JP2007120967A - Microforce measuring instrument and biomolecule observation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To observe dynamic properties such as structural analysis or the like by accurately measuring a biomolecule such as a protein molecule or the like while preventing the lowering of sensitivity. <P>SOLUTION: This microforce measuring instrument 1 is equipped with first and second probes 2 and 3 arranged so as to be opposed to each other and formed into a cantilevered state so that the respective leading ends thereof become free ends to fix the biomolecule P to the main surfaces 2a and 3a, a variable means 4 for allowing the first and second probes to approach or separate relatively while keeping a state that the main surfaces are opposed to each other to vary the mutual distance of the main surfaces, a measuring means 5 for respectively measuring the bendings of the first and second probes and an analyzing means 6 for analyzing the biomolecule on the basis of the measuring result of the measuring means 5. During a period varying the mutual distance of the main surfaces by the variable means 4 in a state that the biomolecule is respectively fixed to the first and second probes, the bending change of the first and second probes is measured by the measuring means. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a micro force measurement apparatus and a biomolecule observation method for performing various observations such as structural analysis of biomolecules such as proteins.

In the post-genomic era, molecular biology approaches are demanding high-resolution and multi-functional observations for morphological observation and functional analysis of biological samples such as DNA and proteins, and other biological molecules and cells. Among them, measuring the length of biomolecules such as DNA, RNA and proteins, and investigating various functions such as geometric structure and mechanical properties are the analysis (clarification) of biomolecules. Therefore, it is very useful in many fields including biochemistry and medicine.
In such an analysis, a biomolecule, for example, a protein having a three-dimensional structure, is stretched from the vicinity of both ends to form a single linear polypeptide, thereby examining the mechanical properties of the protein and the structure. A protein nanomechanical method for performing analysis is known (see, for example, Non-Patent Document 1).

As shown in FIG. 24, this method is a method of stretching protein P using a probe 50 of a scanning probe microscope such as an atomic force microscope (AFM).
First, as shown in FIG. 24, the substrate 51 and the probe 53 provided at the tip of the cantilever 52 are activated with a silanizing agent or the like, and the vicinity of both ends of the protein P using a crosslinking agent or the like Fix by chemical bonding. After fixing, the probe 50 is slowly moved away from the substrate surface. Thereby, the protein P is gradually stretched. At this time, as shown in FIG. 25, the force (tension) acting on the cantilever 52 is measured. Thereby, the mechanical property of the protein P can be investigated and the structural analysis of the protein P can be performed.

  In particular, depending on the type of protein P, a knot (bonded portion) (A and B shown in FIG. 24) in which a polymer chain is locally entangled may be generated in the stage of the three-dimensional structure before being stretched. In this case, when the above-described pulling operation is performed, the knots A and B are separated (bonding is separated). When the knots A and B are separated, the tension acting on the cantilever 52 is instantaneously reduced as shown in FIG. Moreover, the magnitude | size of the coupling | bonding force of each knot A and B etc. can be detected by seeing the reduction | decrease degree of the tension | tensile_strength in this case.

  Thus, by examining the mechanical properties of a protein, the protein can be viewed from a dynamic viewpoint, and more versatile structural analysis and the like can be performed. By the way, in order to examine this mechanical property, it is necessary to detect the bending of the probe. This is because the force applied to the probe appears as bending of the probe.

There are various methods for detecting the deflection, and one of them is a self-detection method (see, for example, Patent Document 1).
This self-sensing method is a method in which a piezoresistor whose resistance value changes as the probe is bent is provided on the probe itself, and the amount of change in the resistance value is detected as the amount of bending of the probe. The force F applied to the probe at this time is expressed by F = k (probe spring constant) × x (probe deflection (displacement)).
JP-A-9-304409 Kunio Takeyasu, “Nanobiology Life Science with Nanotechnology”, First Edition, Kyoritsu Publishing Co., Ltd., July 15, 2004, p.52-61

However, the following problems remain in the conventional self-detection method.
That is, when measuring biomolecules, especially when measuring the stretch of proteins, the spring is not used without using a hard probe with a large spring constant in order to clearly detect the binding portion that is bound by a minute force. It is common to use a soft probe with a small constant. However, in the case of the self-detection method, as described above, the force F applied to the probe is expressed by F = k × x. Therefore, when a probe having a soft spring constant is used, the resistance value changes with respect to the displacement amount. Will decrease. As a result, the sensitivity was inferior and it was difficult to accurately measure biomolecules such as proteins.

  The present invention has been made in view of such circumstances, and its purpose is to accurately measure biomolecules such as proteins while preventing deterioration in sensitivity, and to accurately measure mechanical properties such as structural analysis. It is an object of the present invention to provide a micro force measurement device and a biomolecule observation method that can be observed at once.

In order to achieve the above object, the present invention provides the following means.
The micro force measurement device of the present invention is arranged so as to face each other, and is formed in a cantilever shape so that the tip is a free end, and a first probe and a second probe that can fix a biomolecule to the main surface. A variable means for varying a distance between the probe and the first probe and the second probe by relatively approaching or separating the first probe and the second probe while maintaining the state in which the principal surfaces face each other; Measuring means for measuring the deflection of each of the first probe and the second probe; and an analyzing means for analyzing the biomolecule based on a measurement result by the measuring means. In the state in which the biomolecule is fixed to each of the first probe and the second probe, a change in deflection is measured while the distance is varied by the variable means. That.

  In addition, the biomolecule observation method of the present invention includes a first probe and a first probe that are arranged so as to face each other, are cantilevered so that their tips are free ends, and can fix biomolecules to the main surface. A biomolecule observation method for observing a biomolecule using the probe of No. 2, wherein a first fixing step of fixing one end of the biomolecule to the main surface of the first probe, and the second A second fixing step of fixing the other end of the biomolecule to the main surface of the first probe, and after the first fixing step and the second fixing step, the first probe and the second probe. And a variable step of varying the distance between the first probe and the first probe in the variable step, while maintaining a state in which the principal surfaces face each other. 2 Probe deflection A measuring step of measuring the each, based on the measurement result by said measuring step, and is characterized in that it comprises an analyzing step of analyzing the biomolecule.

