From DRAMs and Pentium chips to calculators and cellular phones, semiconductor devices are prominent. These devices are currently being researched in monitoring the central nervous system and the nicotinic acetylcholine receptor for a long period of time in a nonevasive way. The current use of microelectrodes in patching do not suit in recording for months or for use in non-laboratory environments. A proposal for monitoring the muscarinic acetylcholine G-protein coupled receptor by using CMOS (complementary metal-oxide-silicon) and biological molecules devices will also be discussed. A longer- term recording instrument can monitor the effects of Alzheimer's disease and Myasthenia Gravis by recording the acetylcholine level for years. Also, the toxins for a receptor can be measured for a long time. The fabrication of the semiconductor devices usually start with a silicon or gallium arsenide wafer. Then, the field oxide layer is formed to form the initial isolation between two different devices. Many steps such as oxidations, photolithographies, depositions, drive-ins, and metalizations using modules like wafer cleaning, resist coating, photomasking, oxide etching, polysilicon etching, resist strip, furnace operations, and aluminum evaporation follow. An example of using semiconductor devices is to couple the clonal myoblasts to metal microelectrodes in a nonevasive monitoring of the nicotinic acetylcholine receptor electrical function [1].
In studying the electrical behavior of the nicotinic acetylcholine receptor's lipid bilayer membrane, both patch-clamp and microfabricated extracellular electrodes can be used to give high signal-to-noise ratio. The microfabricated electrode can additionally provide an opportunity to monitor the nAchR for a long period of time. The signal-to-noise ratio increased if the cell forms a high impedance seal with the electrode [1]. The semiconductor device elements can be controlled with a capacitive junction between the membrane and the electrode [2].
In a paper by Quong, et. al, the microelectrode fabricated in monitoring the acetylcholine levels began with a 0.1 micrometer silicon dioxide layer grown in silicon <100> wafer at 1000 degrees C for one hour. Then a small part of the oxide layer was etched following photoresist patterning. In its place, a 0.1 micrometer conducting layer of gold was evaporated. After the removal of the photoresist pattern, a 3 micrometer layer of silicon dioxide was formed by chemical vapor deposition [1].
The two substances used in the experiment were carbachol and alpha-bungarotoxin. Carbachol (CCh) is an agonist of acetylcholine receptor in which the magnitude of the resting potential decreases when carbachol binds to the nAchR. The nicotinic acetylcholine receptor opens and causes transient influx of cations into the cell [1]. Alpha-bungarotoxin inhibits the opening of the nicotinic acetylcholine receptor by forming three disulfide loops in a leaf-like structure which then binds to an extended surface area that is mainly encompassed by the alpha-subunit [3]. The impedance of the microelectrode are modeled with several parallel combinations as shown in figure 1.
The clonal myoblasts formed a high impedance seal with the microcircuit electrode. The outer cell membrane is represented by Rmo and Cmo. Iachr is the flux of cations into the cell elicited by CCh or outer agonist binding to the nAChR. Rme and Cme are the impedances between the inner and the outer cell membrane. Re and Ce form the impedances where the gold electrode contacts with the inner membrane. RL is the leakage resistance where it is the equivalent resistance formed by the parallel combination of the resistance of the seal that forms between the cell, the electrode surface, and the shunt resistance of the silicon dioxide. The Rdag and Cdag are the impedances for the silicon dioxide. Since RL >> Re and RL << Rdag, a simple circuit can be modeled as shown in Figure 2. Vj = Vm * (RL / (Rme + RL)) is the simple voltage divider obtained from the simple circuit [1].
The recording of the cell took place about four days after the plating of the cells. The temperature was at room temperature, and the electrodes were immersed in recording solution that consisted of NaCl (150mM), KCl (5 mM), CaCl2 (2.5mM), MgCl2 (1 mM), HEPES (10mM), and glucose (10 mM). Glass microelectrodes filled with 3 M KCl was used for the intracellular recordings. When stable resting potential was reached, 20 mM of carbachol was transiently introduced with a continuous perfusion of recording solution of 1-2 mL/min. Under current clamp condition, the membrane potential was recorded. Both the intracellular and the extracellular measurement of the continuous perfusion of carbachol showed a decrease in the magnitude of the resting potential as shown in figure 3.
In the second experiment, a steady exposure to carbachol were performed by direct injection of 100 microliter of 20 mM of CCh into 2 mL of recording solution. Again, both the intracellular and the extracellular measurement resulted in a decrease in the magnitude of the resting membrane potential as desired. The measurement is shown in figure 4.
The third experiment studied the inhibition of the nAchR by alpha-bungarotoxin. The cell-electrode system was rinsed at 5 minute interval for three times with 2 mL of recording solution to remove any nAchR-bounded carbachol. Then alpha-bungarotoxin was injected until a final bath concentration of 1e-9 M was reached which is the saturation level. Then, the carbachol response was measured. As desired, the intracellular and the extracellular measurements both showed very little response to carbachol as shown in figure 5 [1].
