JP2937498B2 - Apparatus for capturing biological substances and method for observing biological substances using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to DNA, bacteria,
The present invention relates to a device for capturing biological substances such as fibrinogen and an observation method using the same.

[0002]

2. Description of the Related Art In recent years, a scanning tunneling microscope (Scan) has been proposed.
Ning Tunneling Microscope
e, hereinafter referred to as STM) or atomic microscope (Atomic)
BACKGROUND ART Various objects are observed on the order of sub-nanometers to μm using Force Microscope (hereinafter referred to as AFM). In the study of surface interface properties and the like, a solid surface prepared as a sample is directly observed.
On the other hand, when observing a minute sample such as a biological substance, the sample is first fixed on graphite prepared as a preparation and then observed.

FIG. 6 is a perspective view showing a state in which such a conventional sample is fixed on graphite and observed. As shown in this figure, a sample 2 was placed on a graphite 1 as a preparation, and a probe 3 was moved onto the graphite 1 for observation. As described above, in the conventional method, observation is performed while fixing the sample on the graphite 1 prepared as a preparation. In that case, sample 2
Since the posture and the direction of the sample when is fixed are not controlled, the sample 2 is cluttered on the graphite 1 as shown in FIG. STM and AFM for observing biological materials are required to have a wide field of view (for example, “A small scanning tunnel”).
lng microscope with large
scan range for biologica
l studios "by R.L. Emch, P .; De
scouts et al, Journal of M
icroscopy, Vol. 152, Pt1, Oct
ober 1988, pp. 139-143. 85-92).

[0004]

However, in the above-mentioned conventional observation method, (1) the sample 2 is cluttered on the graphite 1 as a preparation, so that an effective sample is searched from a wide observation area. There must be.
(2) Further, since the sample 2 is cluttered on the graphite 1 as a preparation, crossing of the sample 2 is likely to occur. (3) The capture state changes from time to time, and the accuracy (reproducibility) of identification of the sample 2 is low. In addition, (4)
Since the sample 2 is placed on the graphite 1 as a preparation, the sample 2 is crushed on the graphite 1.
(5) Since the sample is observed from only one direction, the image is rounded by the effective tip radius of the probe (for example, “Sca”).
nunning tunneling microscopic
e of cytoskeleton protein
s: Microtubules and interm
edit Filaments "by Stuar
t Hameroff et al, J. Mol. Vac. Sc
i. Technol. A8 (1), Jan / Feb, 1
990, pp. 687-691). For example, as shown in FIG. 7, S of microtubules 4 as a biological material obtained by X-ray diffraction
The TM image is as shown in FIG.

The present invention has been made to solve the above problems.
By applying an electric field, the manipulation and capture of a sample as a bio-related substance can be performed quickly and accurately, and a bio-related substance capture device that allows the sample to be observed in a more original form And an observation method using the same.

[0006]

In order to achieve the above object, the present invention provides (A) an apparatus for capturing a biological substance, comprising: a preparation; a pair of opposed comb-shaped electrodes formed on the preparation; An electric field is applied between a pair of comb-shaped electrodes, and means for capturing a biological substance is provided between the teeth of the comb-shaped electrodes.

[0007] In addition, a groove is formed in a capturing part of the preparation in which the biological substance is captured, so that the biological substance is captured like a suspension bridge. Further, a plurality of electrodes are arranged at the bottom of the concave groove, and the biological substance is displaced by controlling the potential of the electrodes. (B) In the method for observing a biological substance, an electric field is applied between a pair of opposed comb-shaped electrodes formed on a preparation,
A biological substance is captured between the teeth of the comb-shaped electrode, and the captured biological substance is observed by scanning a probe. (C) In the method of observing a biological substance, a concave groove is formed in a capturing portion of a preparation, and an electric field is applied between a pair of comb electrodes facing each other on both sides of the concave groove, and a biological substance is interposed between teeth of the comb electrode. A substance is captured in a suspension bridge shape, and the captured biological substance is observed by scanning a probe. (D) In the method of observing a biological substance, a concave groove is formed in a capturing portion of a preparation, and an electric field is applied between a pair of comb-shaped electrodes opposed to both sides of the concave groove, and a biological substance is interposed between teeth of the comb-shaped electrode. The substance is captured in a suspension bridge shape, an electrode is provided at the bottom of the groove, the potential of the electrode is controlled, and the probe is scanned and observed while rotating the captured biological substance. is there. (E) In the biological substance capturing apparatus, a pair of capturing needles facing the three-dimensional space, and means for applying an electric field between the pair of capturing needles to capture the biological substance are provided. It is designed to observe a substance three-dimensionally.