  In the micro force measurement device and the biomolecule observation method according to the present invention, first, the main surface of the first probe is activated with a silanizing agent or the like and a cross-linking agent or the like is used to end one end of a biomolecule such as a protein. A first fixing step is performed in which is fixed by chemical bonding. Next, a second fixing step of fixing the other end of the biomolecule to the main surface of the second probe is performed by relatively moving the second probe and the first probe closer to each other by the variable means. Thereby, both ends of the biomolecule are fixed to the main surface of the first probe and the main surface of the second probe, respectively.

  After the biomolecule is fixed, a variable step is performed in which the first probe and the second probe are relatively separated by the variable means while maintaining the main surfaces facing each other. Thereby, for example, a protein having a three-dimensional structure is stretched at both terminals (stretched) to become one linear polypeptide. In addition, while performing this variable process, a measurement process is performed in which the measurement means measures the change in deflection of both probes. And based on the measurement result by this measurement process, the analysis process which analyzes a biomolecule with an analysis means is performed.

Thereby, for example, the tension required to obtain the above-described linear polypeptide can be measured. In addition, proteins usually have knots (binding sites) in which polymer chains are entangled, but these knots are broken when pulled by both probes. At this time, for example, until just before the knots are unwound, the knots pull both probes due to the coupling force, so the deflection of both probes increases. Further, at the moment when the knot is broken, the force acting on both probes instantaneously decreases, so that the bending becomes smaller.
In this way, by performing the measurement process and the analysis process, it is possible to investigate the mechanical properties such as the tension necessary for making a linear polypeptide and the magnitude of the binding force of the knot, and to analyze the structure of the protein. It can be carried out.

In particular, even if soft probes with small spring constants are used as the first probe and the second probe, unlike the conventional case, measurement is performed in a state where the biomolecule is pulled from both ends by two probes. The same force can be applied to both probes. Therefore, unlike the conventional case where measurement is performed with one probe, the sensitivity can be doubled by about two times. Therefore, biomolecules such as proteins can be measured accurately, and mechanical properties such as structural analysis can be observed with high precision. Since both ends of the biomolecule are pulled by two probes, As compared with, it is possible to increase the moving distance when the same force is applied, and to improve the moving resolution.

  Moreover, the micro force measuring device of the present invention is the micro force measuring device of the present invention, wherein at least one of the first probe and the second probe includes a probe on the main surface. It is characterized by.

  In the micro force measuring apparatus according to the present invention, since at least one of the first probe and the second probe has a probe on the main surface, it is aimed at immobilizing biomolecules such as proteins. Only the biomolecules can be reliably fixed to the tip of the probe. Therefore, the amount of the biomolecule that is the measurement target substance can be reduced as much as possible. Therefore, measurement results of a large amount of biomolecules do not overlap, and a force displacement curve that is easier to analyze can be obtained.

  Further, the micro force measuring device of the present invention is the micro force measuring device of the present invention, wherein the conductive films respectively formed on the main surfaces of the first probe and the second probe, and the conductive film A voltage applying means for applying a predetermined voltage between the first probe and the second probe is provided.

  The biomolecule observation method of the present invention is the biomolecule observation method of the present invention described above, wherein the first fixing step and the second fixing step are performed between the first probe and the second probe. A predetermined voltage is applied to the biomolecule, and the biomolecules are dielectrophoresed and fixed to the main surfaces of the first probe and the second probe, respectively.

  In the micro force measurement device and the biomolecule observation method according to the present invention, the first probe and the second probe are interposed between the first probe and the second probe through the conductive film by the voltage application means in the first fixing step and the second fixing step. By applying a predetermined voltage between them, biomolecules such as proteins can be dielectrophoretically drawn to the main surfaces of both probes. Therefore, both ends of the biomolecule can be reliably fixed to both probes at substantially the same timing. Therefore, the biomolecule can be more easily fixed, and the time required for the fixing process can be further shortened. Moreover, even if the protein is a single molecule, it can be easily and reliably fixed to both probes.

  Further, the micro force measuring device according to the present invention is the micro force measuring device according to any one of the above inventions, wherein the first probe and the second probe have the same direction from the proximal side to the distal side. It is characterized by being arranged in parallel so as to face.

  In the micro force measuring device according to the present invention, the first probe and the second probe are arranged in parallel so as to face the same direction. That is, they are arranged so as to be doubled like tweezers. Therefore, the installation space occupied by the first probe and the second probe can be suppressed as much as possible, and downsizing can be achieved. Moreover, since the base end sides of both probes can be aligned on one side, it can be easily moved by the variable means, and the configuration can be simplified.

  Moreover, the micro force measuring device of the present invention includes the microscope device for observing the biomolecule fixed by the first probe and the second probe in any of the micro force measuring devices of the present invention. It is characterized by being.

  In the micro force measurement apparatus according to the present invention, the biomolecule can be observed, for example, by fluorescence using a microscope apparatus such as an inverted microscope while the biomolecule is being pulled by both probes. In this way, in addition to the measurement by the measuring means, various observations (for example, observation of which binding site of the protein is separated, etc.) can be simultaneously performed using a microscope apparatus, so that biomolecules can be observed from various angles. Can do.

  Further, the micro force measurement device of the present invention is the micro force measurement device of the present invention described above, wherein the variable means maintains both the relative positional relationship between the first probe and the second probe. The direction of the probe can be integrally changed.

  In the micro force measurement device according to the present invention, after both ends of the biomolecule are fixed to the first probe and the second probe, respectively, the relative positional relationship between the probes is maintained by the variable means. The direction can be changed integrally. Thereby, a biomolecule can be located in the position where it is easy to observe with a microscope apparatus. And after this position adjustment, it measures, observing with a microscope apparatus. As described above, since the position of the biomolecule can be adjusted before the observation with the microscope apparatus, it is easy to use and the operability is improved and the reliability of the measurement result can be improved.

  Further, the micro force measuring device of the present invention is the micro force measuring device according to any one of the above-described present invention, wherein the measuring means is provided in each of the first probe and the second probe, and according to the deflection amount. A first strain detecting element and a second strain detecting element whose resistance values change, and a calculation circuit for calculating a deflection change of the first probe and the second probe based on a resistance value change of both strain detecting elements; It is characterized by having.

