JP2007010518A - Target substance detection method and detection apparatus using cantilever sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To increase the surface density of a target material that adheres to the surface of a cantilever through binding with a target material recognizing material by avoiding the influence of repulsive force between target materials. Provides a detection method capable of increasing the amount of change in the resonant frequency of the cantilever and detecting the target substance with high detection sensitivity.
When a cantilever and a target material are brought into contact with a target material after applying an external force in advance, the target material is coupled with the target material recognition material in a state where the repulsive force between the target materials is small. After the substance is attached, the external force that has bent the cantilever is removed, and the amount of change in the resonance frequency of the cantilever is measured, so that the detection of the target substance can be performed with higher detection sensitivity and with a simpler procedure.
[Selection] Figure 1

Description

  The present invention relates to a method for detecting a target material using a cantilever sensor, and a target material detection apparatus based on the detection method. In particular, in a probe-type microscope such as an atomic force microscope (AFM), a target material is applied with a high detection sensitivity by applying a cantilever needle (hereinafter referred to as a cantilever) used as a probe for detecting a minute force. And a target substance detection device having high detection sensitivity based on the method.

  In the cantilever sensor, a method of detecting a change in bending stress as a change in bending amount is utilized by utilizing the characteristic that the amount of bending of a cantilever needle (hereinafter, cantilever) is proportional to the bending stress of the cantilever surface. Yes. For example, a detection probe of a probe microscope is used as a means for detecting a small force applied to the tip of a cantilever, for example, a change in atomic force with high sensitivity.

  As a method of detecting a target material by applying a cantilever sensor, a method of inducing a change in bending stress on the cantilever surface by selectively binding the target material to the cantilever surface has been studied. For example, a substance that specifically binds to a target substance (a target substance recognition substance), for example, an antibody molecule having a specific antigen-antibody reactivity to an antigen that is a target substance is placed on the surface of the cantilever. To fix. When this cantilever is brought into contact with a sample containing a target substance (for example, antigen), the target substance (for example, antigen) becomes a substance that exhibits specific binding properties (a target substance recognition substance, for example, a specific antibody). ) And attached to the surface of the cantilever. At that time, due to the load of the target material itself adhering to the surface of the cantilever tip, bending stress applied to the entire cantilever increases, and as a result, the amount of bending of the cantilever tip increases.

  In addition, since the mass m of the entire cantilever is increased due to the load of the target material itself adhering to the surface of the tip of the cantilever, the resonance frequency ω of the cantilever changes. As a method for detecting a target substance using a cantilever by applying such a principle, methods as shown in Non-Patent Documents 1 and 2 have been studied.

  The cantilever is a form of a cantilever spring, and resonates at a specific frequency when forcedly vibrated from the outside. The resonance frequency ω inherent to the cantilever is determined by the mass m of the cantilever and its spring constant k. That is, the resonance frequency ω unique to the cantilever can be expressed as in the following formula 1.

ω = (k / m) ^1/2 Equation 1
When the target material is selectively bonded to the cantilever surface, the mass m of the entire cantilever increases from m → m + Δm due to the load of the target material itself adhering to the surface of the cantilever tip. At that time, if the spring constant k of the cantilever does not change, the resonance frequency of the cantilever decreases by Δω as the mass increases. The increase amount m of the mass m is proportional to the adhesion amount of the target material. In the range where the increase amount Δm is sufficiently small with respect to the mass m of the entire cantilever, the decrease amount Δω of the resonance frequency is approximately , Proportional to the increase Δm. Accordingly, by detecting the decrease Δω of the resonance frequency, it is possible to detect the presence and amount (concentration) of the target substance adhering to the cantilever. In general, when a cantilever used in AFM or the like is used, the ratio of the change Δω in resonance frequency to the change Δm in mass: Δm / Δω is said to be several pg / Hz.
App. Phys. Lett. 69 (19), 1996 p2834-2836 Sensors and Actuators B, 2001 p122-131.

  Although the detection method of the presence and amount (concentration) of the target substance adhering to the cantilever based on the cantilever resonance frequency change Δω has high detection sensitivity as described above, it has the following problems.

  Depending on the target material, when the target materials are close to each other, a strong repulsive force may be generated between the target materials. In that case, the repulsive force between the target materials is too strong, and the target materials cannot be deposited on the cantilever surface with high density. When the concentration of the target material contained in the specimen sample increases, the surface density of the target material adhering to the cantilever surface does not increase any more due to the influence of this repulsive force. That is, the concentration of the target material contained in the specimen sample is not proportional to the surface density of the target material adhering to the cantilever surface, and as a result, an increase in the resonance frequency variation Δω of the cantilever can be suppressed. When the concentration range of the target material is reached, the concentration of the target material cannot be accurately detected.

  In other words, if the repulsive force between the target materials is too strong, when the concentration of the target material contained in the sample exceeds a certain level, the cantilever held flat is coupled with the target material recognizing material. Thus, the surface density of the target material adhering to the surface does not increase any more, and there is a possibility that the concentration of the target material cannot be properly evaluated.

  The present invention solves the above problems, and the object of the present invention is to influence the repulsive force between the target materials even when the target material recognition material is immobilized on the surface of the cantilever with a high surface density. It is possible to increase the surface density itself of the target material to be adhered through the binding with the immobilized target material recognition material, and the load can reduce the amount of change in the resonance frequency of the cantilever. An object of the present invention is to provide a detection method that can be made larger and can detect a target substance with high detection sensitivity, and a highly sensitive target substance detection apparatus based on such a detection method.

  The inventors of the present invention have advanced the research to solve the above-mentioned problems, and have obtained the following knowledge.

  First, even when the target substance recognition substance is fixed on both sides of the cantilever with the same surface density, if the cantilever is bent in advance by applying external force, it is effective between the convex side and the concave side. It was found that there is a difference in the surface density of various target substance recognition substances. That is, compared with the state without deflection, the effective surface density of the target substance recognition substance is relatively sparse on the convex side, while the effective surface density of the target substance recognition substance is on the concave side. Is relatively dense.

  If the target material that binds to the target material recognition material has a reciprocal force between the target materials when the mutual distance falls below a certain value, the target material that binds to the target material recognition material can interact with each other when the cantilever is not bent. The critical state in which repulsive force is generated corresponds to a state in which one target material is attached per target material recognition material Nflat molecule. On the other hand, such a critical state corresponds to a state where one target substance is attached per target substance recognition substance Nconvex molecule on the convex side, and one target substance per target substance recognition substance Nconcave molecule on the concave side. This corresponds to a state in which is attached. At this time, considering the surface density of the effective target substance recognition substance, Nconvex <Nflat <Nconcave, in other words, a larger amount of target substance is attached on the convex side than in the state without deflection. On the other hand, it has been found that only a smaller amount of target material can be deposited on the concave side.

  Furthermore, when the bond between the target substance recognition substance and the target substance has a certain strength or more, the target substance attached to the surface with a high surface density by forming a convex surface deflects the cantilever in the reverse direction, and the concave surface It has been found that a large repulsive force acts between the target substances, but usually does not cause separation. In this state, the surface that was previously concave is convex, and the target material is a target material recognition material Nconvex from the state where one target material is attached per target material recognition material Nconcave molecule. The adhesion surface density can be further increased to a state where one target substance is attached per molecule. Eventually, one target material may be attached to both surfaces of the cantilever per target material recognition material Nconvex molecule. Of course, when the cantilever is returned to a flat state, a repulsive force acts between the target materials. However, once the target material has adhered due to the binding with the target material recognizing material, the separation caused by the repulsive force is usually removed. I found that it did not lead to cause.

  At least the target material recognition material is fixed on both sides of the cantilever, the cantilever is bent in advance, one surface is convex, the target material is bonded to the target material recognition material, and the other surface is subsequently convex. It has been found that by binding the target material to the target material recognition material, it is possible to attach more target material than when the target material is attached with the cantilever in a flat state. That is, when more target materials can be attached to the entire cantilever, the mass increase amount Δm becomes larger, and accordingly, the resonance frequency change amount Δω can also be made larger.

  In addition, on this convex side, in order to reach a critical state in which repulsive force is generated between target substances bonded to a target substance recognition substance, one target substance is attached per target substance recognition substance Nconvex molecule. The target substance concentration Cconvex in the sample in equilibrium with the state is higher than the target substance concentration Cflat in the sample in equilibrium with the state where one target substance is attached per target substance recognition substance Nflat molecule. It was also found that That is, the upper limit concentration (detectable upper limit concentration) at which quantitative measurement of the concentration can be performed by the cantilever sensor is such that the detectable upper limit concentration Cconvex on the convex surface side when the cantilever is bent in advance by applying external force is It was found that the upper limit of the detectable cantilever surface Cflat on the cantilever surface in the absence was higher, and the effective range of quantitative measurement was expanded.

  Based on the series of findings described above, the present inventors have completed the present invention.

That is, the target substance detection method according to the first aspect of the present invention is:
A method for detecting a target substance using a cantilever sensor,
(A) A step of fixing a target substance recognition substance that can be combined with the target substance on the surface of the cantilever constituting the cantilever sensor;
(C) A step of applying an external force to the cantilever so as to make at least one surface convex and hold it in a bent state;
(D) The step of bringing the cantilever held in a bent state into contact with a sample to enable binding of the target material to the target material recognition material;
(E) removing the external force applied to the cantilever;
(F) measuring the resonance frequency of the cantilever after making contact with the sample for the purpose of binding to the target substance;
(G) calculating the difference between the resonance frequency of the cantilever after contact with the sample and the initial resonance frequency of the cantilever before contact with the sample, measured in the step (F);
A method for detecting a target substance.

Moreover, the target substance detection device according to the second aspect of the present invention is:
A detection device that detects a target substance using a cantilever sensor,
(A) a cantilever sensor including a cantilever having a target substance recognition substance fixed on the surface;
(C) means for applying an external force to the cantilever so that at least one surface is convex and held in a bent state;
(D) means for bringing the cantilever held in the bent state into contact with the sample in a state enabling the binding of the target material to the target material recognition material;
(E) means for removing external force applied to the cantilever;
(F) means for measuring the resonant frequency of the cantilever after contact with the sample for the purpose of binding to the target substance;
(H) A target material fixed on the surface of the cantilever based on a resonance frequency difference calculated from the measured resonance frequency of the cantilever after contact with the sample and the initial resonance frequency of the cantilever before contact with the sample. A target substance detection device comprising means for calculating the presence or absence of a target substance bound to a recognition substance or the amount of the bound target substance.

  In the principle of the method for detecting a target material according to the present invention, the cantilever is bent in advance, and then the cantilever and the target material are brought into contact with each other so that the repulsive force between the target materials is small and the target material is placed on the surface of the cantilever. It becomes possible to attach a substance. At that time, by selecting a form for fixing the target substance recognition substance on both sides of the cantilever, and making each cantilever bent in advance for each face, by contacting the cantilever and the target substance, The target material can be attached to the cantilever surface in a state where the repulsive force between the target materials is small. As a result, on the surface of the cantilever, both sides can be put together and a target material can be more adhered, and in this state, by measuring the change in the resonant frequency of the cantilever due to the adhesion of the target material, It is possible to detect a substance with higher sensitivity, or to obtain an effect that enables measurement by a simpler detection means.

