor membrane that is actually on the chips.
So you are measuring cellular ion flux; it’s just through this membrane?
Yes, the membrane gives the electrode specificity, in this case, for calcium only. That’s another main difference between this approach and traditional electrophysiology, such as single-electrode or chip-based patch clamping: Our system is ion-specific. We don’t have to do all kinds of ion replacement studies and ion channel blocker studies on top of the original experiment.
Another term one could use, instead of “ion-selective membrane,” is to say that there is an ion-selective modification or ion-selective chemistry on the surface of the electrode.
What is the cell interface with the membrane like? Do the cells rest at the bottom of wells in contact with these sensors?
For this study, we wanted to have the electrodes at the poles of the cell. That meant that the cell sits down in a pore, and the sensors are built into the side walls of the pore. But for other types of studies, we could simplify the design and just have the electrodes at the bottom of the pore, so the cells basically sit over an array of electrodes or sensors. That would simplify the device for cell electrophysiology for drug discovery, because in that instance you’re not really interested in polarity.
Your paper also mentioned that you can take multiple sensors to allow multiple simultaneous measurements from individual cells.
We collect signals from four sensors at a time in the current device. That means that we can make four different concentration measurements for each individual cell, with 16 cells on a chip. We can also operate sensors in another mode, which we call the dual electrode coupling method, which allows us to look at differentials between sensors. That’s how we’re measuring trans-cellular currents. There are different modalities you can operate the sensors in when you’re using them as potentiometric devices.
That sounds like it would have potential for studying neural transmission or neural networks in vitro.
Yes, instead of building pores to isolate the cells from one another, we could put sensor arrays down in channels, and encourage the cells to grow in channels and interact with each other. We could actually build a larger device where you might put down a whole tissue. We’re doing some work with a group here using the guinea pig nerve cord as a model for nerve cord damage. We’ve thought about putting the sensor arrays in a channel, putting the cord in there, damaging it in silico, and measuring the response of the nerve cord.
There has been a lot of interest recently in alternative methods to patch clamping for electrophysiology.
There was a huge amount of interest in developing patch on a chip. Everybody thought they could overcome all the limitations of this, and bypass the “arts and crafts” aspect of the user skills needed for patch clamping if you could automate it on a chip. It all ultimately still comes down to getting that one-gigaohm seal, which requires an expert, intelligent system that is really only a human being. The patch-on-a-chip systems – the companies say good things about them, but the people that actually use them are not that thrilled. If you can get around all that and get ion specificity to boot, you’re basically leap-frogging over patch clamping to a format that gives you more specific data, and is non-invasive.
Is there a prototype device already developed for drug discovery applications? Are you looking to commercialize this?
I am pursuing commercialization and intellectual property development with the Purdue Research Foundation, which handles all of that. Whether this would be a licensing situation or a start-up company is yet to be decided. I’m not sure what way and how that will progress.
Are you receiving external funding for this currently?
Yes, we had a NASA grant to do this. The fundamental question initially was how these cells sense and respond to gravity. If you look at a lot of bioMEMS development, people will build something, and demonstrate it using a generic platform to test a circuit or two on the device. But we actually built an entire data-acquisition system to interface with the chip, so that we could use the whole chip and do real experiments with it. We actually flew this on a flight experiment with NASA last April. We’re currently working on publishing the scientific results of those studies. The first paper we put out was about the technology and the engineering that went in to building the device.
Are you working with anybody on the drug discovery applications for the technology?
We haven’t really started that phase yet.
What applications are you interested in pursuing first and foremost?
One thing is to take what we have now, modify the geometry of the arrays of electrodes on the chip, and change the ion selectivity, so you can do different types of electrophysiological studies on nerve cells and tissues; for potassium and ammonia; even some heavy metals such as cadmium and lead, using different types of ionophores.
Another thing we are looking at is making this into a multi-analyte detection device, where it doesn’t even have pores on it, but arrays of sensors with different selectivity, where you could put different drops of fluids on it and see what the ion composition is. This could be used for urinalysis or blood chemistry testing. If you really wanted to take that further, we have another version of the chip in progress that would allow us to do amperometry. With that we could do things like oxygen, nitrogen oxide, ascorbate, biosensors for things like glucose, lactase, ethanol, glutamate, et cetera. Then you’d have specialized chips to use for either research applications in those areas, or more medical applications.

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