In the micro force measurement device according to the present invention, a change in deflection (a change in strain) is detected by a self-sensing method using strain detection elements provided in the first probe and the second probe, respectively. That is, when the first probe is bent, the resistance value of the first strain detecting element changes according to the amount of bending. Similarly, when the second probe is bent, the resistance value of the second strain detecting element changes according to the amount of bending. A calculation circuit such as a Wheatstone bridge circuit calculates a change in deflection of both probes based on the change in resistance value of both strain detection elements.
In this way, since the deflection can be measured by a self-detection method instead of a general optical lever method, an optical system for irradiating and receiving laser light becomes unnecessary, the configuration can be simplified, and the size can be reduced. Can be planned.

  The micro force measuring apparatus of the present invention is characterized in that, in the micro force measuring apparatus of the present invention, the strain detecting element is a piezoresistive element.

  In the micro force measuring device according to the present invention, since the strain detecting element is a piezoresistive element, the strain detecting element can be easily and accurately built at a target location using a semiconductor process. Therefore, the deflection (distortion) of both probes can be detected more accurately.

  The micro force measuring device of the present invention is the micro force measuring device of the present invention, further comprising a reference element for correcting environmental conditions in the vicinity of the first strain detecting element and the second strain detecting element. It is characterized by that.

  In the micro force measuring device according to the present invention, since the reference element is provided, it is possible to cancel the influence of electrical characteristics generated by various annular conditions such as temperature conditions. Therefore, the resistance values of both strain detection elements can be more accurately changed according to the amount of deflection without being affected by environmental conditions such as temperature. As a result, the reliability of the measurement result can be improved.

  The biomolecule observation method of the present invention is the biomolecule observation method of the present invention described above, wherein the first fixing step is performed on a probe provided on the main surface of the first probe. One end is fixed.

  In the biomolecule observation method according to the present invention, when fixing one end of a biomolecule such as a protein, only the targeted biomolecule can be reliably fixed to the tip of the probe. Therefore, the amount of the biomolecule that is the measurement target substance can be reduced as much as possible. Therefore, measurement results of a large amount of biomolecules do not overlap, and a force displacement curve that is easier to analyze can be obtained.

  Further, the biomolecule observation method of the present invention is the biomolecule observation method of the present invention described above, wherein the second fixing step is performed on a probe provided on the main surface of the second probe. The other end is fixed.

  In the biomolecule observation method according to the present invention, when fixing the other end of a biomolecule such as a protein, only the targeted biomolecule can be reliably fixed to the tip of the probe. Therefore, the amount of the biomolecule that is the measurement target substance can be reduced as much as possible. Therefore, measurement results of a large amount of biomolecules do not overlap, and a force displacement curve that is easier to analyze can be obtained.

  Further, the biomolecule observation method of the present invention is the biomolecule observation method of any one of the present invention described above, wherein a fluorescent substance is labeled on the biomolecule before the first immobilization step and the second immobilization step. A fluorescent labeling step, and the measuring step includes an observation step of observing the movement of the fluorescent substance during the variable step.

According to the biomolecule observation method according to the present invention, first, before performing the first fixing step and the second step, the fluorescent labeling step of labeling the biomolecule with a fluorescent substance is performed. Thereby, for example, a protein knot is labeled with a fluorescent substance. Thereafter, an observation step of observing the movement of the fluorescent substance with an inverted microscope or the like is performed during the variable step. Thereby, the location where the knot is broken by being pulled by both probes can be clearly determined by the movement of the fluorescent material.
As described above, in addition to the measurement by the measuring means, various observations using the fluorescent substance can be performed, so that the biomolecules can be observed from various angles.

  According to the micro force measurement device and the biomolecule observation method according to the present invention, even if a soft probe having a small spring constant is used as the first probe and the second probe, the biomolecules are obtained using two probes. Since the measurement is performed in a state pulled from both terminal sides, the same force can be applied to both probes by the action and reaction. Therefore, unlike the conventional case where measurement is performed with one probe, the sensitivity can be doubled by about two times. Therefore, biomolecules such as proteins can be accurately measured, and mechanical properties such as structural analysis can be observed with high accuracy.

Hereinafter, a first embodiment of a micro force measurement apparatus and a biomolecule observation method according to the present invention will be described with reference to FIGS. In the present embodiment, a protein is described as an example of a biomolecule.
As shown in FIG. 1, the micro force measuring device 1 according to the present embodiment is disposed to face each other, is formed in a cantilever shape so that the tip is a free end, and the protein is formed on the main surfaces 2a and 3a. The first probe 2 and the second probe 3 capable of fixing P, and the first probe 2 and the second probe 3 are kept relative to each other while maintaining the principal surfaces 2a and 3a facing each other. Based on the measurement results obtained by the measuring means 5, the measuring means 5 for measuring the deflection of the first probe 2 and the second probe 3, respectively. And a personal computer (hereinafter referred to as PC) (analyzing means) 6 for analyzing the protein P.

  The first probe 2 is formed of, for example, an SOI substrate in which three layers of a silicon support layer, an oxide layer, and a silicon active layer are thermally bonded. As shown in FIG. 10 and a support portion 11 that supports the base end side of the lever portion 10 in a cantilevered manner. In addition, an opening 12 is formed on the base end side of the lever portion 10, which is a joint portion between the lever portion 10 and the support portion 11, and the lever portion 10 is more bent and easily bent on the base end side. . That is, the base end side of the lever portion 10 functions as a stress concentration portion where stress concentrates. The number of the openings 12 is not limited to one, and two or more openings may be formed or may not be formed.