  Furthermore, an increase in the maximum amount of the target substance adhering to the cantilever increases the measurement upper limit (saturation detection amount) of the measurement device, and the effect that the detection range is widened is also obtained.

  In addition, it is not necessary to fix the target substance recognizing substance only on one side of the cantilever, in other words, it is possible to omit the steps required to fix the target substance recognizing substance only on one side of the cantilever. There is also. In particular, if the target substance recognition substance is fixed on both sides of the cantilever, the phenomenon of warping of the cantilever due to external factors such as temperature can be avoided, and noise such as warping of the cantilever caused by this kind of external factor In order to calibrate the factors, it is possible to perform measurement with high reliability without using a comparative cantilever and adding calibration software. That is, more than the advantage of omitting an additional process for immobilizing the target substance recognition substance only on one side of the cantilever, troubles caused by external factors such as temperature caused by the difference in the state of both sides of the cantilever In addition, it greatly contributes to greatly reducing the problem of false detection.

The method for detecting a target substance according to the present invention described above,
A method for detecting a target substance using a cantilever sensor,
(A) A step of fixing a target substance recognition substance that can be combined with the target substance on the surface of the cantilever constituting the cantilever sensor;
(C) A step of applying an external force to the cantilever so as to make at least one surface convex and hold it in a bent state;
(D) The step of bringing the cantilever held in a bent state into contact with a sample to enable binding of the target material to the target material recognition material;
(E) removing the external force applied to the cantilever;
(F) measuring the resonance frequency of the cantilever after making contact with the sample for the purpose of binding to the target substance;
(G) calculating the difference between the resonance frequency of the cantilever after contact with the sample and the initial resonance frequency of the cantilever before contact with the sample, measured in the step (F);
Is a method for detecting a target substance characterized by comprising:
At that time, in order to measure the initial resonant frequency of the cantilever before contact with the sample,
Between the step (A) and the step (C),
(B) measuring the initial resonant frequency of the cantilever;
It is preferable to have a configuration in which

Furthermore, between the step (D) and the step (E),
(C ′) An external force in a direction opposite to the external force applied in the step (C) is applied to the cantilever, and the cantilever is held in a state bent in the opposite direction to the direction of the bending in the step (C). The process of
(D ′) a step of bringing the cantilever held in a state of bending in the opposite direction into contact with a sample and allowing the target substance to bind to the target substance recognition substance;
It is desirable to further include

In addition, the target substance detection device of the present invention based on the detection method of the present invention includes:
A detection device that detects a target substance using a cantilever sensor,
(A) a cantilever sensor including a cantilever having a target substance recognition substance fixed on the surface;
(C) means for applying an external force to the cantilever so that at least one surface is convex and held in a bent state;
(D) means for bringing the cantilever held in the bent state into contact with the sample in a state enabling the binding of the target material to the target material recognition material;
(E) means for removing external force applied to the cantilever;
(F) means for measuring the resonant frequency of the cantilever after contact with the sample for the purpose of binding to the target substance;
(H) A target material fixed on the surface of the cantilever based on a resonance frequency difference calculated from the measured resonance frequency of the cantilever after contact with the sample and the initial resonance frequency of the cantilever before contact with the sample. A target substance detection device comprising means for calculating the presence or absence of a target substance bound to a recognition substance, or the amount of the bound target substance,
In particular, in the means for measuring the resonance frequency of the cantilever after the contact with the sample (f), the vibrator for forcibly vibrating the cantilever used for measurement of the resonance frequency is:
(A) It can be incorporated into the cantilever sensor, or can be made to be in contact with the (a) cantilever sensor during measurement, and is generally a desirable form.

  The present invention is described in detail below.

  With reference to the drawings, a detection principle of a target substance detection method using a cantilever sensor according to the present invention will be schematically described.

As shown in FIG. 1A, the target substance recognition substance 12 is fixed in advance on the surface of the cantilever 13 installed so as to protrude from the chip 11. When the cantilever is naturally vibrated in a state where one end of the cantilever is a free end, the tip of the cantilever vibrates at a resonance frequency ω ₀ depending on the mass m of the cantilever and the spring constant k. The resonance frequency indicated by the cantilever in the state shown in FIG. 1A is referred to as “initial resonance frequency” ω _{0i of the} cantilever.

Further, when the oscillator 10 is attached to the chip 11 and the forced vibration of the frequency ω is applied, the cantilever also starts flexural vibration at the frequency ω. At this time, if the difference between the frequency ω of the forced vibration and the resonance frequency ω ₀ inherent to the cantilever is large, the amplitude of the periodic displacement of the tip of the cantilever becomes small. On the other hand, when the forced vibration frequency ω matches the resonance frequency ω ₀ unique to the cantilever, the resonance state is reached, and therefore the amplitude of the periodic displacement of the cantilever tip shows a maximum. That is, as shown in FIG. 4, when the amplitude of the periodic displacement of the tip of the cantilever at that time is plotted against the frequency ω of the forced vibration, the maximum is obtained at the resonance frequency ω ₀ inherent to the cantilever. Shows the peak.

Although the cantilever 13 can be naturally vibrated and the resonance frequency can be measured, an oscillator 10 is attached to the chip 11 and an AC voltage (not shown) is applied to the oscillator 10 to cause forced oscillation of the frequency ω. In addition, from the result of plotting the amplitude of the periodic displacement of the tip of the cantilever at that time against the frequency ω of the forced vibration as shown in FIG. 4, the frequency at which the amplitude is maximum is given to the cantilever. It can also be a measured value of the inherent resonance frequency ω ₀ .

  Next, as shown in FIG. 1B, an external force 14 is applied to the cantilever so that the cantilever on which the target substance recognition substance 12 is fixed is bent into a convex shape. The cantilever 13 and the target material 15 are brought into contact with each other while maintaining the convexly bent state. By setting the cantilever 13 in a bent state in advance, the target material recognition substance fixed on the surface is effectively sparse on the convex surface side of the cantilever as compared to a state without bending.

  As shown in FIG. 1 (c), on the convex surface side of the cantilever, the distance between the target materials combined with the target material recognition material is larger than that in the case where there is no deflection, and the repulsive force 16 between the target materials is small. Thus, the target material adheres to the convex surface of the cantilever. On the other hand, on the convex surface side of the cantilever, the target substance recognition substance fixed to the surface is effectively in a dense state as compared with the case where there is no deflection. For this reason, the target material combined with the target material recognizing material is attached to the cantilever surface only at a lower surface density in order to achieve a distance that is not affected by the repulsive force 16 between the target materials.

  Thereafter, as shown in FIG. 1 (d), except for the previous external force 14, an external force 14 ′ that causes displacement in the opposite direction is applied, so that the surface that is concave on the front side becomes the convex side. The cantilever is bent in the opposite direction. At that time, the surface density of the target material 15 combined with the target material recognition material 12 is high on the concave surface (upper side in the figure), and therefore the interval between the target materials is narrowed. Will be in a working state. However, the target material 15 once attached is in a state of being stably bonded to the target material recognizing material 12, and detachment is not caused by the repulsive force between the target materials.

  On the other hand, as shown in FIG. 1 (d), the target substance recognition substance fixed on the surface is effectively sparse on the newly convex side (lower side in the figure), The distance between the target substances already bonded to the target substance recognition substance is also wide. Therefore, as shown in FIG. 1E, the target material 15 further adheres to the surface that has newly become the convex surface side in a state where the repulsive force 16 between the target materials is small. That is, the target material 15 is in a state of being attached at a high density even on the newly convex surface.

  Finally, as shown in FIG. 1 (f), the external force 14 'that has bent the cantilever is removed, and the cantilever 13 is in a flat state. Of course, since the surface density of the target material 15 combined with the target material recognition material 12 is high, the distance between the target materials is narrowed, and in some cases, a repulsive force is exerted between the target materials. However, the target material 15 once attached is in a state of being stably bonded to the target material recognizing material 12, and detachment is not caused by the repulsive force between the target materials.

The resonance frequency of the cantilever is measured with the target material 15 attached to both surfaces of the cantilever 13 at a high density. The resonance frequency of the cantilever measured after the contact with the sample is referred to as “resonance frequency after target material adhesion treatment” ω _0f . Actually, when the target material 15 adheres to the surface of the cantilever 13 through the binding with the target material recognition material 12, the mass of the cantilever corresponds to an increase in load due to the attached target material 15 from the initial value m. It changes to m + Δm including the mass increase Δm. Accordingly, the “initial resonance frequency” ω _0i is different from the “resonance frequency after the target material deposition process” ω _0f, and the difference Δω ₀ = (ω _0i −ω _0f ) is calculated, and the difference Δω ₀ = ( Based on (ω _0i −ω _0f ), the presence or absence of the target substance on the cantilever surface and the amount of the target substance are detected.

Note that, when the “initial resonance frequency” ω _0i is measured in advance before attaching the target material, the measured value is used to calculate the difference Δω ₀ = (ω _0i −ω _0f ). On the other hand, in the same kind of cantilever in which the target substance recognition substance 12 is immobilized under the same conditions, the “initial resonance frequency” ω _0i is essentially the same value. _Therefore , the “initial stage” measured separately with the same kind of cantilever The value of “resonance frequency” ω _0i can also be used as a representative value.

The effect of bending the cantilever will be described using an antibody that is one of target substance recognition substances as an example. The longitudinal size of the average antibody molecule (Fab) ₂ is about 15 nm, and the opening width of the upper part due to the distance between Fab variable regions V _H and V _L is said to be about 10 to 20 nm. . As shown in FIG. 1 (a), when antibody molecules (Fab) ₂ are closely packed and immobilized, naturally, in the state without deflection, the interval between antibody molecules depends on the opening width of the upper part of each antibody molecule. It is limited to 10 to 20 nm. It is assumed that an external force is applied and a cantilever having a thickness T1 μm and a length L = 500 μm is bent into an arc shape with a radius of curvature r, and the bending amount Δd (displacement amount) at the tip is 50 μm. That is, if the angle of this arc is θ, in the range where θ is sufficiently small,
L = r × θ,
Δd = r−r cos θ≈r × θ × sin θ≈r × θ ²
And can be expressed approximately. Therefore, the radius of curvature r is r≈L ² / Δd.

When the curvature radius r of the deflection due to the external force is about 5000 μm, the distance between the tip portions of the antibody molecules having a longitudinal size of about 15 nm fixed on the cantilever surface is 0.3% in proportion to the deflection. It will expand about%. That is, the distance between the antibody molecule tips is expanded by about 0.3 to 0.6 mm. The interval between the antigen molecules that bind to the variable regions V _H and V _L at the tip of the antibody molecule is larger than the increase in the interval at the tip of the antibody molecule. The extent of the spacing increases as the longitudinal size of the antigen molecule increases. As a result, the repulsive force between the antigens bound to the antibody is weakened by bending the cantilever in advance compared to the case where there is no deflection, and the effect is further increased when the longitudinal size of the antigen molecule is increased. .

  The process of the target substance detection method according to the present invention will be described in more detail.

