JP2001330582A - Cell respiratory activity evaluation method and its device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell respiratory activity evaluation method and its device capable of evaluating metabolic activities such as respiratory activity or photosynthetic activity in a sample solution including a biosample such as a cell, by measuring a local oxygen concentration change near the sample solution. SOLUTION: The sample solution A is set in a capillary tube 1, and an oxygen permeable film 2 is arranged on an capillary tube opening, and the tube is dipped in a measuring solution B. A micro-electrode 6 is installed near the capillary tube opening, and a reference electrode 7 and a counter electrode 8 corresponding to the micro-electrode 6 are arranged. The micro-electrode 6 is used as a working electrode, and potential control and current detection are executed between the reference electrode 7 and the counter electrode 8. The micro-volume sample solution A is prepared, and three-dimensional position control of the capillary tube 1 is executed, and the oxygen concentration in the sample is selectively evaluated by a reduction current.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for evaluating cell respiratory activity for measuring changes in local oxygen concentration near a sample solution.

[0002]

2. Description of the Related Art Recent advances in life science technology have made it possible to elucidate the cell division and cell differentiation mechanisms of animal and plant cells at the molecular and cellular levels. However, in many cases, conventional measurement techniques require a large number of cells,
It is inevitable that the cells are denatured or killed by the treatment or operation before measurement.

[0003] Therefore, there is a need for the development of a technique capable of measuring cell viability and cell differentiation ability at the single cell level while keeping the cells alive.

Specifically, by accurately measuring the metabolic activity of cells, animal and plant cells including fertilized eggs (embryos) and protoplasts can be (1) applied as a method for evaluating viability and quality, and (2) differentiated. It can be used as a technique for measuring the function and production of a physiologically active substance, and (3) applied as a technique for selecting a gene-introduced cell.

[0005] Measurement of a change in oxygen concentration due to metabolic activity of a biological sample such as a cell, particularly respiratory activity, is one of the most basic and simple indicators for evaluating the function of a biological sample. In oxygen concentration measurement, a Clark-type oxygen electrode already exists as a conventional technique, and oxygen concentration evaluation using an oxygen reduction current has been performed. The Clark-type oxygen electrode is composed of an oxygen-permeable membrane, an electrolytic solution, and an electrode, and is widely applicable to not only a cell suspension but also a sample solution in which a change in oxygen concentration is expected. The major advantages of applying the Clark-type electrode to the measurement of the oxygen concentration of a biological sample solution can be summarized in two ways: selective permeation of oxygen and prevention of penetration of electrode active substances and electrode contaminants other than oxygen. It has established itself as a high-quality analytical method.

[0006]

However, it is impossible to evaluate a plurality of samples at a time with one electrode without having a spatial resolution. Although remarkable results have been achieved in miniaturization of electrodes by making full use of microfabrication technology, it is hard to say that a device for minimizing the volume of a sample to be measured is sufficient. Therefore, usually 10
Require a large amount of cells that ⁵ -10 ^7.

An electrochemical microscope (SECM) is a measurement technique for imaging a distribution of a specific chemical species by scanning a microelectrode as a probe and moving the probe close to the sample surface while precisely controlling the position of the probe. [A. J. Bard e
t al. Anal. Chem. 61, 132-138
(1989); C. Engstrom et a
l. Anal. Chem. 58, 844-848 (19
86)].

Although the technique was originally developed for evaluating the characteristics of the electrode surface, it has already been applied to the evaluation of the function of a biological sample and the measurement of oxygen concentration. In the present invention, the measurement principle can be said to be similar to the electrochemical microscope. However, there has been no report of a study in which special measures were taken to enhance oxygen selectivity. That is, in the oxygen concentration measurement using an electrochemical microscope, which has been reported so far, substances that undergo an electrode reaction in a potential region where oxygen reduction occurs in a measurement solution are excluded in advance. At the same time, in the measurement solution, substances such as proteins that contaminate the surface of the probe electrode are excluded in advance. That is, at present, the oxygen concentration can be measured only in a measurement solution having a composition different from that of a culture solution, which is an ideal environment for growing cells and biological tissues of a sample.