A piezoresistive element (first piezoresistive element) 13, which is a strain detection element whose resistance value changes according to the amount of deflection of the first probe 2, is provided on the base end side of the support portion 11 and the lever portion 10. The lever 12 is provided on both sides of the opening 12 in the longitudinal direction. The piezoresistive element 13 is formed by implanting impurities into an SOI substrate by an ion implantation method, a diffusion method, or the like.
Further, a metal wiring 14 such as aluminum is electrically connected to the piezoresistive element 13, and the entire shape combined with the metal wiring 14 is formed in a U shape. Further, the end portions of the metal wiring 14 are electrically connected to the two external connection terminals 15, respectively. That is, the current flowing from one external connection terminal 15 to the metal wiring 14 passes through one piezoresistive element 13, then flows around the opening 12 and flows to the other piezoresistive element 13, and then the other external connection It flows from the terminal 15 to the outside.
An insulating film (not shown) is formed on the piezoresistive element 13 and the metal wiring 14 so as not to be in electrical contact with the outside.

Further, in the first probe 2 of this embodiment, the reference lever portion 16 is supported by the support portion 11 in a cantilever state adjacent to the vicinity of the lever portion 10. The reference lever portion 16 is formed to be slightly shorter than the lever portion 10, and has an opening 12 on the base end side as in the lever portion 10, and a reference piezoresistive element for correcting environmental conditions (see Element) 17 is provided. An external connection terminal 15 is also electrically connected to the reference piezoresistive element 17 via a metal wiring 14.
In the present embodiment, the reference piezoresistive element 17 is used for temperature compensation of the piezoresistive element 13. The reference lever portion 16 is not essential and may not be provided.

Similar to the first probe 2, the second probe 3 is formed of, for example, an SOI substrate in which three layers of a silicon support layer, an oxide layer, and a silicon active layer are thermally bonded, as shown in FIG. Thus, the flat lever portion 20, the probe 20a formed at the tip on the main surface 3a of the lever portion 20, and the support portion 21 that supports the base end side of the lever portion 20 in a cantilever manner. I have. Similarly to the first probe 2, a piezo that is a strain detection element in which an opening 22 is formed on the base end side of the lever portion 20 and the resistance value changes according to the amount of bending of the second probe 3. Resistive elements (second piezoresistive elements) 23 are provided on both sides of the opening 22 in the longitudinal direction of the lever portion 20. Similarly, the piezoresistive element 23 is electrically connected to the metal wiring 24 and the two external connection terminals 25.
An insulating film (not shown) is formed on the piezoresistive element 23 and the metal wiring 24 so as not to be in electrical contact with the outside.

Further, in the second probe 3 of the present embodiment, the reference lever portion 26 is supported in a cantilever state by the support portion 21 and the opening 22 is formed on the proximal end side, similarly to the first probe 2. A reference piezoresistive element 27 for correcting environmental conditions is also provided.
And the 2nd probe 3 comprised in this way is being fixed to the mount frame which is not shown in figure via the support part 21. FIG.

  On the other hand, as shown in FIG. 1, the first probe 2 is placed on an XYZ stage 30 that moves coarsely and finely in the XY and Z directions via a support portion 11. Thereby, the 1st probe 2 and the 2nd probe 3 move relatively with respect to XY direction and Z direction, and a mutual distance changes. The XYZ stage 30 is, for example, a piezoelectric element that can move in three directions, and moves in three directions in accordance with a voltage applied from the drive unit 31. That is, the XYZ stage 30 and the drive unit 31 constitute the variable means 4.

In addition, the external connection terminals 15 and 25 of the first probe 2 and the second probe 3 are electrically connected to the strain detector 32, respectively. The strain detector 32 applies a predetermined voltage to the piezoresistive elements 13 and 23 via the external connection terminals 15 and 25 and the metal wirings 14 and 24, respectively, and changes the resistance values of the piezoresistive elements 13 and 23. A bridge balance circuit (calculation circuit) 33 for calculating the bending change of the first probe 2 and the second probe 3 is provided.
As shown in FIG. 4, the bridge balance circuit 33 is a circuit in which four resistors R1, R2, R3, and R4 are connected and one of two opposing vertices is an input voltage E1 and the other is an output voltage E2. It is. Here, the piezoresistive element 13 of the first probe 2 is connected as a resistor R2, and the piezoresistive element 23 of the second probe 3 is connected as a resistor R4.

The bridge balance circuit 33 outputs an output voltage E2 determined by the input voltage E1 and the resistance values of the four resistors R1, R2, R3, and R4. Therefore, when both the probes 2 and 3 are bent and the resistance values of the piezoresistive elements 13 and 23 are changed, the bridge balance circuit 33 outputs an output voltage E2 corresponding to the resistance change. That is, by monitoring this output voltage E2, the change in deflection of both probes 2, 3 can be calculated.
In particular, the output voltage E2 of the bridge balance circuit 33 is proportional to a value obtained by subtracting (sum of distortions of the resistors R1 and R3) from (sum of distortions of the resistors R2 and R4). In the present embodiment, since both the piezoresistive elements 13 and 23 are connected as the resistors R2 and R4, it is possible to calculate the deflection change of both the probes 2 and 3 with high sensitivity.

  That is, the piezoresistive elements 13 and 23, the metal wirings 14 and 24, the external connection terminals 15 and 25, and the bridge balance circuit 33 constitute the measuring means 5 described above. The measuring means 5 measures the change in deflection while the distance between the first probe 2 and the second probe 3 is varied by the variable means 4 while the protein P is fixed to the first probe 2 and the second probe 3, respectively. It is controlled by PC6. The bridge balance circuit 33 outputs the output voltage E2 to the PC 6.

  On the other hand, the PC 6 calculates the tension (tensile force) and the like necessary for pulling the protein P based on the output voltage E2 that has been sent, and performs structural analysis such as the mechanical properties of the protein P. Yes. The PC 6 comprehensively controls each component such as the variable means 4 and the first probe after the protein P is fixed to the probes 2 and 3 from the moving distance of the XYZ stage 30. The movement distance of 2 can be calculated, and the degree of change in tension with respect to the movement distance can be calculated.

Next, a biomolecule observation method for observing the protein P by the micro force measurement apparatus 1 configured as described above will be described below.
In the biomolecule observation method of the present embodiment, the first fixing step of fixing one end P1 of the protein P to the main surface 2a of the first probe 2 and the probe provided on the main surface 3a of the second probe 3 are performed. After the second fixing step of fixing the other end P2 of the protein P to the needle 20a, the first fixing step, and the second fixing step, the first probe 2 and the second probe 3 are respectively attached to the main surface. A variable step of changing the distance between the two probes by relatively approaching or moving away from each other while maintaining the state in which the surfaces 2a and 3a face each other, and a change in deflection of the first probe 2 and the second probe 3 during the variable step A measurement step for measuring the protein P, and an analysis step for analyzing the protein P based on the measurement result of the measurement step.
Each of these steps will be described in detail below.

