Research Center of SIONTECH Co., Ltd 220, Kungdong, Chungnam National University, Taejon, 305 – 764, Korea _________________________________________________ Dr. Vladimir A. Bobrov 2003-2004 - Brain Pool, KOFST visiting Scientist STUDY OF ION MECHANISM OF MEMORY BIOLOGIC MEMBRANE WITH THE AIM TO CREATE NEW GENERATION BIOLOGIC SENSOR FOR WRITING, READING AND STORING INFORMATION REPORT (2003 – 2004) TAEJEON – 2004

1. Research Center of SIONTECH Co., Ltd
220, Kungdong, Chungnam National University, Taejon, 305 – 764, Korea
_____________________________________________________________________________________
Dr. Vladimir A. Bobrov
STUDY OF ION MECHANISM OF MEMORY BIOLOGIC MEMBRANE
WITH THE AIM TO CREATE NEW GENERATION BIOLOGIC SENSOR
FOR WRITING, READING AND STORING INFORMATION
REPORT (2003 – 2004)
TAEJEON – 2004

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CONTENTS
I. INTRODUCTION 3
II. OBJECTIVES 4
III. MATERIALS AND METHODS
IV. RESULTS AND DISCUSSION
V. SUMMARY
VI. REFERENCES

3. 3
I. INTRODUCTION
Biological systems perceive extra -cellular signals, such as light, smell, nerve-nerve stimuli etc., by the initiation of coupled, cascade - like amplification reactions. In many of these, the initial or intermediate steps in the cascade, involve the opening of membrane – associated ion channels.
In ligand-activated channels, the process is initiated by the binding of the small effectors molecule (neurotransmitter, odorant, or flavor) to a specific receptor that is either structurally or functionally coupled to the channel protein. This induce s conformational changes in the
channel protein that leads to the opening of a pore across the membrane causing a step increase in the membrane's electrical conductance.
By means of the electronic amplification available today, a single channel opening event can be detected.
The conductive path across the membrane is formed by coordinated aggregation of several peptides to create the walls of an aqueous pore. In addition, by modifying their primary sequences, specific channel properties can be altered.
Independently, channel formation can be controlled by restricting the lateral and rotational
mobility of the peptides in the plane of the membrane.
An ion channel is therefore a device that controls the flow of ions through the dielectric formed by the core of the lipid membrane.

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II. OBJECTIVES
The purpose of development
- To study molecular mechanisms of the cell membrane memory at the level of ion channels operation and to develop principles to create new generation biologic sensor.
- To elaborate the technologic base and to create new generation biologic sensor devices for writing, storing and processing the molecular level information.
The new generation biologic sensor devices based on using the biologic macro molecules (ion channels) to write, store and read information, may be used for screening of a potential drugs (in particular, neurotransmitter, odorant or flavor) .
The contents of development
It was supposed to implement the project in the two principal stages:
Stage 1. To study molecular mechanisms of the cell membrane memory at the level of ion transport channels operation and to develop principles to create new generation multi -channel biologic sensor (
2004).
Molecular mechanism of the membrane memory effect at the level of ion transport channels operation, using the voltage – clamp technique, would be established at this stage.

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This technique combined with biochemical and biotechnological techniques for operating the membranes and plant cell allows discovering ion channel character, their sensitivity to membrane
potential, kinetic performances of the activation/inactivation mechanism depending on the structural/functional membrane state and so on.
Based on the data obtained, the kinetic model of the ion channel operation responsible for the membrane memory effect will be created.
The kinetic model in the computer experiment would allow finding optimum conditions for ion channel operation and further would be used as a methodological base while elaborating the principles of new generation multi-channel biologic sensor designed to process biologic level information.
Stage 2. To elaborate the technologic base and to create new generation multi-channel biologic sensor devices based on using the biologic macro molecules (ion channels) to writing, storing and reading information, which will be used for screening of a potential drugs.
The new generation biologic sensor devices based on using the biologic macro molecules (ion channels) for writing, reading deleting and storing of information, will be used for screening of membrane
activity of drugs, in particular, of neurotransmitter, odorant or flavor (2004 - 2005).