(Step A) Step of fixing the target substance recognition substance on the surface of the cantilever Cantilever:
The shape and material of the cantilever are not particularly limited as long as a desired deflection can be generated by a stress about the repulsive force between target substance molecules. In addition, it is preferable to select an optimal material and shape after considering in advance the surface density of the target substance recognition substance to be immobilized and the range of the amount of the target substance to be attached. If the spring constant of the entire cantilever is within the range of about 0.01 to 100 N / m, which is employed in a cantilever type probe of a general probe microscope, it can be normally used without any problem. However, depending on the type of target material to be detected and the material and shape of the cantilever, the spring constant of the entire cantilever can be set outside the above range.

  Further, when the cantilever is charged with static electricity, there is a possibility that the entire cantilever is bent due to a force caused by electrostatic interaction. It is desirable to use a material exhibiting conductivity as the material of the cantilever itself in order to eliminate the cause of the bending that is unrelated to the repulsive force between the target materials. For example, when the cantilever body is made of a semiconductor or an insulating material, the surface is coated with a metal film, or a dopant that improves the conductivity of the semiconductor material is added to the conductive material. A semiconductor material is preferable.

  Furthermore, as exemplified in the description of the process B described later, when detecting the deflection amount (displacement amount) amplitude of the tip of the cantilever by an optical method, it is necessary to reflect the laser beam used for detection on the surface of the cantilever. There is. At that time, if necessary, at least a part of the tip of the cantilever can be subjected to a treatment for improving the light reflectance, for example, a surface coating such as an aluminum metal coating film.

  As an example of a cantilever that satisfies the above-mentioned requirements such as the spring constant, conductivity, and light reflectance of the surface, for example, it is manufactured by applying a semiconductor processing process to an n-type silicon wafer doped with boron or the like. A rectangular cantilever having a thickness (T) of about several μm, a length (L) of about several hundred μm, and a width (W) of about several tens of μm, and a thickness (T) of about several μm from the root to the tip. A V-shaped or U-shaped cantilever having a length (L) of about several hundred μm and a width (W) of several tens of μm can be given. However, since it is difficult to handle with a cantilever alone, it is preferable to integrally process the chip with a side of several mm or more in a shape in which the cantilever is attached. Further, a tip of a tip about 5 mm in length, about 2 mm in width, and about 1 mm in thickness, which is commercially available as a probe for a probe microscope, has a thickness (T) of about 5 μm, a length (L) of about 200 μm, and a width ( W) A tip with a cantilever in which a cantilever of about 35 μm is formed can be diverted. In addition, a structure with a hole in the center of the chip and a cantilever protruding into the hole will damage the cantilever part when handling the chip than a structure with a cantilever protruding from the tip of the chip. The frequency of occurrence of trouble can be greatly reduced. Furthermore, when the chip is mounted on the displacement measuring device, which will be described later, the cantilever is mounted tilted, and the frequency of troubles that erroneously detect the amount of deflection (displacement) at the tip of the cantilever can be greatly reduced.

  Note that when measuring the resonant frequency of the cantilever, the chip portion is vibrated by a vibrator to cause a forced vibration of the frequency ω, but this vibrator can also be incorporated in a chip with a cantilever or measured. It is also possible to adopt a form in which it is incorporated on the device side and brought into contact with the chip. Further, regarding the configuration in which a means for preliminarily holding the cantilever by applying an external force and a means for incorporating a means for detecting the resonance frequency into the cantilever are described in detail in the description of the processes B and C. To do.

Target substance and target substance recognition substance:
The target substance may be an ionic species or a polymer as long as a target substance recognizing substance capable of specifically binding the target substance exists, and its form is not particularly limited. Further, when the target substance and the target substance recognition substance are bonded, a third substance that promotes or bridges the bond between the two may be added. Examples of combinations of target materials and target material recognition materials to which the present invention can be applied include the following.
(1) The target substance is a molecule or ionic species composed of one to several atoms, and the target substance recognition substance. A macromolecule having a complementary shape that exactly matches the molecule or ion shape of the target substance, or a macromolecule containing such a polymer as a functional group.

Specifically, it is known that calix [n] arene specifically binds to various ionic species, fullerenes and the like depending on the side chain and the value of n. 53 No. 3 p26- (1998). In addition, the amino group of chitin and chitosan is specifically bonded with calcium cation species (Ca ²⁺ ) to the carboxyl group part (—COO ⁻ ) of PAA in a state of hydrogen bonding with polyacrylic acid (PAA). Is reported in Chemistry Letters 1999 p199-.
(2) The target substance is an antigen molecule such as an amino acid, a peptide / protein, or a glycoside thereof, or a conjugate with a sugar chain, and the target substance recognition substance is an antigen / antibody specific to the antigen molecule. Reacting antibody. Alternatively, for example, a combination of a ligand molecule and its receptor, such as biotin and avidin.

Various combinations of ligand molecules and their receptors that perform specific binding have been reported. On the other hand, with respect to specific antibodies against antigen molecules, a method for creating a target antibody according to the antigen molecule, particularly a method for isolating and purifying a monoclonal antibody has been established. Furthermore, various monoclonal antibodies are commercially available.
(3) The target substance is a DNA or RNA molecule, and the target substance recognition substance has a base sequence complementary to the base sequence of the single-stranded nucleic acid molecule of the target substance. PNA, a single-stranded DNA that forms a hybrid with a single-stranded nucleic acid molecule.

  For a single-stranded nucleic acid molecule that is the target substance, select a partial base sequence with a length of several tens of bases unique to the target substance from the base sequence, and a base sequence of several tens of bases complementary to it. Single-stranded DNA having can be used as a probe for hybridization reaction. Single-stranded DNA having a base sequence of several tens of bases can be easily synthesized by using a commercially available DNA synthesizer.

Method for immobilizing the target substance recognition substance on the cantilever surface:
The method for immobilizing the target substance recognition substance is not particularly limited, and an appropriate method may be selected in accordance with the characteristics of the target substance recognition substance to be used. For example, when a target substance recognition substance or a macromolecule with a target substance recognition substance in the side chain is dissolved in an organic solvent, etc., a solution in which the target substance recognition substance or the macromolecule is dissolved is dropped onto the cantilever surface and spin A coating method or a dip coating method in which a cantilever is immersed in a solution and then pulled up can be applied. In addition, a method of spraying fine droplets of a solution using an inkjet technique or the like is effective when, for example, another target material recognition substance is applied to each cantilever with respect to an array in which a plurality of cantilevers are arranged. . When the target substance recognition substance is an antibody molecule or single-stranded DNA, a thiol group (-SH) is bonded to one end of the molecule, while a gold or platinum coating layer is coated on the surface of the cantilever, A method of bonding to the gold or platinum coating layer is also a generally used method. For example, when the target substance recognition substance is biotin, a method for producing biotin thiol in which thiol group (-SH) is introduced into biotin and a method for binding to a gold substrate using the introduced thiol group (-SH) Is reported in Science, 262, p1706- (1993). Further, when the surface of the cantilever is composed of a metal or a metal oxide, a method of bonding using biotin silane via reaction of the silane portion is disclosed in Japanese Patent Laid-Open No. 7-260790. And can also be used in the present invention.

(Step B) Measuring the initial resonance frequency of the cantilever Method of vibrating the cantilever:
When measuring the resonant frequency of the cantilever with free vibration, a single pulse external force (impact) is applied from the outside so that the tip of the cantilever bends to induce vibration. The single-pulse external force (impact) used for inducing the free vibration is not particularly limited. For example, it is also possible to use a technique of applying an impact to such an extent that the cantilever is not damaged or deformed by using a means for deflecting the cantilever to be touched in Step C.

  On the other hand, the method of applying forced vibration to the cantilever chip and forcing the cantilever at a predetermined frequency is, for example, piezo oscillation to the cantilever chip, which is generally used in tapping AFM, frequency modulation method AFM, etc. A method can be used in which a child is brought into contact and an AC voltage having a predetermined frequency is applied to the piezo oscillator to forcibly vibrate.

Resonant frequency measurement method:
The method for measuring the resonant frequency of the cantilever is not particularly limited. For example, a method in which a cantilever is freely vibrated and a resonance frequency is measured as a vibration frequency of the free vibration can be used. As a means for inducing free vibration, a method of applying an impact can be used because initial deflection occurs in the cantilever. In addition, when forced vibration is performed, a method of obtaining a resonance frequency based on the frequency dependence of the vibration amplitude can also be used. In the case where this forced vibration is performed, a technique of bending and vibrating the cantilever by minutely vibrating the base portion of the cantilever at a predetermined frequency can be used. For example, with respect to a chip with a cantilever, a method of using an oscillation element to forcibly vibrate the chip portion and flexing and vibrating the cantilever can be considered.

  Whichever measurement method is used, the bending vibration of the cantilever is detected by measuring the amount of periodic bending displacement at the tip of the cantilever. As a method of measuring the deflection displacement amount of the tip of the cantilever, a laser beam is applied to the tip of the cantilever, and a deviation of reflected light caused by the deflection displacement is detected, or an optical fiber is used. The tip of the cantilever is irradiated with a laser beam, the reflected light is guided to the same optical fiber, and the change in the optical path length from the tip of the optical fiber to the tip of the cantilever based on the change in the interference wave between the incident light and the reflected light, that is, the tip of the cantilever An optical detection method such as an interferometry that detects the amount of deflection displacement of the lens can be used. In addition, a strain detection element such as a piezo element is attached to the cantilever to detect the deflection displacement as a change in resistance value due to the piezoelectric effect, or an electrode that is electrically insulated in parallel to the conductive cantilever is installed. In addition, a method of detecting a change in the average distance between the electrodes, that is, a deflection displacement amount at the tip of the cantilever based on a change in the capacitance of the parallel plate type capacitor can be used.

  When measuring the vibration frequency of the free vibration of the cantilever, it is only necessary to detect the amount of periodic deflection displacement at the tip of the cantilever and measure the frequency of the periodic change. When the cantilever is forcibly vibrated by an oscillator, the amount of periodic deflection displacement at the tip of the cantilever is detected, its amplitude is measured, and the frequency dependence of the amplitude is obtained. The resonance frequency of the cantilever is obtained as a frequency at which the amplitude shows a maximum in the frequency dependency of the obtained amplitude.

  Note that when a certain element such as a piezo sensor or an oscillator is attached to the cantilever itself, these elements are formed at the same time when the cantilever is manufactured in the process A. At that time, the pasted element increases the rigidity of the cantilever more than necessary, or because of the difference in thermal expansion coefficient between the cantilever's own material and the pasted element's material. Consideration should be given to the problem that the interface region between the cantilever and the cantilever bends and deforms depending on the temperature as in a bimetal element.

  A dedicated electronic circuit that measures the resonance frequency may be used for the operation of detecting the periodic deflection displacement of the tip of the cantilever and obtaining the resonance frequency based on the detection result. In consideration of the control of the whole measuring device, such as a power supply device that supplies an alternating voltage of a predetermined frequency to an oscillator that induces forced vibration, a personal computer (hereinafter referred to as a personal computer) having an analog signal capturing function such as an AD converter. ) May be used to collectively control the measurement conditions and calculate the resonance frequency based on the detection result.

