JP3200631B2 - Biosensor for measuring effects on cell metabolic activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a biosensor for measuring the influence on the metabolic activity of cells.
More specifically, the present invention relates to a biosensor for detecting environmental hormones or the like by measuring the effect on the metabolic activity of cells.

[0002]

2. Description of the Related Art Environmental hormones are exogenous hormone-like substances. Such a substance is also called an endocrine disrupting substance. In recent years, it has been revealed that such a substance controls abnormal hormones in a living body and thereby disrupts the endocrine system of the living body. Therefore, the effects on humans and wildlife due to the exposure have become a global problem.

Conventionally, such environmental hormones have been detected or quantified by in vitro tests such as a binding test with a hormone receptor and a cell proliferation test, and in vivo tests for anatomically observing genital abnormalities. It has been done. However, these tests have problems such as complicated operation and time-consuming measurement.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an apparatus for simply, quickly and highly sensitively measuring a change in metabolic activity of a cell. In particular, it is an object of the present invention to provide an apparatus for detecting environmental hormones simply and with high sensitivity.

[0005]

Means for Solving the Problems In order to achieve the above object, the present inventors have made intensive studies and found the following means.
A biosensor for measuring the effect of a test substance on the metabolic activity of a cell, comprising a semiconductor layer, an insulating layer laminated on the main surface of the semiconductor, and placed on and in contact with the insulating layer An MIS structure comprising a live cell or a live cell group, and a culture solution layer in which the live cell or the live cell group is immersed and in contact with the insulating layer, and a bias voltage is applied to the MIS structure. Bias electrode means, light source means for irradiating the MIS structure with controlled light in order to generate a photoelectromotive force in the MIS structure, and for adding a test substance to the culture solution layer Means, and means for monitoring a change in photovoltaic power generated in the MIS structure, and the change in the photovoltaic power before and after adding the test substance indicates that the test substance Affect the metabolic activity of the cells A biosensor and measuring the affect.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Apparatus The apparatus of the present invention is a biosensor that combines a biomaterial and a transducer. More specifically, a surface photovoltage device [surface photovoltage; hereinafter also referred to as SPV, and generally, LAPS (Light-Addressable
PotentiometricSensor) or micrometer (Microphy)
A biosensor in which a living cell or a piece of a living tissue is incorporated.

FIG. 1 shows an example of a specific structure. The biosensor of the present invention includes a semiconductor layer 2, an insulating layer 3 laminated on the main surface of the semiconductor layer 2, a living cell or a living cell group 4 disposed on and in contact with the insulating layer, and A MIS structure composed of a culture solution layer 8 in which living cells or a group of living cells are immersed and in contact with the insulating layer; bias electrode means 7 for applying a bias voltage to the MIS structure; A light source means 9 for irradiating the MIS structure with controlled light to generate an electromotive force; a means for adding a test substance to the culture solution layer 8; Means 11 for monitoring the change in the photovoltaic power, and measuring the influence of the test substance on the metabolic activity of the cell from the change in the photovoltaic power before and after the addition of the test substance. Bio Is Nsa.

Here, as the insulating layer 3, any commonly used insulating film such as an oxide film or a nitride film can be used.

The living cells or living cell group 4 is
It is fixed directly to the upper side of the vehicle. The cell group 4 can obtain its nutrient source from the culture solution layer 8 included in the MIS structure.

The culture solution layer 8 can be formed by forming a holding means using a semiconductor layer having an insulating layer and silicon rubber or the like, and adding a solution such as a culture solution to the holding means. In addition to the silicone rubber, a non-corrosive insulating material such as a polyethylene film can be used. The culture solution layer 8 also functions as a conductor that transmits an electric field from the electrode 7 to the MIS structure. Further, the electrode 7 is arranged in contact with the culture solution layer 8. The purpose of the electrode 7 is to maintain a potential by applying a bias voltage.

Further, the inflow and outflow of the solution into and out of the culture solution layer 8 can be performed through the inflow channel 6, and the outflow can be performed through the outflow channel 10. The inflow path and the outflow path can use a non-corrosive insulating material such as a silicone tube, a polyethylene tube, and a fluororesin (FEP) tube. The inflow path and the outflow path may be performed as a single inflow / outflow path. However, since the solution can be continuously poured and the measurement can be continuously performed, it is preferable that the inflow path and the outflow path be separately disposed.

The sample to be detected is a culture solution layer 8
Need to be added. The means for adding the test substance to the culture solution layer 8 may be any means as long as a sample can be added to the culture solution layer 8. Even if it is arranged, any means for adding a culture solution or a buffer solution may be used, but a sample is prepared by previously mixing a test substance with the culture solution or the buffer solution, and the inflow path 6 is formed. It is preferable to add to the culture solution layer 8 after passing through.