Further, in the research example of oxygen concentration measurement using a microelectrode which is a probe of an electrochemical microscope, in addition to the research example using an unmodified electrode as it is as described in the item of the electrochemical microscope, an electrode There are also research examples intended to improve oxygen selectivity and electrode surface contamination prevention ability by modifying and adding functions [Y. Y. Lau et al. Ana
l. Chem. 64, 1702-1705 (199
2); T. Kennedy et al. Ana
l. Chem. 71, 3642-3649 (199
9)]. However, modification of the surface of the microelectrode requires an extremely advanced technique and has a problem in reproducibility.

The present invention has been made in view of the above circumstances, and has as its object to provide a method and apparatus for evaluating cell respiratory activity as described below.

(1) When measuring a substance released / absorbed by a cell using a microelectrode, the electrode is brought into closest contact with the cell. However, according to this measurement principle, the amount of the substance that can be detected by the microelectrode is very small compared to the total amount of the substance actually released / absorbed by the cells. That is, from the viewpoint of detection sensitivity / efficiency, the case where the conventional microelectrode method can be applied to the measurement of a metabolic substance derived from a single cell is very limited. In contrast, the present invention prevents dilution / diffusion of a substance to be measured,
An object of the present invention is to provide a method for realizing a small volume of a sample solution in order to detect a substance efficiently. The miniaturization of the sample volume also plays a role in greatly reducing the number of cells contained in the sample solution, and enables one cell to be confined in the sample container.

(2) Next, the present invention provides a method of separating a sample solution and a measurement solution, and limiting a substance which can move between the two solutions to a substance to be measured. According to the present invention, a protein or an electrochemically active species which is contained in the sample solution side and adversely affects electrochemical detection is prevented from entering the measurement solution side, and at the same time, a cytotoxic substance contained in the measurement solution side Can be prevented from entering the sample solution side. By applying the present invention, the microelectrode itself does not need to be functionalized, and an unmodified microelectrode can be used as it is.

The present invention measures the local oxygen concentration change in the vicinity of the sample solution based on the above (1) and (2), thereby obtaining the metabolic activity, that is, the respiratory activity in the sample solution containing the biological sample such as cells. An object of the present invention is to provide a method for evaluating a cell respiratory activity capable of evaluating a photosynthetic activity and a method for evaluating the concentration of a substance to be measured in a sample solution without directly inserting an electrode that provides the device. .

[0014]

In order to achieve the above object, the present invention provides: [1] In a method for evaluating cell respiratory activity, a sample solution is set in a capillary tube, and an oxygen-permeable membrane is provided in the capillary tube opening. And immersed in the measurement solution, provided a microelectrode near the capillary port, arranged a reference electrode and a counter electrode corresponding to the microelectrode, the microelectrode as a working electrode, between the reference electrode and the counter electrode Control the electric potential and detect the current to prepare a sample solution having a very small volume, control the three-dimensional position of the capillary tube, and selectively evaluate the oxygen concentration of the sample by the reduction current.

[2] The method for evaluating cell respiratory activity according to [1], wherein a spatial distribution of the reduction current is recorded and output as an image.

[3] In the cell respiratory activity evaluation device, a capillary tube in which a sample solution is set, an oxygen-permeable membrane disposed in the capillary tube opening, and a filter tube which is immersed in a measuring solution and which is connected to the capillary tube opening. A microelectrode provided in the vicinity, a reference electrode and a counter electrode corresponding to the microelectrode, the microelectrode as a working electrode, the reference electrode, a potentiostat for performing potential control and current detection between the counterelectrode, A precision positioning device for controlling the three-dimensional position of the capillary tube.

[0017]

Embodiments of the present invention will be described below in detail with reference to the drawings.