(Step C) Step of applying an external force to the cantilever and holding it in a bent state The method of bending the cantilever in advance and holding it in the bent state is not particularly limited. For example, mechanical bending in which a desired external force is applied by a mechanical method such as pushing the tip of the cantilever with a shape and material of the same size as the cantilever or pulling with a needle with a hook Can be used. Further, in the form described in the description of the process B in which the piezo element is bonded to the cantilever, a method of applying a voltage to the piezo element to cause distortion and deforming the cantilever can be used. In addition, a method in which two V-shaped or U-shaped cantilevers having electrical conductivity are arranged so as to be opposed to each other, current is passed through both cantilevers, and they are bent by an electrostatic force generated by a generated magnetic field. A method of bending a magnetic material such as iron at the tip of the cantilever in advance and attracting it with an electromagnet can also be adopted. Furthermore, as will be described in step D described later, a method of spraying a sample solution or gas on the cantilever surface at a constant flow rate / flow velocity / direction to bend can be used.

  Regardless of which method is selected, the external force applied to the cantilever will not be damaged or cause plastic deformation, so that the magnitude and retention of the applied external force will depend on the material and shape of the cantilever. It is necessary to examine in advance the range of the curvature of bending that is to be performed.

(Step D) Step of bringing the cantilever into contact with the sample containing the target material The method of bringing the cantilever held in a bent state in advance into contact with the sample containing the target material is not particularly limited. When the sample is a liquid, the cantilever may be immersed in the sample. When the sample is a gas, the cantilever may be exposed to the sample atmosphere. Further, it is possible to adopt a form in which the sample solution or gas is sprayed onto the surface of the cantilever at a constant flow rate, flow velocity, and direction. At that time, the sample solution or gas is sprayed on the surface of the cantilever at a constant flow rate, flow velocity, and direction, so that the cantilever is bent and contacted with the target material. It is also possible to adopt a form to perform.

  Process D may be performed in a state where the cantilever is incorporated in the displacement measuring device. In addition, after the completion of the process B, the cantilever is detached from the displacement measuring device, and the cantilever is attached to a cassette that is easily attached / detached with a mechanism for holding the cantilever in a bent state, and the process C is performed. In the state, step D can be performed. That is, the process D can be performed using a cantilever attached to the cassette at a place where a sample is sampled, such as a field or a medical site.

  After that, the cassette after the target material adhesion treatment process is collected, removed again from the cassette, and the cantilever on which the target material is adhered is set again on the displacement measuring device, and after the target material is adhered, Measure the resonance frequency. That is, it is possible to collect the collected cassettes in one place and collectively measure the resonance frequency of the cantilever after the target material is attached.

(Step C ′) Step of applying an external force to the cantilever and holding it in a bent state in the reverse direction In Step C ′, a force in the reverse direction to Step C is applied to the cantilever. The cantilever is bent backwards so that the other side becomes a convex surface. In addition, the method of applying the external force in the reverse direction to bend in the reverse direction is not particularly limited as in the process C.

(Step D ′) The step of bringing the cantilever into contact with the sample containing the target material In step D ′, the cantilever is bent in the reverse direction so that the concave side becomes a convex surface by the step C ′. Is further brought into contact with a sample containing the target substance. In the step D ′, the method for contacting the sample containing the target material is not particularly limited as in the step D.

(Step E) Step of removing the external force applied to the cantilever In step C or step C ′, the external force is applied and the cantilever held in a forcibly bent state is applied external force. When this is removed, this forced deflection is eliminated, and the cantilever tip becomes a “released” state where the tip is a free end. When removing the applied external force, if the external force applied to the cantilever is suddenly reduced to zero, the cantilever starts to vibrate due to the stored elastic stress. This vibration gradually attenuates and eventually the cantilever stops.

  However, when the cantilever is held in a bent state, the entire cantilever is convex, so an external force is applied to the tip of the cantilever, but if the amount of bending is large, it is suddenly applied to the cantilever. When external force is removed and free vibration is performed, vibration starts with a large amplitude. That is, if the state in which the cantilever is repeatedly bent with a large amount of bending continues, the cantilever may cause mechanical fatigue due to repetitive bending stress application, and may be damaged. In consideration of this point, it is desirable to gradually reduce the external force applied to the cantilever so that the final applied external force is zero while avoiding the occurrence of vibration. The time required to gradually remove the external force depends on the spring constant of the cantilever, that is, depends on the material and shape of the cantilever. Using a cantilever that is exactly the same material and shape as the actual cantilever used for measurement, it is possible to determine it appropriately after examining the rate of external force reduction suitable for the stage of gradually removing the external force in advance. .

  Also, suddenly, when the external force applied to the cantilever is removed, the amplitude of the generated vibration is suppressed and quickly attenuated, for example, a damper is attached to the base part of the cantilever, the amplitude is limited, and at the same time, A form in which elastic stress is attenuated can also be used. In addition, when the external force applied to the cantilever is suddenly removed, the stopper is operated so as to avoid the state where the tip of the cantilever bends in the opposite direction due to the generated vibration. The use of a stopping method is also conceivable.

  When this damper or stopper is attached, it is necessary to measure the resonance frequency after removing it in advance so as not to be a factor that impedes the free vibration when measuring the resonance frequency of the cantilever.

(Step F) Step of Measuring Resonant Frequency of Cantilever After Target Material Adhesion Treatment After finishing the target material attachment treatment, the resonance frequency of the cantilever is measured. At that time, the resonance frequency of the cantilever after the target material adhesion treatment is measured according to the same measurement procedure as the measurement method used in the step B.

When the target material adheres to the surface of the cantilever and the mass of the entire cantilever increases due to the load of the target material, the measured “resonance frequency after the target material attachment treatment” ω _0f is measured in the above-mentioned step B. This is different from the “initial resonance frequency” ω _0i .

(Step G) A step of calculating a difference between the “initial resonance frequency” ω _0i measured in the step (B) and the “resonance frequency after target material adhesion treatment” ω _0f measured in the step (F). and "initial resonance frequency" omega _0i measured in, calculates the difference Δω between omega _0f "resonance frequency after the target material deposition process" measured in step F ₀ = a (ω _0i -ω _0f) in a personal computer or the like, Based on this difference Δω ₀ , the presence / absence of mass change and the mass change amount derived from the target substance adhering to the cantilever surface are calculated.

Note that the difference Δω ₀ = (ω _0i −ω _0f ) can be calculated using the estimated initial resonance frequency of the cantilever instead of the actually measured value of the “initial resonance frequency” ω _0i .

  In the above description, for the purpose of simplifying the explanation in describing the detection principle of the present invention, a mode using a sensor having one cantilever has been described. However, a sensor configuration having a plurality of cantilevers is used. You can also

For example, by using an array configuration in which a plurality of cantilevers are arranged, the same target substance recognition substance is fixed on the surfaces of the plurality of cantilevers, and the measurement results of the resonance frequency change Δω ₀ of the plurality of cantilevers are averaged. If the form of calculating the amount of the target material adhering based on the value is adopted, it becomes possible to detect the target material contained in the sample with higher accuracy.

In addition, when another target substance recognition substance is fixed to each cantilever using an array configuration in which a plurality of cantilevers are arranged, a plurality of target substances can be loaded at a time based on the measurement result of the resonance frequency change Δω ₀ of each cantilever. It is also possible to adopt a form of detection.

As another example, a pair of two cantilevers arranged in parallel, one is a detection cantilever with a target substance recognition substance fixed, and the other is similar to the target substance recognition substance but is bound to the target substance. When the non-conducting substance is fixed and used as a comparison cantilever, when measuring the resonance frequency change Δω _{0 of the} cantilever, the change Δω ₀ between the two is measured at the same time, and the difference is caused by the change in the spring constant k of the cantilever. In consideration of the change in resonance frequency, it is possible to calculate the presence or absence of mass change and the amount of mass change resulting from the target material adhering to the cantilever surface.

  At this time, the surface of the comparison cantilever is used on the surface where the target material is not adhered, and the difference between the resonance frequency of the measurement cantilever and the resonance frequency of the comparison cantilever is adhered on the measurement cantilever. Reflects the presence or absence of the target substance and the amount of adhesion. On the other hand, the difference between the “initial resonance frequency” and the “resonance frequency after the target material adhesion treatment” in the comparison cantilever is the presence or absence of the change in the spring constant k of the cantilever with the contact with the sample. Can be used as a basis for calculating.

Note that the device configuration of the target material detection device based on the principle of the target material detection method according to the present invention preferably includes the device configuration corresponding to the above steps (A) to (G). In particular,
(A) a cantilever sensor including a cantilever having a target substance recognition substance fixed on the surface;
(B) means for measuring an initial resonance frequency of a cantilever having a target substance recognition substance fixed on the surface;
(C) means for applying an external force to the cantilever to hold one surface on which the target substance recognizing substance is fixed as a convex and hold it in a bent state;
(D) means for bringing the cantilever held in the bent state into contact with the sample in a state enabling the binding of the target material to the target material recognition material;
(E) means for removing external force applied to the cantilever;
(F) means for measuring the resonant frequency of the cantilever after contact with the sample for the purpose of binding to the target substance;
(G) means for calculating a difference between the resonance frequency of the cantilever after contact with the sample and the initial resonance frequency of the cantilever;
(H) A target material fixed on the surface of the cantilever based on a resonance frequency difference calculated from the measured resonance frequency of the cantilever after contact with the sample and the initial resonance frequency of the cantilever before contact with the sample. It is more preferable that the target substance detection apparatus includes a means for calculating the presence or absence of the target substance bound to the recognition substance or the amount of the bound target substance.

  As described above, in the principle of the target material detection method according to the present invention, the cantilever and the target material are brought into contact with each other after the cantilever is bent in advance, so that the repulsive force between the target materials is small. In this state, it becomes possible to attach the target material to the surface of the cantilever. After the adhesion, if the external force that is bending the cantilever is removed, the target material attached to the surface of the cantilever with a high surface density is between the target materials. By the repulsive force, it maintains the attached state without falling off. In this state, by measuring the amount of change in the resonant frequency of the cantilever that reflects the increase in load due to the target material attached at a high surface density, it becomes possible to detect the target material with higher sensitivity, or more easily. Measurement by a simple detection means becomes possible.

  Hereinafter, the present invention will be described in more detail with reference to specific examples. In addition, although the following specific example is an example of the best embodiment concerning this invention, this invention is not limited to the aspect of these specific examples.

(Embodiment 1)
In this embodiment, a case where avidin as a target substance is detected by a cantilever sensor using biotin as a target substance recognition substance and biotin immobilized thereon is shown. In other words, biotin is a coenzyme that specifically binds to avidin, and corresponds to an example in which the target substance protein is attached to the cantilever surface through complex formation with a substrate substance having specific binding ability. To do.

The cantilever sensor used in this mode is configured by a single cantilever. The resonance frequency of the cantilever is measured by forcibly oscillating the cantilever at a predetermined vibration frequency using an attached oscillator, and at that time, the amplitude of the tip of the cantilever (change width of the deflection amount) is measured using an optical lever method. . Under the forced vibration conditions, the amplitude (change width of the deflection amount) of the observed cantilever tip is plotted against the vibration frequency ω, and the vibration frequency ω giving the maximum is the measured value of the resonance frequency ω ₀ of the cantilever. .