The living cell or living cell group usable in the present apparatus may be arranged as a single layer or a multilayer in the insulating layer, or may be a tissue section. For example, cervical cancer-derived HeLa cells, breast cancer cells MCF-7 cells,
T47D cells, various cancer cells such as T-cell lymphoma cells JURKAT cells, and hepatocytes, myocardium, cells of the vascular system,
Normal cell cells such as smooth muscle cells and central cells. Examples of the tissue section may be any tissue section of a living body, such as organ tissues such as liver, heart and kidney, central tissues such as cerebrum and cerebellum, skin tissues, muscle tissues, and vascular tissues. However, the present invention is not limited to these, and it is necessary to determine what kind of cell or cell group or tissue section to use depending on the substance to be detected. For example, when detecting environmental hormones, it is necessary to select cells having receptors for environmental hormones.

The method of fixing the cells can be performed by any method that does not cause the cells to lose their function. For example, it is possible to fix cells by culturing directly on a semiconductor chip. Also, how to fix the tissue
It can be done by any method that does not cause the cells to lose function. For example, after a raw tissue section is produced by a section production apparatus, it is physically placed directly on a carrier such as a sensor surface and fixed.

When cells are fixed, the density of the cells to be fixed is preferably about 1 × 10 ⁴ to 2 × 10 ⁶ per cm ² . The cell layer to be fixed may be a single layer or a plurality of layers. When one layer is used, there is an advantage that the response to the stimulus is fast, and when a plurality of layers are used, there is an advantage that the signal to the stimulus is strong. When a tissue section is used, the response to the stimulus is slow, but the signal is very strong. The thickness of the tissue piece used in the present invention is preferably about 5 μm to about 100 μm, and more preferably about 30 μm.

The device of the present invention can detect a change in the metabolic activity of a cell. In the present invention, a change in the metabolic activity of a cell can be detected by measuring a subtle change in pH around the cell, a change in cell membrane potential, and the like. In addition, if cells having receptors for environmental hormones are used, it is possible to screen or quantify whether the added test substance is an environmental hormone by detecting a change in the metabolic activity of the cells. Is also possible. Furthermore, the detection and quantification of the test substance can be performed by using cells reactive with the desired test substance.

As the culture medium for maintaining the cells or tissue sections, any medium or buffer having a buffering action can be used. Preferably, a solution most suitable for the cell or tissue to be used is used. For example, examples of the medium include RPMI1640, MEM (minimum essential me
dium), L-15 medium (L-15 medium modified) and DM
EM (Dulbecco Modify Eagle Medium) and the like, but are not limited thereto. Preferably, the pH of the medium and buffer is maintained at about 7 to about 8. The temperature is 3
Preferably, it is maintained at 7 ° C.

An example of a light source that can be used in the apparatus of the present invention as light source means for irradiating the MIS structure with controlled light in order to generate a photoelectromotive force in the MIS structure Is a light emitting diode, laser light source,
There are various lamps and the like, and it is possible to arrange one or more of them, but the present invention is not limited to this.
The method for controlling the light from the light source can be performed by a method known per se.

The electrode 7 for applying a bias voltage is
That is, it is an electrode for applying an electric field to the culture solution layer 8,
It is necessary to arrange an electrode or an electrode group capable of controlling the potential. For example, gold, platinum,
Copper, nickel and the like can be used, but the use of gold and platinum electrodes is preferred because they are stable in solution. Further, a reference electrode may be arranged in contact with the culture solution layer 8 in order to keep the surface potential between the culture solution layer 8 and the insulating layer 3 constant. The reference electrode of the present invention may be arranged in the vicinity of the electrode 7 or may be arranged in a waste liquid tank installed before the solution forming the culture solution layer 8 flows out. The provision of the reference electrode is preferable because a change in surface potential caused by the presence of a redox substance such as iron ions can be controlled. Reference electrodes that can be used in the present invention include, but are not limited to, saturated calomel electrodes, silver / silver chloride electrodes, and the like.

The means for monitoring the change in the photovoltaic power generated in the MIS structure is provided by arranging the conductor 1 in contact with the lower surface of the semiconductor layer 2 and contacting the conductor 1 with the conductor 1. It is obtained by arranging the electrodes 11. Various electrodes can be used for the electrode 11 and the conductor 1.
For example, it is aluminum, gold, or the like, and a light-transmitting conductor can be used. Here, the conductor 1
Is desirably arranged so as not to block irradiation light from a light source located thereunder. Further, the electrode 11 and the conductor 1 may be arranged integrally.