One disease that might be able use a biosensor in determining acetylcholine levels for long periods of time is Myasthenia Gravis. People afflicted with Myasthenia Gravis are characterized of having asymmetric weakness and fatiguability of extraocular (causing double vision and droopy eyelids), bulbar (causing difficulty in chewing, swallowing, and talking), neck, and limb muscles [4]. Myasthenia Gravis is an autoimmune disease of the post-synaptic membrane in which antibodies are produced by the immune system to attack the acetylcholine. These antibodies are detected in over 80% of the patients and localized in the post-synaptic membrane. The number of acetylcholine at the axon hillock is normal; however, the number of acetylcholine at the post-synaptic membrane is decreased, causing a decrease at the endplate potentials in response to a nerve stimulation. Thus, the patient is fatigued due to a lower total EPSP at the neuromuscular junction [4,5]. The incidence of Myasthenia Gravis is estimated to be about one in 10,000-20,000. However, the actual rate might be higher due to delay or misdiagnosis. This where the biosensor can help. The biosensor can determine the acetylcholine level for a long extended time which can lead to a better diagnosis of a patient. Since Myasthenia Gravis can be treated, the determination of the disease is vitally important [6]. Once Myasthenia Gravis is diagnosed, the patient can be monitored with the biosensor in determining the best treatment. Myasthenia Gravis can treated by anticholinesterase that inhibit the breakdown of acetylcholine, by immunosuppressive drugs that suppress anti-acetylcholine receptor antibody formation, by plasmapheresis and high dose immune globulin which temporarily reduce the antibody concentration in the serum through poorly understood mechanisms, and by thymectomy which removes an antigenic stimulus for the formation of anti-acetylcholine antibodies [4].
In the paper by Quong, et. al, the electrodes can not be used for muscarine receptors since the measurement depends on the opening of the ion channel [1]. I believe that it is possible to measure the changes of a muscarinic acetylcholine receptor, although the signal-to-noise might be a problem. The change in the shape due to acetylcholine binding in the muscarinic acetylcholine G-protein coupled receptor can be measured by detecting the minute change in the resistance of the inner membrane (Rme in figure 2). If the electrode from the Quong, et. al, paper is used in measuring the muscarinic acetylcholine receptor, the noise would dominate due to the uncertainties of the resistors and capacitors. In measuring the minute resistance change in the configuration of the muscarinic receptor, a more precise electrode would be needed. However, a microsensor made of CMOS (complementary metal-oxide-silicon) can significantly reduce the signal-to-noise ratio since the fabrication of precise capacitors leading to precise measurements of impedances for integrated circuits are possible [7].
As recent as five years ago, the use of CMOS in the cell membrane would have been impossible due to the size of the CMOS. CMOS technology is based on the size of the minimum feature of the integrated circuit. Five years ago, the minimum feature or lambda was around one micron. However, technology has reduced lambda to 0.35 microns at this time. Soon, lambda will decrease to 0.2 microns or even 0.15 microns. This reduction in the size of the CMOS will soon make it possible to implant them into the cell membrane.
In a larger scale, a CMOS was fabricated to measure human blood vessels. The platinum electrodes were fabricated by standard CMOS process with one additional technological step. The platinum was de-sputtered and structured by a left-off process. Above the aluminum layer, the platinum was de-sputtered in order to be immersed with electrolyte in forming the electrode area. The electrodes were initially used to measure conductivity of the electrolytic conductivity of human blood [8]. If the electrode was small enough, the electrode will be able to measure the change of the charges inside the receptor. In relating to the circuit model (figure 1 and 2) presented in the Quong, et. al., paper, Rme and Cme can be measured reliably. Then, the changes of charges due to the conformational change on the receptor can be measured. Combined with the high signal-to-noise ratio of the CMOS, the muscarinic acetylcholine receptor and other G-protein coupled receptors can be monitored.
Another intriguing technology, which will reduce the size of a transistor in measuring acetylcholine levels, might not be silicon based transistors. Currently, biological molecules are being researched in their possible application in computers. The most promising biological molecule is bacteriorhodopsin, which is involved in G-protein coupled receptors. Since the atoms are mobile and predictably changeable in position, the molecules can potentially serve as computer switches. If two discrete states of a molecule can be consistently generated, each state can represent either 0 or 1 as in the CMOS transistor. In the case of bacteriorhodopsin, a structural change occurs in response to light. The resting state of the bacteriorhodopsin is bR which can represent the binary 0 state, while the intermediates such as K and M can represent the binary 1 state. Switching between states can be controlled by a laser beam by noting the change in wavelengths absorption since the resting state and each intermediate state will have different absorption regions. The switches can significantly reduce the size of the transistor since the molecules are very small compared to a semiconductor transistor. The reduction in size can be vital to incorporate these biological electrodes inside the G-protein coupled receptors. Then, the protein changes that occurs when a G-protein coupled receptor becomes activated by a ligand can be measured. Also, biological molecules can be designed one atom at a time which gives the engineers the control that they need to manufacture gates that perform the exact application. The use of biological molecules in practical applications might still be ten to twenty years away; however, biological molecular computers can soon become an intriguing and exciting computer architecture. A hybrid computer that uses the best features of both semiconductors and biological molecules will be the first step in taping into this technology [9].
