Electricity: measuring and testing – Determining nonelectric properties by measuring electric...
Reexamination Certificate
1996-06-13
2001-09-11
Brown, Glenn W. (Department: 2858)
Electricity: measuring and testing
Determining nonelectric properties by measuring electric...
C324S444000, C324S692000, C324S072000, C435S173100, C436S063000, C436S149000, C436S806000
Reexamination Certificate
active
06288527
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a two-dimensional sensor and a measurement system using the sensor for measuring cell activities.
BACKGROUND OF THE INVENTION
Medical research of nerve cells and research for the possibility of using nerve cells as electric devices are being made widely. When nerve cells become active, an action potential is generated. Ion density inside and outside of a nerve cell varies at first due to the alteration of the ion transparency, then the potential of the cell membrane alters. Therefore, it is useful to measure a two-dimensional distribution of the potential of the cell membrane for observing a sample cell or tissue. Measuring two-dimensional distribution of the potential provides a method for determining an active part and a level of the activity.
The inventors have developed an integrated combination electrode as the two-dimensional sensor that can be used for measuring cell membrane potentials of plural spots simultaneously without insertion of glass electrodes or other stimulating electrodes into the cell (Japanese Tokukaihei 6-78889, 6-296595). This integrated combination electrode includes many micro electrodes arranged in matrix and their lead pattern formed on a glass plate using conductive substances, on which a sample cell or tissue can be cultivated. This integrated combination electrode enables measuring potential alterations of plural spots in smaller pitch than glass electrodes or other conventional means. Furthermore, this integrated combination electrode enables long term observation of the sample cell or the tissue that are cultivated on the integrated combination electrode.
However, this integrated combination electrode is not suitable for an extensive use since it has a fixed size and a fixed pitch of measuring electrodes. In other words, it is difficult to use one integrated combination electrode for measuring different samples. In fact, different integrated combination electrodes were made by adjusting the size and pitch of electrodes to different samples.
SUMMARY OF THE INVENTION
A two-dimensional sensor and a measurement system using the sensor are described that are suitable for an extensive use of measuring cell activities of different samples, by improving the above integrated combination electrode and making the size and the pitch of the electrodes changeable.
The two-dimensional sensor according to the present invention has a substrate consisting of three layers made of Si, SiO
2
and Si
3
N
4
, a thin film formed by vapor deposition on the surface of the Si layer on the back side of the sensor substrate, for making an effect electrode, and a fence attached on the Si
3
N
4
layer on a front side of the sensor substrate for containing a sample cell, culture medium and a reference electrode. When a light beam irradiates a spot on the back side of the sensor substrate, a signal is obtained corresponding to a potential alteration at the spot due to an activity of the cell placed in the fence on the sensor.
The two-dimensional sensor for measuring a cell activity according to this invention is based on a LAPS (Light-Addressable Potentiometric Sensor explained in U.S. Pat. Nos. 4,758,786 or 4,963,815) developed by Molecular Device CO., Ltd. in USA. As shown in
FIG. 5
, the LAPS comprises a semiconductor silicon substrate
101
, oxide layer
102
and nitride layer
103
on the substrate. The LAPS is well known as a pH sensor for measuring pH of an electrolyte
104
, such as liquid contacted with the LAPS. The principal of measuring pH of an electrolyte using LAPS will be explained briefly with reference to FIG.
5
.
A bias voltage is applied to an EIS structure consisting of an Electrolyte
104
, an Insulator and a Semiconductor, by using a potentiostat
105
. A light beam modulated with a certain frequency is irradiated at a back side of the EIS structure. Then AC photocurrent flows as shown in FIG.
6
. The time-voltage curve in
FIG. 6
shifts along the horizontal axis (i.e., bias voltage) according to the pH value of the electrolyte. Therefore, the pH can be measured by detecting the AC photocurrent I under the condition where the predetermined bias voltage is applied. The reason that the I-V curve is shifted according to the pH of the electrolyte is considered as followed.
When the voltage is applied to the EIS structure, an energy band bending occurs at the interface between the semiconductor and the insulator. This energy band bending depends on pH of the electrolyte contacting with the insulator. In the surface of the insulator layer, silanol group (Si—OH) and amino group (Si—NH
2
) are formed, and their functional groups combine with protons (H+) selectively, thus an equilibrium between the number of protons in the electrolyte and the number of combined protons is maintained. Therefore, if the pH of the electrolyte changes, electric charge on the insulator varies; then the energy band bending alters. As a result, a width of depletion layer between the semiconductor and the insulator alters. This alteration of the width, i.e., capacitance of the depletion layer causes alteration of the AC photocurrent. The LAPS also uses a photoconductive character of the semiconductor such that the electric conductivity increases by light irradiation.
In the same way as the LAPS, the two-dimensional sensor of the present invention comprises a substrate consisting of three layers made of Si, SiO
2
and Si
3
N
4
as well as a thin film of an effect electrode formed by vapor deposition on the Si layer. The sensor of the present invention further comprises a fence for containing a sample cell, culture medium and a reference electrode. Two-dimensional distribution of the potential alteration generated by the activity of the cell placed in the fence is measured directly. In other words, the sensor of the present invention provides a potential generated directly by the activity of the cell contacted with the insulator layer. This mechanism is different from the pH sensor in the prior art using LAPS which generates a potential on the surface of the insulator by combining protons with the silanol group (Si—OH) and amino group (Si—NH
2
) formed on the surface of the insulator as explained before.
The sensor of the present invention alters the width of the depletion layer between the semiconductor and the insulator. Thus a capacitance of the depletion layer alters. Moreover, electric conductivity at the spot irradiated by the light beam increases. As a result, a signal corresponding substantially to the potential alteration at the spot is obtained from the effect electrode.
The system for measuring a cell activity according to this invention comprises the above two-dimensional sensor, a light beam source for irradiating a spot on the back side of the two-dimensional sensor with a light beam, a DC power source for applying a DC bias voltage between the effect electrode on the back side of the two-dimensional sensor and the reference electrode in the fence on the front side, and means for processing a signal obtained between the two electrodes. It is preferable to use a laser beam source as the light beam source. The laser beam can be easily focused in a small spot, and the location of the beam spot can be controlled precisely. It is also preferable for the system to include means for maintaining an environment for cultivating the sample cell in the fence on the sensor, so as to enable a long-term observation.
In a preferred embodiment, the e system further comprises means for driving the laser with high frequency so as to emit a modulated high frequency laser beam, and the signal processing means detects an amplitude alteration of the AC photocurrent flowing between the effect electrode and the reference electrode. As mentioned before, the alteration of the width (capacitance) of the depletion layer between the semiconductor and the insulator due to the potential alteration generated by the activity of the cell contacted with the insulator is thus detected as the amplitude alteration of the AC photocurrent.
It is als
Iwasaki Hiroshi
Kamei Akihito
Sugihara Hirokazu
Taketani Makoto
Brown Glenn W.
Matsushita Electric - Industrial Co., Ltd.
Morrison & Foerster / LLP
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