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Application of results of development
The drag discovery process is a long, expensive process which often as not doesn't provide a return on investment.
There are a number of techniques which pharmaceutical companies utilize in order to efficiently identify and screen a potential drug candidate and the biosensor may be adapted to any of the processes one might choose.
The new generation biologic sensor devices based on using the biologic macro molecules (ion channels) to write, store and read information, will be used for screening potential drugs, in particular, of
neurotransmitter, odorant or flavor.
One of primary the objectives our study
1. To develop the basic electrical circuit of the multi – channel device for measurement of membrane potentials and electrical currents in a mode of a voltage-clamp on plant cell Chara australis.
2. To study molecular mechanisms of the cell membrane memory and to develop principles to create new generation biologic sensor.

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III. Materials and Methods
Chare australis, dioeciously species of Chara corallina , used throughout this work was cultured indoors in small pots (1.5 liters).
Internodal cells were isolated from neighboring cells. Internodal cell was placed on the polyacrylate vessel composed of 4 chambers as illustrated in FIG. 3.

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Chamber A was filled with artificial pond water ( APW1:O.1 mM KCL, 1.0 mM NaCl , 1.0 mM CaCl2, 0.1 mM MgCl2 and 100.0 mM KCl).
Chamber B, C and D ware filled with artificial pond water (APW2: O.1 mM KCL, 1.0 mM NaCl , 1.0 mM CaCl2, 0.1 mM MgCl2 and osmotic value were adjusted to 200 mM with sorbitol).
Experiments were done at room temperature (2.0 – 27 %C) and under dim light. The light intensity was kept at about 200-250 lux.
For measurement of membrane potential the exocellular method of measurement of membrane potential (without usage of microelectrodes) is utilized, that considerably makes cheaper the equipment (there is no necessity to have the expensive microelectrode equipment and explorers of high proficiency).
IV. RESULT AND DISCUSSION
4.1 The principled unit the scheme WORK STATION for analysis of ion channels on calls fresh water algae’s Chara australis.
Traditional electrophysiology techniques identified ion channels as critical mediators of physiological processes and as targets of many drugs.
These classical techniques, although well suited f or analysis of drag mechanism, are limited as tools for drug discovery, because of expertise requirements, lack of automation.

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Last three years the attention of the explorers in this area is massed on mining of methods and technologies that will revolutionize electrophysiology as a tool for drug discovery and functional
screening.
These include the automated two -electrode voltage clamp and other methods for parallel whole - cell recording and multi – electrode system (work station).
In FIG. 1 The principled unit the scheme WORK STATION for analysis of ion channels on calls fresh water algae’s Chara australis is submit designed by us the general principled unit.

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FIG.1 The principled unit the scheme of the multi-channel device include:
1 - The multi-channel flowing chamber (1) with biologic cell ( Chara australis)
2 - The block of amplifiers.
3 - The multi-channel interface.
4 - The block of the executive devices (peristaltic pump, multi-channel valve and so on).
5 - Data processing (control, treatment and analysis of the information) - external PC,
LabJack data acquisition and two channel digital oscilloscope TDS 210 (5).
4.2 The complete BASIC electrical schematic of the two-channel Potentiostate
and 2 - channel BIOSENSOR
The complete BASIC electrical schematic of the two – channel potentiostate and 2- channel BIOSENSOR is submitted in FIG. 2.
We have used an exocellular way of registration of membrane potential on an outside cellular membrane (plasmalemma) of cell Chara australis.