First, as an initial setting, the main surface 2a of the first probe 2 is activated with a silanizing agent or the like, and preparation is performed in advance so that the protein P is chemically bound using a crosslinking agent or the like. Similarly, the surface of the probe 20a of the second probe 3 is prepared so that the protein P is chemically bound using a silanizing agent, a cross-linking agent, or the like.
Next, as shown in FIG. 5, a first fixing step is performed in which one end P <b> 1 of the plurality of proteins P is fixed on the main surface 2 a of the first probe 2 by chemical bonding.

Next, as shown in FIG. 6, a voltage is applied from the drive unit 31 to the XYZ stage 30 to cause the XYZ stage 30 to move coarsely toward the second probe 3. As a result, the first probe 2 and the second probe 3 approach each other with the main surfaces 2a and 3a facing each other. Then, when the first probe 2 and the second probe 3 approach each other by a certain distance, the coarse movement is switched to the fine movement as shown in FIG. Thus, a desired protein P is selected from the plurality of proteins P fixed on the main surface 2a of the first probe 2, and the other end P2 of the protein P and the probe 20a of the second probe 3 are selected. Can be contacted. As a result, as shown in FIG. 8, the other end P2 of the protein P can be fixed to the tip of the probe 20a. By this second fixing step, the selected protein P is in a state where both ends are fixed to the first probe 2 and the second probe 3.
In addition, other protein P which was fixed on the main surface 2a of the 1st probe 2 and was not selected has the other end P2 in the state which became a free end, as shown in FIG.

After the protein P is fixed, a voltage is again applied from the driving unit 31 to the XYZ stage 30 to finely move the XYZ stage 30 in the Z direction as shown in FIG. 9, and the first probe 2 and the second probe 3 are moved. Are changed while maintaining the state in which the main surfaces 2a and 3a face each other. As a result, the protein P taking a three-dimensional structure can be stretched (stretched) to form a single linear polypeptide.
And while performing this variable process, while performing the measurement process which each measures the bending change of both the probes 2 and 3 by the measurement means 5, based on the measurement result of this measurement means 5, protein P is made by PC6. The analysis process to analyze is performed.

That is, while performing the variable process, both the probes 2 and 3 are pulled by the protein P, so that they are bent. Here, when the first probe 2 is bent, the resistance value of the first piezoresistive element 13 changes in accordance with the amount of bending. That is, the resistance value of the resistor R2 of the bridge balance circuit 33 changes. Further, when the second probe 3 is bent, the resistance value of the second piezoresistive element 23 changes according to the amount of bending. That is, the resistance value of the resistor R4 of the bridge balance circuit 33 changes. As a result, the bridge balance circuit 33 outputs an output voltage E2 corresponding to the deflection amount to the PC 6.
And PC6 can measure the bending change of both probes 2 and 3 from output voltage E2 sent, and can measure the tension required to make protein P into one linear polypeptide. it can.

In addition, as shown in FIG. 9, protein P usually has knots (binding sites) A and B in which polymer chains are entangled, but these knots A and B are pulled when pulled by both probes. Will be solved. At this time, until the knots A and B are unwound, the knots A and B pull the both probes 2 and 3 by the coupling force, so that the bending of both the probes 2 and 3 increases. Further, at the moment when the knots A and B are unwound, the force acting on both probes 2 and 3 instantaneously decreases, so that the bending becomes smaller.
In this way, by measuring the bending change of both probes 2 and 3 during the variable process, as shown in FIG. 10, the mechanical properties such as the magnitude of the binding force of the knots A and B can be examined, Structural analysis of protein P can be performed.

  In particular, according to the micro force measurement apparatus 1 of the present embodiment, even if soft probes having a small spring constant are used as the first probe 2 and the second probe 3, the protein P is obtained by two probes unlike the conventional case. Since measurement is performed in a state of being pulled from both terminals P1, P2, the same force can be applied to both probes 2, 3 by action and reaction. Therefore, unlike the conventional case where measurement is performed with one probe, the sensitivity can be doubled by about two times. That is, among the four resistors R1, R2, R3, and R4 of the bridge balance circuit 33, the resistors R2 and R4 can be used as the resistors of the piezoresistive elements 13 and 23, so that a larger output voltage E2 can be output and the sensitivity can be increased. Can be increased. Therefore, mechanical properties such as structural analysis can be observed with high accuracy.

Further, since the second probe 3 includes the probe 20a and the other end P2 of the protein P is fixed to the tip of the probe 20a during the second fixing step, only the target protein P is reliably detected. It can be fixed to the tip of the needle 20a. Therefore, it becomes easy to fix the protein P, the operability is improved, and the time required for the fixing process can be shortened.
In addition, since the deflection is measured not by a general optical lever system but by a self-detection system using the piezoresistive elements 13 and 23, an optical system for irradiating and receiving laser light becomes unnecessary, and the configuration can be simplified. In addition, downsizing can be achieved.

  Furthermore, since the first probe 2 and the second probe 3 are provided with reference piezoresistive elements 17 and 27, respectively, the influence of electrical characteristics generated by various environmental conditions such as temperature conditions can be offset. it can. The reference piezoresistive elements 17 and 27 are the resistor R1 and the resistor R3 shown in FIG. 5, respectively. That is, although the resistance values of the piezoresistive elements 13 and 23 change depending on the temperature conditions in addition to the bending of the probes 2 and 3, the temperature influences by referring to the reference piezoresistive elements 17 and 27. Can be offset. As a result, the reliability of the measurement result can be further improved.

Next, a second embodiment of the micro force measurement apparatus and biomolecule observation method according to the present invention will be described with reference to FIGS. Note that the same reference numerals in the second embodiment denote the same components as those in the first embodiment, and a description thereof will be omitted.
The difference between the first embodiment and the second embodiment is that the probe 20a is provided only on the second probe 3 in the first embodiment, but in the second embodiment, the first probe 40 is provided. However, as shown in FIG. 11, the probe 41 is provided at the tip of the lever portion 10 as in the first probe 3.

