Fabrication and Utilization of a Low Cost Nerve Stimulation Chamber for the Detection of ATP Using Luciferase Chemoluminescence

Alex Cox*, Joshua Balsam*†, Elizabeth Katz*, Daniel Weinreich*‡, Kim Sapsford** USFDA, Center for Devices and Radiological Health, Office of Science and Engineering Labs † University of Maryland, College Park ‡ University of Maryland, Baltimore

Neurostimulation has become an ever increasing area of research and is used to treat a multitude of medical conditions. Many devices in the market use neurostimulation to heal patients or ameliorate their symptoms. Histology has long been the “gold standard” for pre clinical demonstrations of the safety of peripheral neurostimulation devices. However, histology has a few major limitations, including high costs for veterinary care and difficulties in implanting “human-sized” devices into animal subjects.

One of these limitations is that histology does not address the fact that the stimulation of peripheral nerves causes the release of neuroactive substances such as acetlycholine and histamine. The neurotransmitters (NT) are released in minute quantities ( pico-nano molar). There are some biochemical tools that are used for detecting NT’s such as luciferase for ATP detection. Employing chemoluminescence methods for NT detection also has its drawbacks, including the high cost of the biochemicals and lab setup. From a purely economical perspective, the high cost of research can be restrictive.

We created an inexpensive nerve chamber out of polymethylmethacrylate (PMMA) to use instead of the expensive machined polycarbonate chambers often used in experimental setups. We wanted to test nerves for secreted adenosine triphosphate (ATP), and needed something that could be cheaply disposed if we had used toxic substances in it.

To test the PMMA chip we stimulated the nerves and evoked an ATP release, which is of particular importance as a marker for neuropathology. ATP receptors mediate excitatory neurotransmission; excessive ATP neurotransmission can result in cell death. By testing for this molecule we hoped to prove the lab on a chip nerve chamber worked, and to determine the amount of the ATP released from stimulated nerve. Our goal was to create a stimulation chamber where we can cheaply and accurately determine the amount of ATP released by the nerve while also reducing solution waste.

1. By employing the laser cut nerve chambers we are able to create an experimentally viable stimulation chamber in which we may test the nerve for chemical secretions and determine the neurotoxicity of various substances on peripheral neurophysiology.

2. The chamber is cheap and easy to create, which enables us to make a chamber to accommodate a specific type of nerve, and to easily and cost effectively create many prototypes to ascertain which configuration is best for our purposes.

3. If we are working with neurotoxins that might be difficult to completely clean out of the chamber we are able to simply throw away the chamber as toxic waste without needing to worry about the expenses and difficulties that could otherwise be involved in getting a new chamber.

This project was funded in part by the FDA Critical Path initiative.

ATP Detection

Nerve Stimulation ResultsMethodsAbstract

Conclusions

The interaction between ATP, O2 , Mg, luciferin, and luciferase ultimately gives off light. The amount of light given off can be detected and used to determine how much ATP was used in the reaction to produce that light. The ATP degradation enzyme, apyrase, can be used to demonstrate that an observed luminescence signal is ATP dependent., and therefore, not an artifact.

ATP Chemoluminescence

ATP + D-luciferin

+ O2

Luciferase+ Mg 2+

Oxyluciferin + AMP

+ 2Pi + CO2 + light

http://www.sciencedirect.com/science/article/pii/S1471491412000081

2 (400 µL), luciferase (25 µL, Sigma L L1759; 10 mg/ml in 0.5M Tris buffer), D-luciferin sodium salt (BD Biosciences 556875; 11.1 mg/ml in d H20)

Design the Chip on Corel Draw X4

1 In Mm: NaCl (117), KCL (3), MgSO4 X H20 (1), NaH2PO4 (0.5), NaH2PO4 (0.5), NaHCO3 (32), D-Glucose (15), CaCl2 x 2 H20 (2), pH 7.4 ± .1, 320 mOsm ± 10.

Print the Chip on a sheet of PMMA with an Epilog Legend

laser cutter

Take and lay out chip components

Weld chip plates together using dichloromethane

Create the base of the chip Place a needle in the clear PMMA cover holes

Glue the needle into the holes to create the solution input and

outputs

The cover is then ready Add platinum iridium wires to the chip to stimulate the nerve during the

experiment

Secure the wires and the cover groove with Plasti-dip to create a

water tight seal

The nerve stimulation chip is finished and ready to use

Peripheral nerves come in several dimensions. Here, rabbit sciatic

nerves are ready to be tested for ATP release in the chip

Sciatic Nerve

Vagus Nerve

Sympathetic Nerve

Add the nerve to the chip. Put a Vaseline barrier and mineral oil on the electrodes side and Ringer’s solution1

on other side of the barrier

Put the nerve in the CCD camera to get baseline light emissions

Begin stimulating the nerves at 1, 8, 10, and 20 Hz with 15 mA and 15,

45, 70 mA at 20Hz

Add enough firefly “cocktail”2 to cover nerve. Continue stimulating

and recording

Add Apyrase after the light emissions are peaking in order to determine that the emitted light is a result of

ATP

Stop stimulation and recording. Analyze results

Luciferin +Luciferase Apyrase

Introduction

We are developing an economical method to detect neurostimulation release of ATP using the lab on a chip platform. This new platform will enable us to determine the concentrations of ATP (≥ nano molar), and to develop biomarkers for detecting unsafe levels of neurostimulation. The chips versatile and economical attributes simplifies research that can be used to explore neurostimulation induced release of neuroactive substances, and hence, contribute to our understanding of safe neurostimulation parameters. The nerve-on-a-chip technology can be used to determine the concentration at which excessive levels of certain neurotransmitters such as ATP are unsafe, and contributes to the CDRH objective of analyzing medical device performance

20Hz 70mA + apyrase

20Hz 70mA+ apyrase +30mins

Region of interest

Figure 1 A and B depicts the rabbit sciatic nerve in a chip with the stimulating electrodes attached. Only 100 µL of firefly cocktail was added to the nerve.

Figure 2. CCD images of the nerve before and after stimulation.Figure 3. Degradation of luminescence after the addition of two units of apyrase.Figure 4. Luminescence of firefly cocktail in the chip in presence of 20 and 200 nM ATP.

SignificanceConventionalSet Up

Nerve on ChipSet Up

Cost Considerations

CCD Camera System $45,000-65,000 $700-1500

Nerve Chamber $500 + $.018 Other ConsiderationsReusability √ √Disposability X √Versatility X √

This chip and its associated setup costs are greatly reduced in comparison with current methods. The chip is also more versatile than current nerve chambers and, unlike those chambers, it is disposable as well as reusable.The cheaper platform enables researchers to utilize small volume of research biochemicals, reducing solution waste and providing a greater flexibility for accommodating nerves of different dimensions.

Figure 2

1Hz 15mA

10Hz 15mA

20Hz 15mA

B

D

C

A

A

B

In situ image of rabbit sciatic nerveIn chip

Figure 1

Figure 4

Pre-stimulation

A

Figure 3

20 nM ATP

200 nM ATP

B

B

A

Human apyrase protein design and structure

http://en.wikipedia.org/wiki/Apyrase