[0008] Further, a radial electrode is formed around the biological substance. (F) In the method of observing a biological substance, an electric field is applied between a pair of capturing needles facing the space to capture the biological substance, and the biological substance is three-dimensionally observed with a probe. Things. (G) In the method of observing a biological substance, an electric field is applied between a pair of capturing needles facing the space to capture the biological substance, and a radial electrode centering on the biological substance is provided. By applying the potential, the biological substance is three-dimensionally observed with a probe while rotating.

[0009]

According to the present invention, (1) an electric field is applied along a sample capturing surface (preparation surface) to capture samples of biological substances in a row. Therefore, the reproducibility of the observation result of the sample is verified by orderly capturing the same sample in a row, and the accuracy of sample identification can be improved.
(2) A pair of needles for capturing a sample that is a bio-related substance is prepared, and a linear sample is captured between the pair of needles by applying an electrostatic field therebetween. Thus, the sample can be observed without using the preparation, and the sample can be observed in a form closer to the original shape.
(3) The sample captured between the capturing needles is rotated by applying a rotating electrostatic field. Thereby, the influence of the rounding of the image due to the effective radius of the tip of the observation probe can be reduced.

[0010] Therefore, it is possible to quickly and accurately capture biologically relevant substances, to improve the reliability of observation of biologically relevant substances, which is increasing more and more, and to automate them.

[0011]

Embodiments of the present invention will be described below in detail with reference to the drawings. FIG. 1 is a view showing a method for observing a biological substance according to a first embodiment of the present invention.
FIG. 1A is a perspective view of a capture preparation used for the sample, FIG. 1B is a view showing a sample captured on the capture preparation,
It is a perspective view which shows the state which performs observation.

Here, the capturing preparation is manufactured by applying a semiconductor integrated circuit manufacturing technique, and fine opposing comb-shaped electrodes are formed. First, when observing biological substances (DNA, bacteria, fibrinogen, etc.), FIG.
As shown in FIG. 1A, a capturing comb electrode 12 and a capturing comb electrode 13 are formed on a capturing slide 11 so as to face each other, and a terminal 1 is provided between the comb electrodes 12 and 13.
A voltage is applied via 5 and 16, and an electric field (for example, 10 ⁶ V / m) is applied between opposing comb-shaped electrode teeth 12a and 13a. Here, the applied voltage is usually an alternating current, but a direct current may be used.

Next, a solution in which a sample 14 as a bio-related substance is dispersed is introduced onto the capture preparation 11 with a voltage applied between the capture comb electrodes 12 and 13. Then, as shown in FIG. 1B, when the sample 14 is captured, the sample 14 is arranged under the influence of the electric field on the surface of the capturing preparation 11, and is captured between each tooth 12a and each tooth 13a of the comb-shaped electrode. Is done. Thereafter, the observation is performed in the liquid as it is, or the liquid is removed and the observation is performed with the probe 15 of STM or AFM in a dry state.

The voltage and frequency applied between the comb electrodes 12 and 13 can be appropriately set according to the type and conditions of the sample 14 to be captured. In addition, the comb-shaped electrode 12
The tips of the teeth 12a and 13a of and
It is desirable to make it easy to capture FIG. 2 is a view showing an apparatus for capturing a biological substance and an observation method using the same according to a second embodiment of the present invention.