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For this purpose the chamber was made (FIG. 2 and Fig. 3), consisting from four compartments (A, B, C, D).
The internodal cell Chara australis was placed in the chamber and the compartments (A, B, C, D) were isolated (insulated) among themselves with the help of silicone lubrication.
Testable and control solutions were given from containers (not shown) in B and C compartments by means of the peristaltic pump through the multi-channel valve.
Membrane potential - difference of electrical potentials between measurement (E2-E3) and reference (E1) electrodes.
The body of reference (E1) and measurement (E2, E3) electrodes was made from glass.
The inner reference electrode was mad e from silver/silver chloride wire (Ag/AgCl) having a diameter of 0.2 mm.
Working electrodes (Ag1, Ag2, Ag3) for the drive of current through two channel (B and C) in the mode a voltage – clamp (compartments B and C electrically insulated one from other) was made from the Ag/AgCl wire by the diameter 0.1 mm.
Information (membrane potential and electrical current from two independent channels B and C) enters in the two channel digital oscilloscope TDS 210.
In these conditions the membrane potential on the part of butt end of a cell became to equal zero point (0 mV).
Thus, the potential electrode E1 appeared inside a cell and the difference of electric potentials between electrode E1 and E2 equaled to membrane potential in a compartment B (1-channel).
The current through a channel B was skipped between electrode Ag1 and Ag2 (Ag/AgCl wire).

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Similarly, the difference of electric potentials between electrode E1and E3 equaled to membrane potential in a compartment C (2 - channel).
The current through a channel C was applied between current electrodes Ag1 and Ag3 (Ag/AgCl wire).
The membrane potential in a compartment B and C was measured on a differential circuit (FIG. 2) with the help of amplifiers U1, U2, U3 and fur ther went on an operational amplifier U5, where was compared to a command potential (voltage - clamp mode, potentiostate), going with an output of the summator U7.
4.3 Stable square wave generator, Monostable generator and
Constant voltage generator.
The command potentials moved on an input of the summator U7 with the help of the generators, designed and made by us – Stable square wave generator (FIG. 4), Monostable generator (FIG. 5), and
Constant voltage generator (FIG. 6).
Stable square wave generator (FIG. 4) and Monostable generator (FIG. 5) were made on the basis of four real - time clocks LM555 that has allowed considerably reducing expenditures on mining and manufacturing of the equipment.
The laboratory breadboard WORK STATUON is submitted on FIG. 7.
Was stipulated, hereinafter, for the second year of the Project, to use the low-cost equipment for automatic input of command impulses of voltage and loading data in a PC about value of voltage and current on two channels (B and C) with the help Data Acquisition LabJack U12.

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It is the cheapest system for loading information in the personal computer.

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Fig. 7
The laboratory breadboard WORK STATUON is submitted on FIG. 7.
1. 2-channel BIOSENSOR. 2. 2-channel voltage-clamp potentiostate.
3. Internodal cell Chara australis in multi-channel flowing camber.
4. Multi-channel valve. 5. Peristaltic pump. 6. Two channel digital oscilloscope TDS 210.
7. LabJack data acquisition

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4.4 To study ion mechanisms of the memory biologic membrane.
The research and development of the memory materials are extensively being conducted.
In recent years, applications of memory materials are the most important in electronic industries such as computers and related apparatuses, video discs, and digital audio discs.
The performances required for memory materials depend on the application field thereof.
The general concept of utilizing electrically writable and erasable phase change memory materials (i.e., materials which can be electrically switched between generally amorphous and generally crystalline states) for electronic memory applications is well known, as is disclosed, for example, in U.S. Pat. No. 3,271,591 to Ovshinsky, issued Sep. 6, 1966 and in U.S. Pat. No. 3,530,441 to Ovshinsky, issued Sep. 22, 1970.
As disclosed in the Ovshinsky patents, such phase change materials can be electrically switched between structural states of generally amorphous and generally crystalline.
The early memory materials described by the Ovshinsky patents could also, if required, be switched between just the two structural states of generally amorphous and generally crystalline local order to
accommodate the storage and retrieval of single bits of encoded binary information.