  The cantilever manufactured in the same process has improved manufacturing accuracy and reproducibility of characteristics so that the mass m and spring constant k of the cantilever itself are essentially the same. The resonance frequency has almost the same value. Therefore, after measuring the initial resonance frequency of the cantilever used for detecting avidin, the measurement of the initial resonance frequency can be omitted for the cantilever used for comparison.

(1) Immobilization of biotin on the surface of a cantilever The cantilever sensor used for detection is pre-coated with a gold coat on the surface of the cantilever, and then utilizes the reactivity of the sulfanyl group (-SH) with respect to gold, -SH) -immobilized biotin (biotinthiol) is immobilized on a gold-coated film.

  FIG. 2 (a) schematically shows the structure of a cantilever chip itself manufactured from a silicon substrate using a normal semiconductor manufacturing process. A cantilever 33 is formed at the center of a square tip 31 having a length and width of 10 mm and a thickness of about 1 mm. As shown in FIG. 2B, the cantilever 33 has a cantilever structure having a thickness of 1 μm, a length of 500 μm, and a width of 100 μm, and is manufactured by etching a silicon substrate. A portion removed by this etching process becomes a hole 32 having a diameter of 600 μm at the center of the square chip 31. The cantilever 33 protrudes from the center of the hole 32. After this processing step, the cantilevers with chips A and B are coated with a gold vapor deposition film on both the upper and lower surfaces of the cantilevers a and b.

  A tip with a cantilever coated with a gold-deposited film is immersed in an ethanol solution (0.5 mM) of biotinthiol into which sulfanyl group (—SH) is introduced with respect to biotin for 30 minutes. After the biotin is immobilized on the gold coating surface, the biotin thiol is removed from the ethanol solution and washed three times with ethanol to remove unreacted biotin thiol. After washing and drying, a dicing process is performed from the silicon substrate with the above size to separate the cantilevered chip.

  As a result, the cantilevers with chips A and B are essentially the same, both-side biotin-fixed cantilever sensors.

(2) Detection of avidin using biotin-fixed cantilever sensor Next, a procedure for detecting avidin using a double-sided biotin-fixed cantilever sensor will be described.

FIG. 3 schematically shows the configuration of an apparatus used for measuring the resonance frequency of the cantilever. This measuring device is composed of a displacement measuring device using the optical lever method, which is used to detect the deflection amount of the cantilever tip, and a piezo element type oscillator for forcibly vibrating the cantilever at a predetermined frequency ω. Yes. At that time, in a state where the cantilever is forcibly vibrated at a predetermined frequency ω, the change width (amplitude) of the deflection amount of the cantilever tip is measured, the amplitude of the cantilever vibration is plotted for each vibration frequency ω, and the maximum is given Let the vibration frequency be the measured value of the resonance frequency ω ₀ of the cantilever.

  The stage 41 that fixes the tip 31 with the cantilever has a surface that is in contact with the tip that is recessed according to the shape of the tip, and the surface that contacts the tip is mirror-finished to be a flat surface. When the chip 31 is fixed to the stage 41 with the clamp 42, the mounting position of the chip 31 itself is the same position with high accuracy and reproducibility. On the other hand, the laser beam 44 emitted from the semiconductor laser transmitter 43 is applied to the tip of the cantilever through the lens 45. The reflected light 46 reflected at the tip of the cantilever reaches the detector 47. The detector 47 is a so-called two-divided sensor composed of two upper and lower photodiodes (not shown). The signals from the two upper and lower photodiodes are input to the personal computer 48 as digital information via the 2-channel AD conversion board.

  A piezo oscillator 40 is embedded in the stage 41 so as to be in contact with the chip 31. By oscillating the piezo oscillator 40 at a predetermined frequency ω, the vibration is propagated through the chip 31, and as a result, the cantilever 33 is forced to vibrate at the vibration frequency ω.

When the amount of bending of the tip of the cantilever changes periodically due to this forced vibration, the incident angle θ of the laser beam 44 with respect to the tip changes, and correspondingly, the reflection angle θ of the reflected light also changes. The trajectory periodically reciprocates between a shift state such as 46 ′ and a shift state such as 46 ″. In a state in which the cantilever 33 is stationary, the reflected light is incident on the two-divided sensor detector 47 central axis, while the angle of incidence of the laser beam 44 theta is the initial angle theta _0, two upper and lower The semiconductor laser transmitter 43 and the detector 47 are aligned so as to be positioned on the boundary line of the photodiode. In general, in the state in which the zero point is adjusted, alignment is performed so that output signal intensities proportional to incident light intensities on two upper and lower photodiodes are equal.

When the amount of deflection of the cantilever tip is changed, the angle of incidence of the laser beam 44 theta is, when displaced from the initial angle theta _0, finally, the two central axis of the reflected light incident on the divided sensor, two upper and lower It is displaced from the boundary line of the photodiode to a position that is biased to one of the two upper and lower photodiodes. As a result, a difference occurs in the output signal intensity proportional to the incident light intensity to the upper and lower two photodiodes. Based on the difference between the two output signal intensities, the amount of displacement of the central axis of the reflected light incident on the two-divided sensor from the boundary line between the two upper and lower photodiodes is calculated, and the laser beam with respect to the cantilever tip An amount Δθ in which the incident angle θ of 44 is displaced from the initial angle θ ₀ is calculated. The series of arithmetic processing is performed in the personal computer 48 according to the data analysis program.

In the forced vibration state, the amount of bending at the tip of the cantilever fluctuates periodically. Correspondingly, the amount Δθ displaced from the initial angle θ ₀ also fluctuates periodically. That is, the amount of deflection δ at the tip of the cantilever is based on the displacement Δθ (maximum value and minimum value) at two points in time when the locus of the reflected light is shifted to 46 ′ and shifted to 46 ″. A maximum value δmax and a minimum value δmin are calculated for each, and the difference (δmax−δmin) is taken as a measured fluctuation range (amplitude) of the deflection amount of the tip of the cantilever.

The oscillation frequency of the piezo oscillator 40 coincides with the frequency of the high-frequency voltage supplied from the oscillator 49 which is the driving power source. The frequency of this high-frequency voltage is continuously changed from ω ₁ to ω ₂ , and the fluctuation width (amplitude) actual measurement value of the bending amount of the cantilever tip at each vibration frequency ω is measured. When the fluctuation range (amplitude) actual measurement value of the deflection amount is plotted against the vibration frequency ω, the amplitude shows a maximum in the vibration frequency region corresponding to the resonance frequency ω _{0 of the} cantilever as shown in FIG. In other words, the fluctuation frequency (amplitude) measured value (δmax−δmin) of the bending amount is numerically differentiated as a function of the vibration frequency ω, and the vibration frequency at which the differential coefficient is d (δmax−δmin) / dω = 0. calculated the omega, the measured value of the resonance frequency omega ₀ of the cantilever: the omega _0-obs.

In the operation of calculating the deflection amount δ of the tip of the cantilever from the amount Δθ displaced from the initial angle θ ₀ , the cantilever tip 31 with the cantilever tip, for example, the length of the cantilever 33, the laser beam 44 on the cantilever 33, Based on the initial irradiation position, the initial deflection amount (or radius of curvature), and the amount Δθ displaced from the initial angle θ ₀ calculated from the detection result, the deflection amount δ of the cantilever tip is calculated. From the amount Δθ displaced from the initial angle θ ₀ , an operation for calculating the deflection amount δ of the cantilever tip, and further, the fluctuation amount (amplitude) measured value (δmax−δmin) of the deflection amount as a function of the vibration frequency ω, Numerical calculation processing such as operation for differentiating numerically and calculating an actual measurement value ω _0-obs of the resonance frequency ω ₀ of the cantilever is also performed in the personal computer 48 according to the corresponding data analysis program. In addition, the frequency of the high-frequency voltage supplied from the oscillator 49 is continuously changed from ω ₁ to ω ₂ , and the maximum value Δθmax of the amount Δθ displaced from the initial angle θ _{0 at} each vibration frequency ω The operation for measuring the minimum value Δθmin is also made an automated measurement routine according to the control program by the personal computer 48.

  Before detecting avidin using the double-sided biotin-fixed cantilever A, the tip A with the biotin-fixed cantilever is set in the displacement measuring device, and the irradiation spot position of the laser beam 44 at the tip of the cantilever However, the optical axis of the incident light source system composed of the laser transmitter 43 and the condensing lens 45 is finely adjusted so that the predetermined position is obtained. At the same time, the position of the two-divided sensor type detector 47 constituting the light receiving system for the reflected light is finely adjusted so that the central axis of the reflected light incident on the two-divided sensor is positioned on the boundary line between the upper and lower two photodiodes. Like that. Specifically, the position of the two-divided sensor detector 47 is finely adjusted so that the output signal intensity is proportional to the intensity of light incident on the two upper and lower photodiodes constituting the two-divided sensor.

Subsequently, the frequency of the high frequency voltage supplied from the oscillator 49, the omega ₁ to omega _2, by continuously changing, at each vibration frequency omega, and measured the cantilever tip deflection amount of fluctuation width (amplitude), the Based on the actual measurement value (δmax−δmin), the initial resonance frequency actual measurement value: ω _0-obs-Ai of the biotin-fixed cantilever a is calculated.

  After the actual measurement of the “initial resonance frequency” is completed, the tip A with the cantilever is removed, and the tip 51 having the cantilever 52 with the probe 53 for the probe microscope is inserted through the spacer 54 as shown in FIG. Then, the probe 53 is attached to the upper surface of the tip 31 and the probe 53 is brought into contact with the tip of the cantilever 33. As a result, due to the external force applied to the contact point of the probe 53, the cantilever 33 is bent with its upper surface convex. Here, an abutment condition (in particular, the thickness of the spacer 54) is that the cantilever 33 having a cantilever structure having a thickness of 1 μm, a length of 500 μm, and a width of 100 μm has a deflection amount of about 100 μm. Is selected.

  With the tip 51 having the cantilever 52 with the probe 53 attached, the tip A with the cantilever is immersed in an avidin aqueous solution. After a predetermined time has elapsed, the cantilevered tip A is taken out of the avidin aqueous solution and washed with distilled water. Avidin and the like physically adsorbed on the cantilever surface are removed by washing with distilled water, except for avidin that stably adheres through binding to biotin immobilized on the cantilever surface. After washing with distilled water, the tip A with a cantilever having avidin attached to the surface is naturally dried.

The cantilever tip A to which the avidin is stably attached is re-set to the resonance frequency measuring apparatus used for the measurement of the “initial resonance frequency” actual measurement value through the binding with biotin, and the avidin is attached to the surface. The resonance frequency of the attached cantilever a is measured. The difference between the measured resonance frequency of the cantilever a having avidin attached to the surface: ω _0-obs-Af and the measured “initial resonance frequency” of the biotin-fixed cantilever a: ω _0-obs-Ai , Δω _{0 -obs-A} = (ω _0-obs-Ai −ω _0-obs-Af ) is calculated.

On the other hand, with respect to the tip B with the cantilever, the biotin-fixed cantilever b is made essentially the same as the biotin-fixed cantilever a, that is, the actually measured “initial resonance frequency” of the biotin-fixed cantilever b is: The initial resonance frequency measurement value of the biotin-fixed cantilever a: the same value as ω _0-obs-Ai . Actually, when the “initial resonance frequency” of the biotin-fixed cantilever b was measured, the “initial resonance frequency” of the biotin-fixed cantilever b was measured: ω _0-obs-Bi was the “initial resonance frequency of the biotin-fixed cantilever a. “Frequency” measured value: ω _0-obs-Ai and within the measurement error.