As shown in FIG. 1, in this embodiment, (1) the sample is two-dimensionally crushed, which is a conventional disadvantage. (2) A four-probe AFM is configured to solve problems such as that an image is rounded by the effective tip radius of the probe because observation is performed from only one direction. That is, as shown in FIG. 2, a pair of capturing needles 21 and 22 are opposed to each other, a voltage is applied between the capturing needles 21 and 22 via terminals 25 and 26, and an appropriate electric field is applied. Then, the sample 23 which is a biological substance is captured.

Thus, the sample 23 can be captured without being constrained two-dimensionally. Therefore, if the observation by STM or AFM is performed by moving the probe 24, three-dimensional observation can be performed in a shape close to the original shape of the sample. FIG. 3 is a view showing an apparatus for capturing a biological substance and an observation method using the same according to a third embodiment of the present invention.

As shown in this figure, in this embodiment, a radial electrode 27 whose axis is in the direction in which a sample is captured is further arranged in the biological substance capturing apparatus of FIG. The sample 23 is captured by applying a voltage that rotates about the sample 23 to the capture needles 21 and 22.
Can be rotated about its axis while being captured by the Thereby, rounding of the image due to the effective radius of the tip of the probe 24 can be reduced, and accurate three-dimensional observation can be performed.

FIG. 4 is a view showing an apparatus for capturing biological substances and an observation method using the same according to a fourth embodiment of the present invention. As shown in this figure, in this embodiment, each tooth of the comb-like electrode for capture is arranged in an overhang state in the space. That is, a concave groove 32 is formed in the sample capturing portion of the capturing preparation 31, and the teeth 33 a of the capturing comb-shaped electrode 33 and the teeth 34 a of the capturing comb-shaped electrode 34 project from both sides of the concave groove 32 and face each other. I have. Then, an appropriate voltage is applied between these catching comb electrodes 33 and 34 via terminals 36 and 37, and the catching comb electrodes 33 and 34 are applied.
An electric field is applied between the respective teeth 33a and 34a. Therefore, the sample 35 is captured without being two-dimensionally crushed between the teeth 33a and 34a of the capturing comb electrodes 33 and 34.

Therefore, the solution in which the sample is dispersed is placed in the groove 32.
To be introduced. Then, when being captured, the samples 35 are arranged under the influence of the electric field on the surface, and are captured between each tooth 33a and each tooth 34a of the comb-shaped electrode. After that, the observation is performed in the liquid as it is, or the liquid is removed and the observation is performed with a probe 38 of STM or AFM in a dry state. FIG. 5 is a view showing an apparatus for capturing biological substances and an observation method using the same according to a fifth embodiment of the present invention.

As shown in this figure, in this embodiment, the trapping device for the biological substance shown in FIG.
A plurality of strip-shaped electrodes 39 are formed on the bottom of the substrate. Therefore, by controlling the potential applied to these electrodes 39, the captured sample 35 is rotated (displaced) all at once, and the sample 35 is observed by the probe 38 in the same posture (rotation angle). be able to.

Further, it is possible to confirm that the sample has been captured by the above-described biological substance capturing apparatus as follows. (1) Regarding FIGS. 2 and 3, the sample 23 is
In order to confirm that it has been captured between the
In a state where the force detection function as the probe of the AFM is provided to the first and second 22, the capturing needles 21 and 22 are vibrated minutely, and it can be seen that the capturing is performed when the amplitude changes greatly. it can. (2) Alignment of Capture Needles As for the pair of capture needles in FIGS. 2 and 3, the smaller the sample, the more strict mutual alignment needs to be performed. Therefore, the capturing needle is provided with a force detecting function as an AFM probe. Therefore, by observing the other capturing needle using one capturing needle, the vertexes of the two can be matched. This allows for strict alignment,
Samples can be captured in known very small areas.

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

[0023]

As described above in detail, according to the present invention, an electric field is applied along a capturing surface (preparation surface) of a sample which is a biological substance, and the same linear sample is captured in a row. Since the same sample is orderly captured in a row, the reproducibility of the observation result of the sample is verified, and the accuracy of sample identification can be improved.