2. Measurement Principle of Biosensor The biosensor of the present invention utilizes the fact that when a proper bias voltage is applied to the MIS structure, light is applied to the lower interface to generate a photoelectromotive force.

Here, the application of the bias voltage can be preferably performed by disposing the electrode 7 in contact with the culture solution layer 8.

FIG. 2 shows an example of an apparatus for measuring metabolic activity using a biosensor. The electrode 7 of the present biosensor is connected to a voltage application device, and the electrode 11 connected to the conductor 1 is connected to a measurement meter to detect a current amount or a change in potential. The measurement meter is further connected to a personal computer (indicated as a personal computer in FIG. 2) or the like, and can perform measurement condition management, data processing, and the like. Although not shown in the drawing, the reference electrode can be arranged in the culture solution layer 8 or in the waste liquid tank.

The biosensor can be fed into and out of the culture solution layer 8 by using a means such as a peristaltic pump, a syringe pump, and a plunger pump.
In addition, the biosensor of the present invention can be used in a batch system that does not perform liquid feeding, if desired.

In order to obtain the response of the present biosensor, it is possible to carry out a method of detecting a change in photoelectric flow with a constant bias voltage and a method of detecting a change in potential at an inflection point. For real-time measurement, a method of fixing the bias voltage is preferable.

Samples that can be measured by the biosensor of the present invention are all synthetic compounds and all natural substances. In addition, by using the biosensor of the present invention, it is possible to measure the influence of a test sample on metabolic activity in a living cell or a cell group or a living tissue section. Further, a detection substance can be selected by selecting a cell or a cell group or a tissue section to be used. For example, when detecting environmental hormones, it is necessary to select cells having receptors for environmental hormones. Thereby, it is possible to screen or determine whether the substance is an environmental hormone.
Furthermore, by using cells or cell groups or tissues having specific reactivity to other desired detection substances, it is possible to specifically detect and quantify the desired substances.

3. Example 3-1. Apparatus [Surface Photoelectric Device] Preferred embodiments of the present invention are described below. Microphysiometer (Technolog SE1030), semiconductor chip for Technolog SPV (manufactured by Shindengen), control computer (NEC 9801 ns / t), measurement program (Technolog SPV Measurement System), peristaltic pump It was used.

[Cell culture and immobilization] T47D breast cancer cells were used as a living cell group. Since breast cancer cells T47D are adherent cells, they will grow by adhering to the bottom of the culture vessel.
Therefore, the semiconductor chip was placed on a 60 mm dish, a suspension of breast cancer cells T47D was added, and RPMI1640 medium containing 10% fetal bovine serum (hereinafter referred to as RPMI1640-10).
% FBS medium), and the breast cancer cells were adhered to a semiconductor chip by culturing the dish for 48 hours at 37 ° C. and 5% CO ₂ . After this culture, cell adhesion was confirmed with a microscope.

[Biosensor] Using the SPV device and cells described above, a measuring device equipped with a biosensor was assembled according to FIGS. 1 and 2.

Platinum was used as a reference electrode, and gold was used as a counter electrode for applying a bias voltage. The bias current applied to the device was arbitrarily set between 0 and 4V. As the light source, one light emitting diode (hereinafter, also referred to as LED) having a peak emission wavelength of 940 nm and a frequency of 750 Hz was used.

[Measurement Method of Biosensor] First, a solvent was flowed at a constant flow rate, and then a response curve when the bias voltage was increased was measured. The bias voltage value at the inflection point at this time is calculated. The following tests were performed by maintaining the bias voltage at the inflection point voltage under these conditions and measuring the change over time in the photoelectric flow rate observed when the measurement sample was passed.

[Measurement of pH change using buffer] SPV
The device can measure small changes in pH as changes in photocurrent. Then, the relationship between the photocurrent and the pH was confirmed by the following method.

A semiconductor chip on which cells, tissue sections and the like are not fixed is set on an SPV device, and the flow rate is set to 0.1 m.
A phosphate buffer (pH 7) was flowed at 1 / min. Next, the flow was switched to a borate buffer (pH 9), and the change in photocurrent with pH was measured.

[Measurement of Photoelectric Flow Using Phosphate Buffer Solution as Solvent] A semiconductor chip on which breast cancer cells T47D were immobilized was
It was set in a PV device, and a phosphate buffer solution (hereinafter, referred to as PBS) was flowed at a flow rate of 0.1 ml / min. This flow rate was determined by examining the rate at which adherent cells did not detach before and after the measurement (data not shown).