When the protein P is observed using the first probe 40 configured as described above, first, the surface of the probe 41 of the first probe 40 is used with a silanizing agent, a crosslinking agent, or the like. Then, the protein P is prepared in advance so as to be chemically bonded. Next, as shown in FIG. 11, the first probe 40 is finely moved by the XYZ stage 30 to approach the protein P, and the first end P1 of the protein P is fixed to the probe 41 as shown in FIG. 1 fixing process is performed.
Next, as shown in FIG. 13, the first probe 40 is coarsely moved by the XYZ stage 30 to approach the second probe 3 by a certain distance. Then, as shown in FIG. 14, the first probe 40 is finely moved to bring the other end P <b> 2 of the protein P closer to the probe 41 of the first probe 40. Then, as shown in FIG. 15, the other end P2 of the protein P is fixed to the tip of the probe 41 of the first probe 40. As a result, the protein P is fixed to the tips of the probes 20a and 41 of both probes 40 and 3, respectively. After that, as shown in FIG. 16, the first probe 40 is finely moved so that both the probes 40 and 3 are separated from each other, and the protein P is pulled to start the measurement.

  Thus, by providing the probes 20a and 41 on the probes 40 and 3 respectively, both ends P1 and P2 of the protein P can be more reliably fixed. Therefore, the amount of protein P that is a measurement target substance can be reduced as much as possible. Therefore, the measurement results of a large amount of protein P do not overlap, and a force displacement curve that is easier to analyze can be obtained.

Next, a third embodiment of the micro force measurement apparatus and biomolecule observation method according to the present invention will be described with reference to FIGS. Note that the same reference numerals in the third embodiment denote the same parts as in the second embodiment, and a description thereof will be omitted.
The difference between the third embodiment and the second embodiment is that in the second embodiment, when the protein P is fixed to the probe 41 of the first probe 40, the first probe 40 is finely moved by the XYZ stage 30. While the probe 41 and the one end P1 of the protein P are brought close to each other while being moved, the fixation is performed. However, in the third embodiment, the protein P is dielectrophoretically attracted to both the probes 20a and 41.

  That is, the two probes 40 and 3 of the present embodiment are provided with conductive films (not shown) from the main surfaces 2a and 3a to the probes 20a and 41, respectively, so that the probes 20a and 41 have conductivity. . In addition, the micro force measuring apparatus according to the present embodiment applies a predetermined voltage between the two probes 20a and 41, that is, between the first probe 40 and the second probe 3 via a conductive film (not shown). Voltage application means is provided.

  In the case of observing the protein P with the micro force measuring apparatus configured as described above, first, as shown in FIG. 17, the first probe 40 is roughly moved by the XYZ stage 30 to move the second probe. 18, the first probe 40 is moved while a predetermined voltage is applied between the first probe 40 and the second probe 3 by the voltage applying means, as shown in FIG. Move it slightly. Then, the protein P is attracted to the probes 41 and 20a of both probes 40 and 3 by dielectrophoresis. As a result, as shown in FIG. 19, both ends P1 and P2 of the protein P can be reliably fixed to both the probes 20a and 41 at substantially the same timing.

Therefore, the protein P can be more easily and reliably fixed. Therefore, the operability can be improved and the time required for the fixing process can be further shortened.
Then, after the protein P is fixed, as shown in FIG. 20, the first probe 40 is finely moved so that both the probes 40 and 3 are separated from each other, and the measurement is started by pulling the protein P.

Next, a fourth embodiment of the micro force measurement apparatus and biomolecule observation method according to the present invention will be described with reference to FIGS. Note that in the fourth embodiment, identical symbols are assigned to configurations identical to those in the third embodiment and descriptions thereof are omitted.
The difference between the fourth embodiment and the third embodiment is that, in the third embodiment, the first probe 40 and the second probe 3 are arranged to face each other so that the proximal end sides are opposite to each other, Whereas only the first probe 40 is configured to be movable by the XYZ stage 30, the micro force measurement device of the fourth embodiment is configured such that the first probe 40 and the second probe 3 are proximal to each other. They are arranged side by side in parallel so that the direction from the tip to the tip side faces the same direction, and each of them can be moved in the XYZ directions by the XYZ stage 30.

That is, as shown in FIG. 21, the first probe 40 and the second probe 3 of this embodiment are arranged side by side in a state of being aligned in parallel so as to face the same direction. That is, they are arranged so as to be doubled like tweezers. Both probes 40 and 3 are configured to be movable in the XY directions by the XYZ stage 30.
The micro force measurement apparatus according to the present embodiment includes an inverted microscope (microscope apparatus) 45 that observes the protein P fixed by the first probe 40 and the second probe 3. The inverted microscope 45 is disposed above the first probe 40 and the second probe 3. The first probe 40 and the second probe 3 may be disposed below the first probe 40 and the second probe 3.

  A case where the protein P is observed with the micro force measuring apparatus configured as described above will be described below. Further, the biomolecule observation method of the present embodiment includes a fluorescent labeling step for labeling the protein P with a fluorescent substance before performing the first fixing step and the second fixing method, and the measuring step is in the variable step. An observation process for observing the movement of the fluorescent material is provided.

  First, the fluorescent labeling step is performed before the first fixing step and the second fixing step. Thereby, each knot of protein P is labeled with the fluorescent substance. After the labeling is completed, as shown in FIG. 21, both probes 40 and 3 are moved slightly to approach each other with the protein P sandwiched therebetween. At the same time, a voltage is applied between the probes 20a and 41. As a result, as shown in FIG. 22, the protein P can be dielectrophoretically attracted to both the probes 20a and 41, and both ends P1 and P2 of the protein P can be fixed to the probes 20a and 41, respectively.