A pair of needles for capturing a sample are prepared, and a linear sample is captured between the pair of needles by applying an electrostatic field therebetween. Thus, the sample can be observed without using the preparation, and the sample can be observed in a form closer to the original shape. Further, the sample captured between the capturing needles is rotated by applying a rotating electrostatic field. Thereby, the influence of the rounding of the image due to the effective radius of the tip of the observation probe can be reduced.

Therefore, according to the present invention, it is possible to quickly and accurately capture biologically relevant substances, to improve the reliability of observation of biologically relevant substances, which is increasing, and to achieve automation thereof. The effect provided by the present invention is remarkable.

[Brief description of the drawings]

FIG. 1 is a view showing a method for observing a biological substance according to a first embodiment of the present invention.

FIG. 2 is a view showing a biological substance capturing apparatus and an observation method using the same according to a second embodiment of the present invention.

FIG. 3 is a view showing a device for capturing biological substances and an observation method using the same according to a third embodiment of the present invention.

FIG. 4 is a view showing a biological substance capturing device and an observation method using the same according to a fourth embodiment of the present invention.

FIG. 5 is a view showing a biological substance capturing apparatus and an observation method using the same according to a fifth embodiment of the present invention.

FIG. 6 is a perspective view showing a state where a conventional sample is fixed on graphite and observed.

FIG. 7 is a perspective view of a microtubule as a biological material obtained by conventional X-ray diffraction.

FIG. 8 is a diagram showing an STM image of a conventional biological material.

[Explanation of symbols]

 11, 31 Capture preparation 12, 13, 33, 34 Comb electrode 12a, 13a, 33a, 34a Tooth of comb electrode 14, 23, 35 Sample (biological substance) 15, 16, 25, 26, 36, 37 Terminal 17, 24, 38 Probe 21, 22 Capture needle 27 Radial electrode 32 Groove 39 Strip electrode

Claims (10)

(57) [Claims] An electric field is applied between (a) a preparation, (b) a pair of opposing comb-shaped electrodes formed on the preparation, and (c) a pair of comb-shaped electrodes, between the teeth of the comb-shaped electrodes. Means for capturing a biologically-related substance. 2. The bio-related substance capturing device according to claim 1, wherein a groove is formed in the bio-related substance capturing portion of the preparation, and the bio-related substance is captured in a suspension bridge shape. 3. A plurality of electrodes are arranged at a bottom of the groove,
The bio-related substance capturing device according to claim 1, wherein the bio-related substance is displaced by controlling a potential of the electrode. 4. An electric field is applied between a pair of opposed comb-shaped electrodes formed on a preparation, a biological substance is captured between the teeth of the comb-shaped electrode, and a probe is scanned with the captured biological substance. Observation method of biological substances to observe. 5. A groove is formed in a capturing part of the preparation,
An electric field is applied between a pair of comb-shaped electrodes opposed to both sides of the concave groove to capture a biological substance between the teeth of the comb-shaped electrode in a suspension bridge shape, and scan the probe with the captured biological substance. Observation method of the biological substance to be observed. 6. A groove is formed in a capturing part of the preparation,
An electric field is applied between a pair of comb-shaped electrodes opposed to both sides of the concave groove, a biological substance is trapped between the teeth of the comb-shaped electrode like a suspension bridge, an electrode is provided at the bottom of the concave groove, and the electric potential of the electrode is provided. , And scanning the probe while rotating the captured biological substance to observe the biological substance. 7. A pair of capturing needles facing a three-dimensional space, and (b) means for applying an electric field between the pair of capturing needles to capture a biological substance, and (c) An apparatus for capturing a biological substance, wherein the biological substance is observed three-dimensionally. 8. The biological substance-trapping device according to claim 7, wherein a radial electrode is formed around the biological substance. 9. A method for observing a biologically relevant substance in which an electric field is applied between a pair of capturing needles facing a space to capture a biologically relevant substance, and the biologically relevant substance is three-dimensionally observed with a probe. 10. An electric field is applied between a pair of capturing needles facing a space to capture a biological substance, a radial electrode centered on the biological substance is provided, and a potential is applied to the radial electrode. A method of observing a biologically related substance in which a biologically related substance is three-dimensionally observed with a probe while rotating the biologically related substance.