  As shown in FIG. 5, a tip 51 having a cantilever 52 with a probe 53 for a probe microscope is attached to the top surface of the tip 31 via a spacer 54, and the cantilever tip B is attached to the cantilever tip B. The probe 53 is brought into contact with the tip. As a result, due to the external force applied to the contact point of the probe 53, the cantilever 33 is bent with its upper surface convex. Here, an abutment condition (in particular, the thickness of the spacer 54) is that the cantilever 33 having a cantilever structure having a thickness of 1 μm, a length of 500 μm, and a width of 100 μm has a deflection amount of about 100 μm. Is selected.

  With the tip 51 having the cantilever 52 with the probe 53 attached, the tip B with the cantilever is immersed in pure water. After a predetermined time has elapsed, the tip B with the cantilever is taken out from the pure water and naturally dried.

A chip B with a cantilever that has been subjected to a pure water immersion treatment is set in the resonance frequency measuring device, and the resonance frequency of the cantilever b to which no avidin is attached is measured. Measured resonance frequency of cantilever b with no avidin attached to the surface: ω _0-obs-Bf Measured value of “initial resonance frequency” of biotin-fixed cantilever b: ω _0-obs-Bi ≈ω _0-obs The difference from _-Ai , [Delta] [omega] _0-obs-B = ([omega] _{0-obs-Ai- [} omega] _0-obs-Bf ) is calculated.

From Δω _0-obs-B , the amount of fluctuation of the resonance frequency when avidin cannot be detected can be obtained.

Specifically, this Δω _0-obs-B = (ω _0-obs-Ai −ω _0-obs-Bf ) is a state where the biotin-fixed cantilever is brought into contact with a sample solution not containing avidin, that is, There is no avidin attached to the surface of the biotin-fixed cantilever and there is no increase or decrease in the mass m of the entire cantilever, but due to immersion in an aqueous solvent, the spring constant of the cantilever is changed from k to (k− By changing to Δk), the initial resonance frequency: ω _0-i = (k / m) ^1/2 , and the resonance frequency after immersion treatment: ω _0-Bf = ([k−Δk] / m) ¹ Reflects the change to ^{/ 2} .

From Δω _0-obs-A , the fluctuation amount of the resonance frequency derived from the detection of avidin can be obtained.

Specifically, this Δω _0-obs-A = (ω _0-obs-Ai −ω _0-obs-Af ) is a state where the biotin-fixed cantilever is brought into contact with the sample solution containing avidin, that is, As a result of avidin being attached to the surface of the biotin-fixed cantilever, the mass of the entire cantilever increases from m to (m + Δm), and at the same time, the spring constant of the cantilever is By changing to (k−Δk), from the initial resonance frequency: ω _0-i = (k / m) ^1/2 , the resonance frequency after avidin attachment: ω _0-Af = ([k−Δk] / [M + Δm]) Reflects a change to ^1/2 .

Based on the above correspondence,
Δω _0-obs-B = (ω _0-obs-Ai −ω _0-obs-Bf )
≒ [(k / m) ^1/2 -([k-Δk] / m) ^1/2 ]
Δω _0-obs-A = (ω _0-obs-Ai −ω _0-obs-Af )
≒ [(k / m) ^1/2 -([k-Δk] / [m + Δm]) ^1/2 ]
Δω _0-obs-A −Δω _0-obs-B ≈ {([k−Δk] / m) ^1/2 − ([k−Δk] / [m + Δm]) ^1/2 }
ω _0-obs-Ai ≒ (k / m) ^1/2
From the above relational expression, [m + Δm] / m can be finally calculated, that is, it is possible to estimate the mass increase Δm caused by avidin attached to the surface of the biotin-fixed cantilever.

(Embodiment 2)
In the present embodiment, an example is shown in which calcium ions (Ca ²⁺ ) of a target material are detected by a cantilever sensor using chitosan immobilized as a target material recognition material. That is, chitosan once hydrogen bonds with polyacrylic acid (PAA), and PAA in a hydrogen bond state with chitosan uses the characteristic of having a specific binding property with calcium ion, and uses PAA as an auxiliary agent. This corresponds to the case where chitosan is used as a target substance recognition substance.

In place of chitosan (β-1,4-poly-D-glucosamine), β-D-mannuronic acid (β-D-mannopyruronic acid) and α-L-guluronic acid (α-L-glopyranuronic acid) are used. The resulting polyuronic acid, alginic acid, is used as a negative control. Like chitosan (β-1,4-poly-D-glucosamine), which is a sugar-like fibrous polymer, alginic acid, a kind of viscous polysaccharide, is a fibrous polymer that is hardly soluble in water at room temperature. However, it does not form a complex with PAA by hydrogen bonding. Therefore, alginic acid does not exhibit the function of immobilizing calcium ions (Ca ²⁺ ).

(1) Immobilization of chitosan or alginic acid on the cantilever surface The cantilever sensor used for detection is a set of two cantilevers, and the detection of the difference in deflection at the tip of the two cantilevers Employs a method of irradiating each cantilever tip with a demultiplexed laser beam using an optical fiber, measuring the interference wave due to the reflected light, and calculating the deflection amount difference (optical path length difference). .

  Sensor chips C and D having a pair of cantilevers in which chitosan is immobilized on the surface of one cantilever and alginic acid is immobilized on the other cantilever are prepared according to the following procedure. To do.

  FIG. 6A schematically shows the structure of a cantilever chip itself manufactured from a silicon substrate using a normal semiconductor manufacturing process. A pair of cantilevers 63 and 63 'are formed at the center of a square chip 31 having a length and width of 10 mm and a thickness of about 1 mm. The cantilevers 63 and 63 'are both cantilever structures having a thickness of 1 μm, a length of 500 μm, and a width of 100 μm, and are arranged in parallel at an interval of 50 μm. As shown in FIG. 6B, the cantilevers 63 and 63 'are manufactured by etching the silicon substrate so that the upper surface thereof coincides with the upper surface of the chip. A portion removed by this etching process becomes a hole 32 having a diameter of 600 μm at the center of the square chip 31. A set of two cantilevers 63 and 63 ′ protrudes from the center of the hole 32.

  In the cantilever chip C manufactured by this processing step, the cantilever chips 63 and 63 'corresponding to a pair of cantilevers 63 and 63' are denoted as cantilevers c and c ', respectively. Further, in the chip H with a cantilever, those corresponding to a pair of cantilevers 63 and 63 'are denoted as cantilevers d and d', respectively.

  Next, the substrate on which the chips C and D with cantilevers are fabricated is set on a spin coater, and a chitosan acetic acid aqueous solution in which 1.0% chitosan is dissolved in 1.0% acetic acid aqueous solution with respect to the cantilevers c and d. And is spin-coated under the condition of 2000 rpm for 20 sec. By drying after spin coating, the cantilever c and cantilever d are made cantilevers whose surfaces are coated with chitosan. On the other hand, the substrate is set on a spin coater, and a 1.0% alginic acid aqueous solution dissolved by heating to 60 ° C. is dropped on the cantilever c ′ and cantilever d ′, and spin-coated under the condition of 2000 rpm for 20 sec. By drying after spin coating, cantilever c 'and cantilever d' are coated with alginic acid on the surface to form a cantilever.

  After applying different coats to the surface of each pair of cantilevers, dicing treatment is performed from the silicon substrate with the above size to separate the pair of cantilevers with a pair.

(2) Detection of calcium ions using a cantilever sensor including a chitosan-fixed cantilever and an alginate-fixed cantilever Next, a procedure for detecting calcium ions using a cantilever sensor will be described.

First, as in Example 1, the initial resonance frequency of the cantilevers c and c ′ of the cantilevered chip C is measured. At that time, the measurement method of the resonance frequency is substantially the same as in Example 1, and the change width (amplitude) of the deflection amount of each cantilever tip is measured in a state where each cantilever is forcibly vibrated at a predetermined frequency ω. The amplitude of each cantilever vibration with respect to each vibration frequency ω is plotted, and the vibration frequency giving the maximum is taken as an actual measurement value of the resonance frequency ω ₀ of each cantilever.

  On the other hand, the amplitude of the cantilever vibration is measured by irradiating the tip of the cantilever with a laser beam with an optical fiber, and receiving the reflected laser beam with the same optical fiber, and the change in the amount of bending of the cantilever tip (change in optical path length) The phase change of the reflected light caused by the change in quantity (change in optical path length) is measured as interference light. The intensity of the interference light repeatedly varies according to the frequency ω of the forced vibration. Based on the measured intensity of the interference light and its periodic change, the change width (amplitude) of the deflection amount of each cantilever tip is calculated. The frequency at which the calculated amplitude is maximized is defined as the initial resonance frequency. In a state where no forced vibration is made at the frequency ω, that is, in a state where the tip of the optical fiber is stopped, the relative position between the end face of the optical fiber and the tip of the cantilever is finely adjusted in advance, and the cantilevers c and c 'A laser beam is applied to each tip.

Subsequently, the frequency of the high-frequency voltage supplied from the oscillator, which is the driving power source of the piezo oscillator, is continuously changed from ω ₁ to ω ₂ and the fluctuation range of the deflection amount of the cantilever tip at each vibration frequency ω. (Amplitude) is measured, and based on the measured value (δmax−δmin), the initial resonance frequency measured values of cantilevers c and c ′: ω _0-obs-ci and ω _0-obs-c′i are calculated, respectively. To do.

  FIG. 7 shows a cantilever sensor 31 provided with a pair of cantilevers in which a surface coated with chitosan or alginic acid is convex and bent, and then an aqueous solution of calcium carbonate to which PAA is added. The structure of the apparatus for immersing in 71 is shown typically. As shown in FIG. 7A, the pair of cantilevered tips C is inserted into a flow path 73 in which a calcium carbonate aqueous solution 71 to which PAA is added circulates at a constant flow rate by rotation of a paddle 72. At this time, since the calcium carbonate aqueous solution 71 to which PAA is added passes through the hole 32 of the cantilever sensor 31, the pair of cantilevers 74 receives an external force due to this flow, and as shown in FIG. It is held in a bent state with a predetermined radius of curvature. In this state, it is left in the flow path 73 for a certain time.

  Next, the paddle 72 is stopped once, and after the flow of the aqueous solution in the flow path 73 is stopped, the paddle 72 is rotated in the reverse direction. Since the rotation direction of the paddle 72 is reversed, the calcium carbonate aqueous solution 71 to which PAA is added circulates in the reverse direction at a constant flow rate in the flow path 73. At this time, since the calcium carbonate aqueous solution 71 to which PAA is added passes through the hole 32 of the cantilever sensor 31, the pair of cantilevers 74 receives an external force due to this flow and is opposite to the direction shown in FIG. It is held in a state of bending with a predetermined curvature radius. In this state, the cantilevered tip C is allowed to stand in the flow path 73 for a certain time.

  Then, after the paddle 72 is stopped and the flow of the aqueous solution in the flow path 73 is stopped, a pair of two cantilever tips C are taken out from the aqueous solution. The cantilever sensor 31 taken out is washed with distilled water several times and then naturally dried.