  After the completion of the first fixing step and the second fixing step, as shown in FIG. 23, a variable step of pulling the protein P by moving the probes 40 and 3 so as to be separated from each other by the XYZ stage 30 is performed. Do. And while performing this variable process, while performing a measurement process and an analysis process, the observation process which observes the motion of a fluorescent substance with the inverted microscope 45 is performed. Thereby, the location where the knots A and B are broken by being pulled by the probes 40 and 3 can be clearly determined by the movement of the fluorescent material. Thus, in addition to the measurement by the measurement means 5, since the fluorescence observation using the fluorescent substance and the inverted microscope 45 can be performed, the protein P can be observed from various angles.

  In particular, since the deflection of both probes 40 and 3 is measured by the self-detection method, it is not necessary to install an optical system such as a laser in the vicinity of both probes 40 and 3 unlike the optical lever method. Therefore, the inverted microscope 45 can be freely installed in the vicinity of both the probes 40 and 3. Therefore, the characteristics of the inverted microscope 45 can be sufficiently extracted, and the degree of freedom in design can be improved.

In the above embodiment, the variable means 4 is configured to integrally change the directions of the probes 40 and 3 while maintaining the relative positional relationship between the first probe 40 and the second probe 3. It doesn't matter.
In this way, after the protein P is fixed by the first probe 40 and the second probe 3, the orientation is changed integrally while maintaining the relative positional relationship between the probes 40 and 3, and the protein P P can be positioned at a position where it can be easily observed with the inverted microscope 45. Thus, since the position of the protein P can be finely adjusted before starting the measurement, it is easy to use and the operability is improved and the reliability of the measurement result can be improved.

  The technical scope of the present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.

For example, in each of the above embodiments, a protein is taken as an example of a biomolecule, but is not limited to a protein.
In addition, the probe is provided on at least one of the first probe and the second probe. However, the present invention is not limited to this, and both probes are probed together like the first probe of the first embodiment. You may comprise only the flat lever part without a needle | hook. Even in this case, the same effect can be obtained.

  The probe deflection is measured by a self-sensing method using a piezoresistive element. However, the present invention is not limited to this, and the probe deflection may be measured by an optical lever method using a laser beam. .

It is a lineblock diagram of the micro force measuring device concerning a 1st embodiment of the present invention. It is a perspective view which shows the 1st probe of the micro force measuring apparatus shown in FIG. It is a perspective view which shows the 2nd probe of the micro force measuring apparatus shown in FIG. It is a figure which shows the bridge balance circuit which the distortion detection part shown in FIG. 1 has. FIG. 2 is a process diagram showing an example of a biomolecule observation method for analyzing a protein structure using the micro force measurement apparatus shown in FIG. 1, and shows a state in which one end of the protein is fixed to the main surface of the first probe. It is. FIG. 6 is a process diagram illustrating an example of a biomolecule observation method for analyzing the structure of a protein using the micro force measurement apparatus illustrated in FIG. 1, and finely moves the first probe toward the second probe from the state illustrated in FIG. 5. It is a figure which shows the state made to move. FIG. 7 is a process diagram showing an example of a biomolecule observation method for analyzing the structure of a protein using the micro force measurement apparatus shown in FIG. 1, and the protein is brought into proximity to a second probe from the state shown in FIG. It is a figure which shows the state which has moved the 1st probe finely so that the other end of may contact the probe of a 2nd probe. FIG. 8 is a process diagram showing an example of a biomolecule observation method for analyzing the structure of a protein using the micro force measurement apparatus shown in FIG. 1, and from the state shown in FIG. 7, the probe of the second probe and the other end of the protein It is a figure which shows the state which fixed. FIG. 9 is a process diagram showing an example of a biomolecule observation method for analyzing the structure of a protein using the micro force measurement device shown in FIG. 1, and the first probe and the second probe are separated from the state shown in FIG. 8. It is a figure which shows the state which has moved the 1st probe finely so that protein may be extended. It is a figure which shows the displacement of the tensile force (tensile force) which acts on a probe, when extending | stretching protein. It is process drawing which showed an example of the biomolecule observation method which performs the structural analysis of protein with the micro force measuring device which concerns on 2nd Embodiment of this invention, Comprising: The probe of a 1st probe is made to contact one end of protein Thus, it is a figure which shows the state which has moved the 1st probe finely. FIG. 12 is a process diagram showing an example of a biomolecule observation method for analyzing a protein structure using the micro force measurement apparatus according to the second embodiment of the present invention, and from the state shown in FIG. 11, the probe of the first probe It is a figure which shows the state which fix | immobilized one end of protein. FIG. 13 is a process diagram showing an example of a biomolecule observation method for analyzing the structure of a protein by the micro force measurement device according to the second embodiment of the present invention, and the second probe is inserted into the second probe from the state shown in FIG. It is a figure which shows the state which is finely moved toward the probe. FIG. 14 is a process diagram showing an example of a biomolecule observation method for analyzing a protein structure by the micro force measurement device according to the second embodiment of the present invention, approaching a second probe from the state shown in FIG. 13. FIG. 5 is a diagram showing a state in which the first probe is finely moved so that the other end of the protein contacts the probe of the second probe after being moved. FIG. 15 is a process diagram showing an example of a biomolecule observation method for analyzing a protein structure by the micro force measurement device according to the second embodiment of the present invention, and from the state shown in FIG. 14, the probe of the second probe It is a figure which shows the state which fixed the other end of protein. FIG. 16 is a process diagram showing an example of a biomolecule observation method for performing structural analysis of a protein by a micro force measurement device according to a second embodiment of the present invention. From the state shown in FIG. It is a figure which shows the state which moved the 1st probe finely so that it may space apart, and is extending the protein. It is process drawing which showed an example of the biomolecule observation method which performs the structure analysis of protein with the micro force measuring device which concerns on 3rd Embodiment of this invention, Comprising: A protein is pinched | interposed into a 2nd probe. It is a figure which shows the state which is moving the 1st probe coarsely toward. FIG. 18 is a process diagram showing an example of a biomolecule observation method for analyzing a protein structure by a micro force measurement device according to a third embodiment of the present invention, and after the state shown in FIG. FIG. 6 is a diagram showing a state in which the first probe is finely moved while applying a voltage to and the protein is attracted to both probes by dielectrophoresis. FIG. 19 is a process diagram showing an example of a biomolecule observation method for analyzing a protein structure using the micro force measurement apparatus according to the third embodiment of the present invention, and after the state shown in FIG. It is a figure which shows the state which fixed the both ends of protein. FIG. 20 is a process diagram showing an example of a biomolecule observation method for performing structural analysis of a protein by a micro force measurement device according to a third embodiment of the present invention, from the state shown in FIG. 19, the first probe and the second probe; It is a figure which shows the state which moved the 1st probe finely so that it may space apart, and is extending the protein. It is process drawing which showed an example of the biomolecule observation method which performs the structural analysis of protein with the micro force measuring device which concerns on 4th Embodiment of this invention, Comprising: A fluorescently labeled protein is pinched | interposed into tweezers form. It is a figure which shows the state which applies a predetermined voltage between both the probes, finely moves both the probes arranged in parallel, and draws the protein between the two probes by dielectrophoresis. FIG. 21 is a process diagram showing an example of a biomolecule observation method for analyzing a protein structure by a micro force measurement apparatus according to a fourth embodiment of the present invention, and after the state shown in FIG. It is a figure which shows the state which fixed the both ends of protein. FIG. 23 is a process diagram illustrating an example of a biomolecule observation method for performing structural analysis of a protein using the micro force measurement device according to the fourth embodiment of the present invention, and includes a first probe and a second probe from the state illustrated in FIG. 22. It is a figure which shows the state which is extending the protein, moving the 1st probe finely so that it may space apart, and observing the movement of a fluorescent substance with an inverted microscope. FIG. 2 is a diagram for explaining a conventional nanomechanical method for structural analysis of a protein, in which one end of the protein is fixed to a probe at the tip of a cantilever and then the protein is stretched by separating the probe. is there. It is a figure which shows the displacement of the tensile force (tensile force) which acts on a probe when protein P is extended.