When the photocurrent response in PBS was stabilized, the influent was switched to a 50 μM-estradiol 17β solution prepared in PBS to exchange the solution in the culture solution layer 8 and the change over time in the photocurrent response was measured. As a control, a semiconductor chip on which no cells were fixed was also measured in the same manner.

3-2. Measurement results [Culture and immobilization of animal cells] Cell adhesion on the semiconductor chip was observed with a microscope (FIG. 3). The upper diagram of FIG. 3 shows a used 35 mm × 18 mm semiconductor chip,
Five regions of mm × 1.5 mm are insulating films in which a silicon oxide film and a silicon nitride film are coated on a silicon substrate, and these portions play a role of sensing a potential change. FIG.
The photo below shows cells fixed in this area. From this result, it was confirmed that cells could be cultured on the semiconductor chip.

[Measurement of pH change using buffer] pH 7
When the solvent was changed from the buffer to the buffer of pH 9, a change in photoelectric flow of about 30 nA was observed (FIG. 4). From this, it was confirmed that the present SPV device can measure a change in pH as a change in photoelectric flow.

Here, an increase in the photoelectric flow rate means that it has changed to alkaline, and a decrease in the photoelectric flow rate means that it has changed to acidic.

[Measurement of Photoelectric Flow Rate Using Phosphate Buffer Solution as Solvent] The results are shown in FIG. In the figure, “without cells” indicates the temporal change of the photoelectric flow due to only the silicon chip, and “with cells” indicates the temporal change of the photoelectric flow when the chip having cells fixed to the silicon chip is used as described above. Show. Further, at the time of the arrow, the solution of the culture solution layer 8 was changed to the estradiol 17β solution, and this time was set to time 0 minute, and the change with time of the photoelectric flow rate was measured.

Without the cells, there was no change before and after the injection of estradiol 17β, and the photoelectric flow increased continuously over time. This means that there is no pH difference between PBS and estradiol in PBS. It also shows that there is no adsorption of estradiol to the semiconductor chip.

On the other hand, with cells, the photoelectric flow decreases from about 15 minutes. That is, this indicates that the metabolism of the cell group was activated by the added estradiol.

When the presence or absence of cell detachment due to the flow rate before and after the photoelectric flow measurement was observed with a microscope, the influence of the flow on cell adhesion was not remarkable.

[0043]

According to the apparatus of the present invention, the effects of various substances on the metabolic activity of cells can be measured simply, promptly and with high sensitivity. Also, by using cells that have receptors for environmental hormones,
It is possible to detect and quantify environmental hormones with very high sensitivity, quickly and easily. In addition to the detection of environmental hormones, by using cells having reactivity to a desired substance, a desired substance is measured by measuring a change in the metabolic activity of the cell, thereby placing the cell. The nutritional status of the environment can also be easily analyzed. Therefore, the device of the present invention, for example, environmental monitoring for detecting pesticides, heavy metals and carcinogens,
Further, for example, application to food monitoring for detecting or quantifying amino acids, nucleic acids, vitamins, and the like is also possible.

[Brief description of the drawings]

FIG. 1 is a diagram showing a biosensor of the present invention.

FIG. 2 is a view showing a biosensor of the present invention.

FIG. 3 is a micrograph showing cells fixed on a semiconductor chip.

FIG. 4 is a graph showing the results of measuring pH using the apparatus of the present invention.

FIG. 5 is a graph showing the results of measuring the effect of estrogen on the metabolic activity of cells using the device of the present invention.

[Explanation of symbols]

1. Conductor 2. Semiconductor 3. Insulating layer 4. 6. Living cells or living cell group Electrode 8. Culture layer 9.
Light emitting diode 10. Outflow channel 11. electrode

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12M 1/34 C12Q 1/00 G01N 27/416 G01N 33/48-33/98

Claims (1)

(57) [Claims] 1. A biosensor for measuring the effect of a test substance on metabolic activity of cells and detecting environmental hormones, comprising: a semiconductor layer; an insulating layer laminated on a main surface of the semiconductor; An MIS structure comprising a living cell or a living cell group sensitive to an environmental hormone disposed thereon in contact with the layer, and a culture solution layer in which the living cell or the living cell group is immersed and in contact with the insulating layer Body, bias electrode means for applying a bias voltage to the MIS structure, and light source means for irradiating the MIS structure with controlled light to generate photovoltaic power in the MIS structure And a means for adding a test substance to the culture solution layer, and a means for monitoring a change in photovoltaic power generated in the MIS structure, wherein the test substance is added. Before and after Biosensor characterized from the change in the photovoltaic, said test substance to measure the effect on metabolic activity of the cells.