FIG. 1 is a configuration diagram of an apparatus for electrochemically detecting an object to be measured according to an embodiment of the present invention.

As shown in this figure, a cell suspension is used as a sample in a culture solution base, and is filled in a glass capillary tube 1 having an inner diameter of 0.55 mm. When the sample solution height in the capillary tube 1 is 40 mm, the sample volume is estimated to be 10 μL. One side of the mouth of the capillary tube 1 is covered with an oxygen-permeable membrane 2 and plays a role of restricting a substance that can flow between the sample solution A side and the measurement solution B side to only the oxygen to be measured.

The position of the capillary tube 1 is defined as an XYZ stage,
Control was performed by a precision positioning device 3 including a three-axis stepping motor and a stage controller. The positioning accuracy is 0.1 μm. A measuring solution (phosphate buffer) B is immersed in the liquid tank 9, and a platinum microelectrode (electrode radius: 2.0 μm, a total radius of 1 including the glass seal portion 6A) is applied to the probe.
5 μm) 6 and fixed in the measurement solution B such that the electrode surface faces upward. In the electrochemical measurement, the microelectrode 6 was used as a working electrode, the silver-silver chloride electrode 7 was used as a reference electrode, and a platinum wire was used as a counter electrode 8,
The potential control and the current detection were performed by the potentiostat 5.

The potential of the microelectrode 6 was fixed at -0.6 V with respect to the silver-silver chloride electrode serving as the reference electrode 7, and the reduction current of oxygen was measured. Capillary tube 1 on which oxygen permeable membrane 2 is fixed
By scanning the horizontal direction with the position of the opening close to 100 μm near the microelectrode 6, the oxygen concentration distribution near the opening of the capillary tube 1 can be measured, and the oxygen concentration in the sample solution A can be evaluated. The computer (PC) 4 simultaneously controlled the electrochemical measurement system and the capillary drive system, and recorded the measurement results.

Animal cells (HeLa-S3 cells) were used as samples. HeLa-S3 cells are RITC80-
7 (manufactured by Asahi Techno Glass Co.) in 5% fetal bovine serum (IC
N Biomedical).
The cells were cultured for 4 to 5 days in a T-25 flask (manufactured by Corning Incorporated) to make them almost saturated. The cells were dispersed with a 0.05% trypsin solution (GIBCO-BRL) to give a single cell suspension.

The number of cells was measured with a hemocytometer, and the viable cell ratio was measured by trypan blue staining immediately before the measurement of respiratory activity.
Experiments were performed on cell suspensions of ≧%. Cell concentration 1.4 × 10 ⁶ and 2.0 × 10 ⁵ cells / mL
Was filled into the capillary tube 1.

FIG. 2 is a diagram showing an image of the two-dimensional distribution of the oxygen reduction current in the vicinity of the sample according to the embodiment of the present invention, and shows a decrease in the oxygen concentration as the color shifts from white to black.

In this figure, animal cells (He
La-S3) Oxygen reduction current distribution (1020 μm × 1020 μm) near the capillary tube mouth when using the suspension, and increase and decrease of the oxygen reduction current are shown by color bars. The cell concentration of the suspension was 14000 cells / 10 μL [see FIG. 2 (a)] and 2000 cells / 10 μL.
[(Refer to FIG. 2 (b).] In both FIG. 2 (a) and FIG. 2 (b), a decrease region of the oxygen reduction current is observed in the capillary tube 1 due to a decrease in the oxygen concentration derived from cell respiration. The larger the number of cells, the greater the amount of oxygen consumed by respiration, and the greater the amount of decrease in oxygen reduction current.

That is, a remarkable decrease in the oxygen reduction current value due to the respiration of the animal cells in the capillary was observed. The magnitude of the current decrease clearly reflects the difference in the cell concentration of the sample suspension, and it is also possible to quantitatively compare the oxygen consumption of the sample. Since the sample volume is extremely small as 10 μL, the cell concentrations are 1.4 × 10 ⁶ and 2.0 ×
At 10 ⁵ cells / mL, the total number of cells was 140
Oxygen concentration derived from metabolism of a very small number of cells was 00 and 2000.