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The memory element thus formed perform s electrical switching action of memory between high - resistance state and a low - resistance state.
Biological systems perceive extra -cellular signals, such as light, smell, nerve-nerve stimuli etc., by the initiation of coupled, cascade - like amplification reactions. In many of these, the initial or intermediate steps in the cascade, involve the opening of membrane - associated ion-channels.
In conducted by us of experiment, we managed to show, as on a biological membrane of cell Chara australis it is possible to write, to read and to remove one bit of the binary information.
Below, in a FIG.1 the example of reading, record and shelf - life of one bit of the binary information is adduced.
Optimum time of reading, writing and storage information (DT1 - time of reading, DT2 - time of writing and DT3 - time of storage information) is adduced on Fig.1.

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Kinetics of changing a current (J, μ A/sm2) getting through the compartment B in conditions voltage - clamp was registered simultaneously with membrane potential (ψ, mV).
From the Fig. 1 seen that membrane potential forbears at a rate of ψ = - 140 mV and at current J = 0.5 μ A/sm2.
At shift of membrane potential from - 140 mV before - 70 mV ion current J increases before
-3 μ A/sm2 and stays on constant level through 50 ms.
This time ΔT1 is approximately 50 ms and was marked as a time of reading information (time of reading of 1 bit information).
Under the following shift of membrane potential from - 140 mV before - 70 mV ion current J increases approximately before -23 μ A/sm2 during 400 ms.
This time ΔT2 is approximately 400 ms and was marked as a time of writing information (time of writing).
At the shift of membrane potential on the source level (ψ = - 140 mV) ion current quickly returns before the value J = 0.5 μ A/sm2.
However under the following shift of membrane potential within 50 ms (time of reading ) before the level - 70 mV ion current increases before the value approximately - 23 μ A/sm2.

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Such condition a membrane with the high conductivity (on the drawing marked as read (1)) is well tested following short (50 ms) by pulses of voltage during approximately 110 S.
This time T 3 is approximately 110 – 120 S and was define a time of storage information (time of storage information).
Optimum time of reading, writing and deleting information (ΔT1 - time of reading, ΔT2 - time of writing and ΔT4- time of deleting information).
Example of such experiments is submitted on the FIG. 2 and FIG. 3

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From the drawing 2 seen that in 1850 ms (after writing one bit information (read(1)) membrane potential was shifted from the level - 140 mV before the level - 210 mV within 550 ms (ΔТ4 - time of deleting).
For this time ion current returned approximately to the value of ion current at the potential - 140 mV.
The following reading information in 2570 ms (read (0)) has shown that recorded before one bit information (read (1)) was removed described above by the procedure.
Thus, the new type of a multi - channel digital biosensor grounded on a voltage-clamp method of recording of the binary information on and reading from biological membrane it is possible briefly to define so:
A multi - electrode biosensor includes a biological plant cell, separated on a plurality of electrical independent parts, a plurality of working and measuring electrodes on the same plant cell, and a reference measuring and a reference working el electrodes in one
electrical independent part of the plant cells.

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V. SUMMARY
1. Developed the basic electrical circuit of the multi-channel device for measurement of membrane potentials and electrical currents in a mode of a voltage-clamp on cell Chara australis.
2. The optimum conditions for writing, reading, deleting and storage of the binary information on of biological membrane is determined.
3. Developed principles to create new generation biologic sensor.
The new type of a multi - channel digital biosensor grounded on a voltage-clamp method of recording of the binary information on and reading from biological membrane it is possible briefly to define so:
A multi-electrode biosensor includes a biological plant cell Chra australis, separated on a plurality of electrical independent parts , a plurality of working and measuring electrodes on the same plant cell, and a reference measuring and a reference working electrodes in one electrical independent part of the plant cells Chare australis.
By the obtained results the patent for patenting in Korea will be prepared:
«Method and device for writing, reading, deleting and storage of the binary information on a biological membrane»

27. REFERENCES
in U.S. Pat. No. 3,271,591 to Ovshinsky, issued Sep. 6, 1966 and in U.S. Pat. No. 3,530,441 to Ovshinsky, issued Sep. 22, 1970.