Finally, the resonance frequencies of the cantilevers c and c ′ are measured again, and the measured resonance frequencies: ω _0-obs-cf and ω _0-obs-c′f after immersion in the aqueous solution of calcium carbonate with PAA added are calculated. To do. The difference between the measured value of “initial resonance frequency” and the measured value of “resonance frequency after immersion treatment” is expressed as Δω _0-obs-c = (ω _0-obs-ci −ω _0-obs-cf ), Δω, respectively. _{Let 0-obs-c ′} = (ω _0-obs-c′i −ω _0-obs-c′f ).

Similarly, for the cantilevers d and d ′ of the tip D with the cantilever, the fluctuation range (amplitude) of the deflection amount at the tip of the cantilever at each vibration frequency ω is measured in advance, and the measured value (δmax−δmin) is used as a basis. Then, the actual resonance frequency values of the cantilevers d and d ′: ω _0-obs-di and ω _0-obs-d′i are calculated, respectively. Then, according to the procedure of the immersion treatment applied to the tip C, the tip D is inserted into a water channel through which pure water flows at a constant flow rate, subjected to pure water immersion treatment, and then naturally dried.

Finally, the resonance frequencies of the cantilevers d and d ′ are measured again, and the resonance frequency measured values after the pure water immersion treatment: ω _0-obs-df and ω _0-obs-d′f are respectively calculated. The difference between the measured value of “initial resonance frequency” and the measured value of “resonance frequency after immersion treatment” is expressed as Δω _0-obs-d = (ω _0-obs-di −ω _0-obs-df ), Δω, respectively. _{Let 0-obs-d '} = (ω _0-obs-d'i- ω _0-obs-d'f ).

When immersed in an aqueous solution or pure water, the chitosan layer coated on the surface of the cantilever and the alginate layer swell as a result of absorbing water. Along with this swelling, the spring constant k of the cantilever changes from k → (k−Δk) in the initial dry state and the swollen state. Therefore, in the cantilevers d and d ′ subjected to the pure water immersion treatment, no adhesion has occurred to the chitosan layer and the alginic acid layer, but the “initial resonance frequency” measured value and “after the immersion treatment” The difference from the “resonance frequency” measured value, Δω _0-obs-d and Δω _{0-obs-d ′} are not zero. However, since no adhesion has occurred, the difference between Δω _0-obs-d and Δω _{0-obs-d ′} is a small value.

In addition, even in the cantilever c ′ having the alginic acid layer, the attachment of PAA in a state where calcium ions are trapped does not occur. However, the spring constant k of the cantilever is k → ( k−Δk). Therefore, also in the cantilever c ′, the difference Δω _{0-obs-c ′} between the actually measured value of “initial resonance frequency” and the actually measured value of “resonance frequency after immersion treatment” is not zero.

On the other hand, in the cantilever c provided with the chitosan layer, both sides of the cantilever are attached with PAA in a state of capturing calcium ions, and the mass m of the entire cantilever is swollen from the initial dry state. In the meantime, it increases as m → (m + Δm). Of course, even in the cantilever c, the spring constant k of the cantilever changes from k → (k−Δk) between the initial dry state and the swollen state, and this is due to the increase in the mass m of the entire cantilever. There is also. This two factors, observed in cantilever c, and the actually measured value "initial resonant frequency", the difference between the actual measurement value "resonance frequency after immersion process", the _{Δω 0-obs-c, Δω} 0 cantilever c ' Compared with _-obs-c, it shows a clear difference proportional to the amount of PAA deposited with calcium ions trapped.

For example, based on the above correspondence,
ω _0-obs-ci = ω _0-obs-di ≒ (k / m) ^1/2
Δω _0-obs-d = (ω _0-obs-di −ω _0-obs-df )
≒ [(k / m) ^1/2 -([k-Δk] / m) ^1/2 ]
Δω _0-obs-c = (ω _0-obs-ci −ω _0-obs-cf )
≒ [(k / m) ^1/2 -([k-Δk] / [m + Δm]) ^1/2 ]
Δω _0-obs-d −Δω _0-obs-c ≈ {([k−Δk] / m) ^1/2 − ([k−Δk] / [m + Δm]) ^1/2 }
From the above relational expression, [m + Δm] / m can be finally calculated, that is, PAA attached to the surface of the cantilever c provided with the chitosan layer and capturing calcium ions. It is also possible to estimate the mass increase Δm caused by the adhesion of the material.

At least, in the cantilever c in which the chitosan layers are formed on both sides of the cantilever, the both sides of the cantilever are preliminarily bent, and PAA is attached in a state where calcium ions are captured. The adhesion amount can be increased, and the mass increase Δm can be increased. The difference in resonance frequency change between the cantilever c and the cantilever c ′, or between the cantilever c and the cantilever d ′, which reflects the adhesion amount of PAA in the state where calcium ions are captured, Δω _{0-obs-c ′} − Δω _0-obs-c or Δω _0-obs-d −Δω _0-obs-c can be easily measured.

(Embodiment 3)
In the present embodiment, an example is shown in which DNA of a target substance is detected by a cantilever sensor using a single-stranded DNA having a specific base sequence as a target substance-recognizing substance and using the single-stranded DNA immobilized thereon. That is, using the probe hybridization reaction, only the detection target DNA is selectively selected through the formation of a hybrid between the single-stranded DNA for the probe and the detection target DNA containing a complementary base sequence. This corresponds to the case of adhering to the cantilever surface.

  In addition, the cantilever sensor is a so-called multi-probe array type sensor in which a plurality of types of DNA probes are immobilized on the individual cantilever surfaces and the plurality of probe-fixed cantilevers are arranged in an array. For example, it can be used to detect whether the base sequence of the DNA to be detected contained in a sample has a base sequence portion complementary to the base sequences of these multiple types of DNA probes. You can also.

(1) Immobilization of single-stranded DNA on the surface of a cantilever In a multi-probe array type sensor used for detection, a pair of upper and lower electrically insulated V-shaped cantilevers is provided for each probe. Arranged in an opposing structure. As shown in FIG. 8, when an electric current is passed through a pair of upper and lower V-shaped cantilevers arranged opposite each other and made of a conductive material, an electromagnetic force is generated, that is, an attractive force according to the direction of the electric current. Or repulsion occurs. In addition, the conductive layer included in the pair of upper and lower V-shaped cantilevers constitutes a capacitor, and a change in the interval causes a change in the capacitance of the capacitor.

  Therefore, by utilizing this feature, when currents in opposite directions are passed through a pair of upper and lower V-shaped cantilevers, an attractive force is generated and the V-shaped cantilevers can be bent. Further, when a change in the interval is caused due to a change in the amount of deflection of the tip of the V-shaped cantilever, it can be detected as a change in the capacitance of the capacitor.

(Making a chip with a pair of upper and lower V-shaped cantilevers)
FIG. 8 schematically shows the structure of a sensor chip in which a pair of upper and lower V-shaped cantilevers with electrodes formed on the upper and lower surfaces of an insulating substrate are provided in an arrangement facing each other. The chip 81 is manufactured by using an insulating substrate, and a slit 82 having a vertical width of 5 mm for arranging V-shaped cantilevers in an array shape is provided at the center of the chip 81.

  On the upper surface of the chip 81, electrodes 85-1 and 85-used to supply current to the conductive wires 84-1 and 84-2 formed integrally with the V-shaped cantilever 83 on the upper surface side. 2 is provided. Correspondingly, a current is supplied to the lower surface of the chip 81 to the conductors 84-3 and 84-4 formed of a conductive layer integrated with the V-shaped cantilever 83 ′ on the lower surface side. Electrodes 85-3 and 85-4 used for the above are provided. FIG. 8A shows only the conductive wires 84-1 and 84-2 on the upper surface side of the chip 81 and the electrodes 85-1 and 85-2, but the lower conductive wires 84-3 and 84 are shown. -4 and the electrodes 85-3 and 85-4 are arranged at corresponding positions.

  The V-shaped cantilever 83 on the upper surface side and the V-shaped cantilever 83 ′ on the lower surface side are provided in a shape protruding into the slit 82 with an interval of 100 μm therebetween. That is, in the chip 81, the insulating substrate having a thickness of 1 μm corresponding to the spacer portion that gives an interval of 100 μm is left along one long side of the slit 82, and the insulating substrate is etched. An area is provided. In the capacitor structure composed of a pair of upper and lower V-shaped cantilevers, a portion having a major contribution to the capacitance is a pair of upper and lower V-shaped cantilever portions protruding into the slit 82. The conductive wires 84-1 and 84-2 and the conductive wires 84-3 and 84-4 are disposed with an insulator layer having a thickness of 1 μm interposed therebetween. When detecting a change in capacitance caused by a change in the amount of deflection at the tip of a pair of upper and lower V-shaped cantilevers, the contribution of the portion where the upper and lower conductors sandwich an insulator layer with a thickness of 100 μm is suppressed. It is desirable to keep it. Therefore, an insulating layer having a predetermined thickness corresponding to a spacer portion for adjusting the gap between a pair of upper and lower V-shaped cantilevers has a high insulating property and at the same time selects an insulating material having a low dielectric constant. It is preferable. Further, it is desirable that the chip 81 itself be made of an insulating material having high insulating characteristics and at the same time having a low dielectric constant. For example, a quartz substrate is preferably used.

  The V-shaped cantilevers 83 and 83 ′ are manufactured by etching n-type silicon, which is a conductive material, into a V-shape having a thickness of 5 μm, a distance from the root to the tip of 500 μm, and a width of 100 μm. Conductive wires 84-1 and 84-2 and conductive wires 84-3 and 84-4 are also integrated with V-shaped cantilevers 83 and 83 ', respectively, and fabricated by etching n-type silicon. In addition, the V-shaped cantilever and the conductor to be integrated have a gold / n-type silicon / gold laminated structure by adding a gold deposited film having a thickness of about 100 nm on the upper and lower surfaces of the n-type silicon layer. ing.

  Gold and n-type silicon have different thermal expansion coefficients. When the ambient temperature fluctuates, the two-layer structure of gold / n-type silicon induces surface stress and causes bending, but gold / n-type silicon / gold In the three-layer structure, the stresses induced on the upper surface and the lower surface cancel each other, and as a result, the occurrence of bending due to temperature fluctuation is suppressed. In addition, the upper and lower surfaces of the V-shaped cantilever part are covered with a gold vapor deposition film, and single-stranded DNA is bound by utilizing the reactivity of the sulfanyl group (—SH) to gold. It is possible. Of course, when a three-layer structure of gold / n-type silicon / gold is used, the gold deposited film on the upper surface and the lower surface also has a function of improving the overall conductivity.

  Further, GdFe is deposited on the tip of the V-shaped cantilever 83 (not shown). Using this magnetic material GdFe, the cantilever 83 is affected by a magnetic field.

  When constructing a multi-probe array type sensor using four types of DNA probes, four sets of V-shaped cantilevers with a pair of upper and lower electrodes; (e1, e′1) to (e4, e′4) These are arranged in an array in the horizontally long slits 82.