The reason why a minute change in oxygen concentration can be measured in a system having a small total number of cells is that, in the present invention, the sample volume is small and the change in oxygen concentration is concentrated at one location (one end of the capillary). It is due to being. By using a capillary with a thinned tip as a container for the sample solution to further reduce the sample volume, the effect can be expected to appear more remarkably.

The total number of cells in the sample can be controlled by the concentration of the suspension, and a change in oxygen concentration derived from a single cell can be measured. Hanemo (Bryopsis) as a sample
plumosa) protoplasts were used. Janemo was collected in July 1972 at Murotohama, Miyadojima, Naruse-cho, Momou-gun, Miyagi Prefecture, and has been subcultured under continuous irradiation of 1.3 klx white fluorescent light. The honey was cut 1 cm from the tip and the cytoplasm was squeezed out of the small pieces. When left in seawater for 20 to 30 minutes, the cytoplasm aggregates spherically to form protoplasts.
The size and number of protoplasts filled in the capillary were directly observed with an optical microscope.

FIG. 3 shows the results of imaging the oxygen concentration distribution in the vicinity of the sample by using the measuring method of the present invention using one protoplast of 200 μm in diameter as a sample showing an example of the present invention. That is, FIG. 3A is a diagram showing an oxygen reduction current distribution (1020 μm × 510 μm) near the mouth of the capillary tube 1 when one protoplast of plant cell Hanemo is used as a sample. (B) shows a state without irradiation.

As shown in FIG. 3A, under non-irradiation,
Since plant cells generate oxygen by photosynthesis, the oxygen concentration increases in the capillary and an area where the oxygen reduction current increases is observed, whereas, as shown in FIG. A reduced region of the oxygen reduction current is observed due to the resulting oxygen consumption.

As a result, an increase in oxygen concentration due to photosynthesis under light irradiation and a decrease in oxygen concentration due to respiration under no light irradiation could be imaged.

As described above, according to the present invention, a change in oxygen concentration accompanying the metabolism of a biological sample such as a cell can be measured with a small sample volume and with high selectivity. (1) Use a capillary tube to handle a small volume sample. According to the present invention, a sample solution of about 10 μL can be handled with almost negligible reliability such as evaporation.

(2) An oxygen permeable membrane is fixed to one end of the capillary tube. As a result, the sample solution and the measurement solution can be separated from each other, and substances that can be transferred between the sample solution and the measurement solution can be limited to only oxygen.

(3) The microelectrode is moved from the measurement solution side toward the oxygen-permeable membrane at the boundary between the measurement solution and the sample solution, and is scanned in parallel with the oxygen-permeable membrane. Measure the concentration distribution of oxygen. The balance of oxygen moving between the sample solution and the measurement solution is evaluated by measuring the oxygen reduction current with a microelectrode. The spatial arrangement between the probe and the sample is controlled by a precision positioning device.

Although the probe side is fixed and the sample side is scanned in the present invention, the spatial arrangement between the probe and the sample can be similarly controlled in the opposite manner (fixing the sample / scanning the probe). Since the present invention has a spatial resolution, it is possible to evaluate a plurality of samples.

The present invention is not limited to a biological sample in principle and can be widely applied to evaluation of a solution sample in which a change in oxygen concentration is expected. By using a membrane that selectively transmits a specific molecular species instead of the oxygen-permeable membrane, measurement other than oxygen is also possible.

Techniques such as drug diagnosis using metabolic activity at the cell level as an index have attracted attention as a screening method that can replace current animal experiments. A system that can measure efficiently and easily is expected to contribute to the advancement of fields such as biochemistry and clinical medicine where new methods for evaluating cell functions and diagnostic methods are desired to be developed.

