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A new biosensor developed at Purdue University can measure whether neurons are performing correctly when communicating with each other, giving researchers a tool to test the effectiveness of new epilepsy or seizure treatments.
Marshall Porterfield, an associate professor of agricultural and biological engineering and biomedical engineering, postdoctoral researcher Eric McLamore, and graduate student Subhashree Mohanty developed the self-referencing glutamate biosensor to measure real-time glutamate flux of neural cells in a living organism. The nanosensor not only measures glutamate around neural cells, it can tell how those cells are releasing or taking up glutamate, a key to those cells' health and activity.
"Before we did this, people were only getting at glutamate indirectly or through huge, invasive probes," said Porterfield, whose research was published in the early online version of the Journal of Neuroscience Methods. "With this sensor, we can 'listen' to glutamate signaling from the cells."
The firing of neurons is involved in every action or movement in a human body. Neurons work electrically, but ultimately communicate with each other through chemical neurotransmitters such as glutamate. One neuron will release glutamate to convey information to the next neuron's cell receptors.
Once the message is delivered, neurons are supposed to reabsorb or clear out the glutamate signal. It is believed that when neurons release too much or too little glutamate and are not able to clear it properly, people are prone to neurological diseases.
A Self-Referencing Glutamate Biosensor for Measuring Real-Time Neuronal Glutamate Flux
E.S. McLamore, S. Mohanty, J. Shi, J. Claussen, J.L. Rickus, S.S. Jedlicka, D.M. Porterfield
Quantification of neurotransmitter transport dynamics is hindered by a lack of sufficient tools to directly monitor bioactive flux under physiological conditions. Traditional techniques for studying neurotransmitter release/uptake require inferences from non-selective electrical recordings, are invasive/destructive, and/or suffer from poor temporal resolution. Recent advances in electrochemical biosensors have enhanced in vitro and in vivo detection of neurotransmitter concentration under physiological/pathophysiological conditions. The use of enzymatic biosensors with performance enhancing materials (e.g., carbon nanotubes) has been a major focus for many of these advances. However, these techniques are not used as mainstream neuroscience research tools, due to relatively low sensitivity, excessive drift/noise, low signal-to-noise ratio, and inability to quantify rapid neurochemical kinetics during synaptic transmission. A sensing technique known as self-referencing overcomes many of these problems, and allows non-invasive quantification of biophysical transport. This work presents a self-referencing CNT modified glutamate oxidase biosensor for monitoring glutamate flux near neural/neuronal cells. Concentration of basal glutamate was similar to other in vivo and in vitro measurements. The biosensor was used in self-referencing (oscillating) mode to measure net glutamate flux near neural cells during electrical stimulation. Prior to stimulation, the average in?ux was 33.9 � 6.4 fmol cm-2s-1). Glutamate efflux took place immediately following stimulation, and was always followed by uptake in the 50-150 fmol cm-2s-1 range. Uptake was inhibited using threo-▀-benzyloxyaspartate, and average surface flux in replicate cells (1.1 � 7.4 fmol cm-2s-1) was significantly lower than uninhibited cells. The technique is extremely valuable for studying neuropathological conditions related to neurotransmission under dynamic physiological conditions.
Jenna Rickus, an associate professor of agricultural and biological engineering and biomedical engineering who oversaw the study's neurological aspects, said researchers need more information about how neurons work to create more effective treatments for neurological disorders.
"Understanding neurotransmitter dynamics has implications for almost all normal and pathological brain function," Rickus said. "The reason we don't have good information is because we haven't had a good measurement tool before."
Porterfield and McLamore's sensor exploits conductive carbon nanotubes and is only 2 micrometers in diameter, or about 50 times smaller than the diameter of a human hair. They also use an enzyme, called glutamate oxidase, on the end of the probe that reacts with glutamate to create hydrogen peroxide. The carbon nanotubes enhance the conductivity of the hydrogen peroxide, and a computer can calculate the movement of glutamate relative to the cell surface.
The sensor oscillates and samples the concentration activities of glutamate at various positions relative to the neurons in culture. Those measurements at different distances can tell researchers whether the glutamate is flowing back toward the neurons or dissipating in many directions.
Current sensor technology allows for sensing in vitro, but those probes are large and invasive, Porterfield said, and they don't measure the movement of the chemicals.
McLamore said the sensor also is valuable because it is able to hone in on only glutamate using just one probe and custom software that filters out variations in the signals that are read, which removes signal noise due to other compounds.
"There are many compounds present near the neurons which can potentially create noise, but this sensor should be selective for one compound. We filter out all of the background noise," McLamore said. "It's the same thing modern hearing aids do. They're filtering out ambient noises, and that's the same thing you get when you oscillate a biosensor."
The sensor also could be adapted to measure other chemicals by changing the enzyme used on its tip.
Rickus said the sensor's versatility would be valuable for understanding the effects of therapies for epilepsy, Parkinson's disease, damage caused by chemotherapy, memory loss and many other conditions. The sensor will give valuable data on how damaged neurons function and how drugs or therapies affect those cells.
Porterfield said the next step is to make small improvements to the sensor and adapt it to use other enzymes. The Office of Naval Research funded the research.
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Writer: Brian Wallheimer,
Sources: Marshall Porterfield,
Jenna Rickus, 765-494-1197,
Eric McLamore, 806-239-9556,
Ag Communications: (765) 494-8415;
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