Explanation of symbols

P protein (biomolecule)
P1 One end of the protein P2 The other end of the protein 1 Microforce measuring device 2, 40 First probe 2a Main surface of the first probe 3 Second probe 3a Main surface of the second probe 4 Variable means 5 Measuring means 6 PC (Analysis means)
13 Piezoresistive element (first strain detecting element, first piezoresistive element)
17, 27 Piezoresistive element for reference (reference element)
20a Probe of second probe 23 Piezoresistive element (second strain detecting element, second piezoresistive element)
33 Bridge balance circuit (calculation circuit)
41 Probe of the first probe 45 Inverted microscope (microscope device)

Claims (14)

A first probe and a second probe, which are arranged to face each other, cantilevered so that the tip thereof is a free end, and can fix a biomolecule to the main surface;
Variable means for varying the distance between the first probe and the second probe by relatively approaching or separating the first probe and the second probe while maintaining the state in which the main surfaces face each other;
Measuring means for measuring the deflection of each of the first probe and the second probe;
Analyzing means for analyzing the biomolecule based on the measurement result by the measuring means;
The measurement means measures a change in deflection while the distance between the first probe and the second probe is varied by the variable means in a state where the biomolecule is fixed to the first probe and the second probe, respectively. A micro force measuring device. In the micro force measuring device according to claim 1,
At least one of the first probe and the second probe includes a probe on the main surface. In the micro force measuring device according to claim 1 or 2,
Conductive films respectively formed on main surfaces of the first probe and the second probe;
A micro force measuring apparatus comprising voltage applying means for applying a predetermined voltage between the first probe and the second probe via the conductive film. In the micro force measuring device according to any one of claims 1 to 3,
The microscopic force measuring apparatus, wherein the first probe and the second probe are arranged in parallel so that a direction from a base end side to a tip end side is in the same direction. In the micro force measuring device according to any one of claims 1 to 4,
A micro force measuring device comprising a microscope device for observing the biomolecule fixed by the first probe and the second probe. In the micro force measuring device according to claim 5,
The micro force measuring apparatus characterized in that the variable means can integrally change the orientation of both probes while maintaining the relative positional relationship between the first probe and the second probe. In the micro force measuring device according to any one of claims 1 to 6,
The measurement means is provided in each of the first probe and the second probe, and a first strain detection element and a second strain detection element whose resistance values change according to the amount of deflection, and both strain detection elements And a calculation circuit for calculating a change in deflection of the first probe and the second probe on the basis of the change in resistance value. In the micro force measuring device according to claim 7,
The strain detecting element is a piezoresistive element. In the micro force measuring device according to claim 7 or 8,
A micro force measuring apparatus comprising a reference element for correcting environmental conditions in the vicinity of the first strain detecting element and the second strain detecting element. Using the first probe and the second probe, which are arranged so as to face each other and are cantilevered so that the tip is a free end, and can fix the biomolecule to the main surface, A biomolecule observation method for observing,
A first fixing step of fixing one end of the biomolecule to the main surface of the first probe;
A second fixing step of fixing the other end of the biomolecule to the main surface of the second probe;
After the first fixing step and the second fixing step, the first probe and the second probe are relatively approached or separated while maintaining the state in which the main surfaces face each other. Variable process for changing the distance between each other,
A measuring step of measuring a change in deflection of each of the first probe and the second probe during the variable step;
A biomolecule observation method comprising: an analysis step of analyzing the biomolecule based on a measurement result of the measurement step. The biomolecule observation method according to claim 10,
In the first fixing step, one end of the biomolecule is fixed to a probe provided on the main surface of the first probe. The biomolecule observation method according to claim 10 or 11,
In the second fixing step, the other end of the biomolecule is fixed to a probe provided on the main surface of the second probe. The biomolecule observation method according to any one of claims 10 to 12,
In the first fixing step and the second fixing step, a predetermined voltage is applied between the first probe and the second probe to cause dielectrophoresis of the biomolecule, and thereby the first probe. And a method for observing a biomolecule, wherein the method is fixed to the main surface of each of the second probes. The biomolecule observation method according to any one of claims 10 to 13,
Before performing the first fixing step and the second fixing step, a fluorescent labeling step for labeling the biomolecule with a fluorescent substance,
The biomolecule observation method, wherein the measurement step includes an observation step of observing the movement of the fluorescent substance during the variable step.

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