It should be noted that the present invention is not limited to the above embodiment, but various modifications are possible based on the spirit of the present invention, and these are not excluded from the scope of the present invention.

[0039]

As described above, according to the present invention, the following effects can be obtained.

(A) By measuring the change in local oxygen concentration near the sample solution, it is possible to evaluate the metabolic activity, that is, the respiratory activity and photosynthetic activity in the sample solution containing the biological sample such as cells. Fields in which measurement with a small volume of solution as a sample and measurement of changes in oxygen concentration caused by a small number of cells or a single cell are desired include cells in parts of animals and plants that cannot be obtained in large quantities as tissue specimens, such as reproduction. It is expected to be applied to cell activity evaluation.

(B) Further, the present invention can be applied to the case of evaluating the respiratory activity of cells and microorganisms having high mobility, which was difficult to measure by the conventional microelectrode method. Since the change in the entire oxygen concentration within a limited volume can be evaluated, even if the sample moves around, there is no problem in the measurement.

(C) When the oxygen concentration of a plurality of samples is evaluated, the present invention is applied to the case in which the measurement system represented by the microelectrode is simply replaced with the capillary tube and the oxygen-permeable membrane, and the oxygen concentration of the sample is exchanged. It is expected that many samples will be evaluated. Furthermore, it is expected that the oxygen concentration of a plurality of samples can be evaluated in one image by using one having two solution spaces in one capillary (theta tube) or using a multi-barrel capillary tube.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of an apparatus for electrochemically detecting an object to be measured according to an embodiment of the present invention.

FIG. 2 is a diagram showing an image of a two-dimensional distribution of an oxygen reduction current near a sample, showing an example of the present invention.

FIG. 3 shows a sample of 200 μm in diameter according to an embodiment of the present invention.
FIG. 6 is a diagram showing the results of imaging the oxygen concentration distribution near the sample by using the measurement method of the present invention using one m-protoplasm of Hanemo.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Capillary tube 2 Oxygen permeable membrane 3 Precision positioning device 4 Computer (PC) 5 Potentiometer 6 Microelectrode (Platinum microelectrode) 6A Glass seal part 7 Reference electrode (silver-silver chloride electrode) 8 Counter electrode 9 Liquid tank Solution A Sample solution (Cell suspension) Solution B Measurement solution (phosphate buffer)

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) G01N 27/46 301M (72) Inventor Hiroyuki Abe 3-5-27 Kagota, Yamagata City, Yamagata Prefecture (72) Inventor Hiroshi Hoshi 5-10-5-C102 F-term (reference) 2J045 BB04 CB01 DB30 FA19 FB05 GC20 HA14 JA01 JA04 JA07 4B063 QA01 QQ20 QR72 QR73 QR77 QR78 QR82 QX05

Claims (3)

[Claims] 1. A sample solution is set in a capillary tube, (b) an oxygen permeable membrane is arranged in the capillary tube, and (c) a sample solution is immersed in the measurement solution, and the sample solution is placed near the capillary tube. (D) arranging a reference electrode and a counter electrode corresponding to the micro electrode, (e) using the micro electrode as a working electrode, performing potential control and current detection between the reference electrode and the counter electrode, and (f) 3.) A method for evaluating cell respiratory activity, comprising preparing a sample solution having a small volume, controlling the three-dimensional position of the capillary tube, and selectively evaluating the oxygen concentration of the sample by a reduction current. 2. The method for evaluating cell respiratory activity according to claim 1, wherein the spatial distribution of the reduction current is recorded and output as an image. 3. A capillary tube in which a sample solution is set, (b) an oxygen-permeable membrane disposed in the capillary tube, and (c) a capillary tube immersed in a measurement solution. (D) a reference electrode and a counter electrode corresponding to the micro electrode, and (e) a potential control and current detection between the reference electrode and the counter electrode with the micro electrode as a working electrode. And (f) a precision positioning device for controlling the three-dimensional position of the capillary tube.