One application for the use of biosensors in measuring the level of acetylcholine in muscarinic receptor would be in determining Alzheimer's disease in a patient. A patient with Alzheimer's disease has extensive central nervous system cell loss, neurofibrillary tangle plaques, a decreased acetylcholine level, and decreased acetylcholine receptor number. In a study of Alzheimer's disease, the cholinergic innervation of the mediodorsal nucleus matrix which derives mainly from the laterodorsal tegmental nucleus and the mediodorsal nucleus patches from the substantia innominata were studied. In the controls, the cholinergic innervation of mediodorsal nucleus of the thalamus was distributed heterogeneously in densely labelled patches surrounded by less heavily stained matrix. However, in patients with senile Alzheimer's dementia, the choline acetyltransferase had positive varicosities decreased by 34% in the matrix and by 46% in the patches [10]. A biosensor can help diagnose a person who might be suffering from Alzheimer's. Currently, there are not many treatments to counteract Alzheimer's, although an acetylcholinesterase inhibitor named tacrine is used to treat Alzheimer's [11]. Pharmaceutical companies can use a biosensor, which detects acetylcholine levels, to test their drugs in measuring the acetylcholine level in Alzheimer's patients. A biosensor can be one of the many tools used before human testing to find an effective treatment for Alzheimer's disease.
The future of biological instrumentation will correlate with the advancement in biology. For instance, the patch clamp techniques made it possible to study ion channels on cell membrane which led to the explosion in the knowledge of cellular signaling. [12] The use of semiconductor devices and possibly biological molecular devices will also soon significantly increase our knowledge in biology. These devices are more rugged than the materials used in the patch clamp techniques; thus, these devices can be incorporated to monitor neurotransmitter levels for long periods. Myasthenia Gravis can be diagnosed earlier and correctly, and the best treatment of Myasthenia Gravis can be monitored with these biosensor devices by measuring the acetylcholine level. In the future, the CMOS and biological molecules based microelectrode can be used in measuring the acetylcholine level in the muscarinic acetylcholine receptor, a G-protein coupled receptor, to monitor the long-term effects of Alzheimer's disease. As more research takes place in this area of microscale and nanoscale biomedical instrumentation, more advancement of biology in both clinical and research applications will take place.
1. Quong, Judy N., Fare, Thomas L., Howard, Bryant J., &
Stenger, David A. (1993) Measurement of Acetylcholine Receptor
Function in Microcircuit-Coupled Myoblasts. IEEE Transactions
on Biomedical Engineering 40 (11): 1122-1125.
2. Fromherz P., Offenhausser A., Vetter T., & Weis J. (1991) A
Neuron-Silicon Junction: A Retzius Cell of the Leech on an
Insulated-Gate Field-Effect Transistor. Science 252:
1290-1293.
3. Taylor, P. & Brown J. H. (1994) Basic Neurochemistry:
Molecular, Cellular, and Medical Aspects, 5th Edition, Raven
Press Ltd., New York, p. 252.
4. Gendron, D. (1996) Myasthenia Gravis and Other Disorders of
the Neuromuscular Junction. McGill University. Edited by R.
Funnell.
Http://funsan.biomed.mcgill.ca/~funnell/medcurr/unit1/1genmya.html.
5. Marshall, Debbie. (1995) Causes of MG. The MG
Communicator. Myasthenia Gravis Association of Colorado.
Http://www.infohiway.com/way/mgacolorado/feb95/causes.html.
6. Myasthenia Gravis Association of Western Pennsylvania, Inc.
(1990s) Agency Profile.
Http://www.pitt.edu/~uklst1/mginfo.html.
7. Muller, Richard S. & Kamins, Theodore I. (1986) Device
Electronics for Integrated Circuits, 2nd Edition. John Wiley
& Sons, New York, p. 379.
8. Kordas N, Manoli Y, Mokwa W & Rospert M. (1994) The
Properties of Integrated Micro-Electrodes for CMOS-Compatible
Medical Sensors. Sensors and Actuators A-Physical 43 (N1-3): 828-829.
9. Birge, Robert R. (1995) Protein-Based Computers.
Scientific American 272 (3): 90-95.
10. Brandel JP, Hirsch EC, Malessa S, Duyckaerts C & Cervera P,
Agrid Y. (1991) Differential Vulnerability of Cholinergic
Projections to the Mediodorsal Nucleus of the Thalamus in Senile
Dementia of Alzheimer Type and Progressive Supranuclear Palsy.
Neuroscience 41 (1): 25-31.
11. Presti, David & Barrie, John. (1996) Molecular Biology of Acetylcholine
Receptors.
Http://sulcus.berkeley.edu/mcb165/mcb165_sp96.html.
12. Neher, Erwin & Sakmann, Bert. (1992) The Patch Clamp
Technique. Scientific American 266 (3): 44-51.