(Production of single-stranded DNA for DNA probes)
As the single-stranded DNA for the DNA probe, a chemically synthesized oligo DNA having a predetermined base sequence is used. Single-stranded DNA can be synthesized using an automatic DNA synthesizer (ABI, 381A). A sulfanyl group (—SH) used for binding onto the gold vapor deposition film is introduced into the 5 ′ end of the synthesized single-stranded DNA. A thiol modifier (Thio-Modifier: manufactured by Glen Research) is used to introduce a thiol group at the 5 ′ end during synthesis by an automatic DNA synthesizer. After synthesis, normal deprotection treatment is performed to recover chemically synthesized oligo DNA. Next, purification is performed using high-performance liquid chromatography to obtain a single-stranded DNA preparation having a target base sequence and a thiol group introduction treatment at the 5 ′ end.

  Four DNA probes shown in Table 1 are prepared: Probes E1 to E4.

  DNA probe: Probe E1 is a single-stranded DNA having a base sequence (SEQ ID NO: 1) corresponding to the EcoRI and SacI recognition sites in the multicloning site portion of plasmid pUC18, which is a commercially available cloning vector. is there.

  DNA probe: Probe E2 is a single-stranded DNA having a base sequence (SEQ ID NO: 2) corresponding to the BamHI and XbaI recognition sites in the multiple cloning site portion of plasmid pUC18.

  DNA probe: Probe E3 is a single-stranded DNA having a base sequence (SEQ ID NO: 3) corresponding to the PstI and SphI recognition sites in the multicloning site in the plasmid pUC18.

  DNA probe: Probe E4 is a single-stranded DNA having a base sequence (SEQ ID NO: 4) corresponding to the SphI and HindIII recognition sites in the multicloning site of plasmid pUC18.

(Immobilization of single-stranded DNA on cantilever surface)
Four types of single-stranded DNA (DNA probes): Probes E1 to E4 are each dissolved in 10 mM Tris-HCl buffer so that the final concentration is 1 μM to prepare four types of DNA probe aqueous solutions. To do. Using a micropipette, move the Probe E1 aqueous solution into the upper and lower set of cantilevers (e1, e'1), Probe E2 aqueous solution, the upper and lower set of cantilevers (e2, e'2), and the Probe E3 aqueous solution The Probe E4 aqueous solution is dropped into the pair of cantilevers (e3, e′3) and the pair of upper and lower cantilevers (e4, e′4). The dropped DNA probe solution droplets wrap around a pair of upper and lower cantilevers so that they do not mix with adjacent droplets. In this state, after standing for a while, 1M NaCl / 50 mM phosphate buffer (pH 7.0) is dropped several times to wash away unfixed single-stranded DNA. In this step, single-stranded DNA is immobilized on the outer surfaces of each pair of upper and lower cantilevers.

(3) Preparation of target DNA The following 6 types of chain DNA; target DNA1-target DNA6 are prepared.

  Target DNA 1 is a chain DNA obtained by digesting plasmid pUC18 with digestive enzyme EcoRI. That is, in the plasmid pUC18, the EcoRI recognition site is only present at the multiple cloning site portion, and as a result of digestion at this EcoRI recognition site, the resulting strand DNA no longer has an EcoRI recognition site. Absent.

  Target DNA 2 is a chain DNA obtained by digesting plasmid pUC18 with the digestive enzyme BamHI. That is, in the plasmid pUC18, the BamHI recognition site is present only at the multiple cloning site portion, and the resulting chain DNA no longer has a BamHI recognition site as a result of digestion at this BamHI recognition site. Absent.

  Target DNA 3 is a chain DNA obtained by digesting plasmid pUC18 with digestive enzyme PstI. That is, in the plasmid pUC18, the PstI recognition site exists only at the multicloning site part, and as a result of digestion at this PstI recognition site, the resulting strand DNA no longer has a PstI recognition site. Absent.

  Target DNA 4 is a chain DNA obtained by digesting plasmid pUC18 with the digestive enzyme HindIII. That is, in the plasmid pUC18, the HindIII recognition site exists only at the multiple cloning site, and as a result of digestion at this HindIII recognition site, the resulting strand DNA no longer has a HindIII recognition site. Absent.

  Target DNA 5 is a chain DNA obtained by digesting plasmid pUC18 with the digestive enzyme KpnI. That is, in the plasmid pUC18, the KpnI recognition site is present only at the multicloning site portion, and the resulting chain DNA no longer has a KpnI recognition site as a result of digestion at this KpnI recognition site. Absent. However, EcoRI, BamHI, PstI and HindIII recognition sites contained in the multicloning site part remain.

  Target DNA 6 is obtained by digesting plasmid pUC18 with digestive enzymes EcoRI and HindIII to obtain two fragments, and then collecting and purifying only a linear DNA fragment having a large molecular weight by electrophoresis. When digested with the digestive enzymes EcoRI and HindIII, the multicloning site in the plasmid pUC18 becomes a chain DNA fragment having a small molecular weight. Therefore, the chain DNA fragment having a large molecular weight does not include EcoRI, BamHI, PstI, and HindIII recognition sites present in the multiple cloning site.

(4) Target DNA detection using a multi-probe array type sensor The above four types of DNA probes: Probe E1 to E4 are used to make the above six types using the multi-probe array type sensor. A procedure for detecting the chain DNA; target DNA 1 to target DNA 6 will be described.

For the multi-probe array type sensor fabricated in advance, the “initial resonance frequency” ω _{0-obs-e1i to} ω of each of the upper and lower cantilevers (e1, e′1) to (e4, e′4) Measure _0-obs-e4i .

  First, as shown in FIG. 9A, a DC power supply 91 is connected to the electrodes 85-1, 85-2, a pulsed DC current is passed through the conductors 84-1, 84-2, and a pair of upper and lower cantilevers. Among 83 and 83 ′, GdFe provided at the tip of the upper V-shaped cantilever 83 is magnetized. This magnetization operation is performed on the upper cantilevers e1 to e4. Next, the DC power supply 91 is removed, the high frequency oscillator 49 is connected to the electrodes 85-3 and 85-4, and the high frequency current is supplied to the conductors 84-3 and 84-4 formed in the lower V-shaped cantilever 83 ′. Shed. The high-frequency oscillator 49 is controlled by the personal computer 48, and the control sets the frequency ω of the supplied high-frequency current and sweeps the frequency ω. A magnetic field that oscillates at a frequency ω is induced by the high-frequency current flowing through the conductors 84-3 and 84-4 of the lower V-shaped cantilever 83 '. A magnetic force is generated between the magnetized GdFe provided at the tip of the upper V-shaped cantilever 83 and the oscillating magnetic field induced in the lower V-shaped cantilever 83 '. As a result, an attractive force / repulsive force is generated according to the frequency ω of the oscillating magnetic field, and the upper V-shaped cantilever 83 and the lower V-shaped cantilever 83 ′ vibrate in a targeted manner. When the capacitance meter 92 is connected to the electrodes 85-1 and 85-3 and the capacitance between the pair of upper and lower cantilevers 83 and 83 ′ is measured, the pair of upper and lower cantilevers 83 are accompanied by vibration at the frequency ω. , 83 ′, the periodic capacitance change due to the variation in the gap between them is measured. The amplitude of this periodic capacitance change corresponds to the sum of the amplitudes of flexural vibrations of the pair of upper and lower cantilevers 83, 83 'caused by forced vibration.

  The pair of upper and lower cantilevers 83 and 83 'have essentially the same configuration, and their resonance frequencies are equal, and are forced to vibrate by the electromagnetic force, and the amplitude of the periodic capacitance change at the frequency ω. ΔC (ω) is measured. The frequency ω at which the amplitude ΔC (ω) of the capacitance change becomes a maximum, that is, the frequency ω at which the sum of the amplitudes of the flexural vibrations of the pair of upper and lower cantilevers 83 and 83 ′ is the maximum, 'Resonance frequency' measured value.

The “resonance frequency” measured values of the pair of upper and lower cantilevers (e1, e′1) to (e4, e′4) measured in the initial state are respectively measured as the “initial resonance frequency” measured values ω _{0-obs- e1i to} ω _0-obs-e4i .

  Next, as shown in FIG. 9 (b), with respect to a pair of upper and lower V-shaped cantilevers, the current supplied from the high-frequency oscillator 49 to the electrodes 85-3 and 85-4 is a direct current, and the upper V-shaped cantilever. The current direction is selected so that the electromagnetic force generated between the magnetized GdFe provided at the tip of the cantilever 83 and the magnetic field induced in the lower V-shaped cantilever 83 ′ becomes an attractive force. By this attractive force, V-shaped cantilevers 93 and 93 'are bent inward and bent with the outer surface of the cantilever convex.

  The aqueous solution containing the target DNA is heated in advance to 90 ° C., and the contained double-stranded DNA is denatured and separated into two single-stranded DNAs. A pair of upper and lower V-shaped cantilevers (e1, e ′) that are bent inward with a micropipette using an aqueous solution containing a single-stranded DNA that has been subjected to the denaturer treatment. 1) to (e4, e′4) are dropped. Each droplet encloses a pair of upper and lower cantilevers (e1, e'1) to (e4, e'4). After standing in this state for a certain time, the direction of the direct current supplied to the electrodes 85-3 and 85-4 is reversed. As a result, the electromagnetic force generated between the magnetized GdFe provided at the tip of the upper V-shaped cantilever 83 and the magnetic field induced in the lower V-shaped cantilever 83 ′ becomes a repulsive force. At this time, as shown in FIG. 9C, this repulsive force results in V-shaped cantilevers 94 and 94 'that are bent outward and bent so that the inner surface of the cantilever is convex. In this state, after leaving still for a fixed time, the supply of current is stopped. Finally, 1M NaCl / 50 mM phosphate buffer (pH 7.0) was dropped several times on each of the upper and lower sets of cantilevers (e1, e′1) to (e4, e′4), The bound single-stranded DNA is washed away and left to dry.

Hybridization reaction with four types of DNA probes immobilized on each of the V-shaped cantilevers (e1, e′1) to (e4, e′4) of the multi-probe array type sensor: Probes E1 to E4 After the above procedure is performed, the respective resonance frequencies are measured again. The resonance frequency is measured in the same procedure as described above. The resonant frequency measured after the hybridization reaction, and each "resonant frequency after the immersion treatment" Found _{ω 0-obs-e1f ~ω 0} -obs-e4f. For each pair of upper and lower cantilevers (e1, e′1) to (e4, e′4), “resonance frequency after immersion treatment” measured values ω _{0-obs-e1f to} ω _0-obs-e4f and “initial” the difference between the resonant frequency "Found _{ω 0-obs-e1i ~ω 0} -obs-e4i: Δω 0-obs-g1 = (ω 0-obs-e1i -ω 0-obs-e1f) Δω 0-obs- from _e4 = to calculate the _{(ω 0-obs-e4i -ω} 0-obs-e4f).

When the target single-stranded nucleic acid is hybridized to the DNA probe by the hybridization reaction, the mass m of the entire cantilever is proportional to the amount of the single-stranded nucleic acid to be hybridized before and after the hybridization reaction. It increases as (m + Δm). On the other hand, the spring constant k of the cantilever also increases as k → (k + Δk) before and after the hybridization reaction when there is a repulsive force between the hybrid bodies. For example, when hybrid formation occurs in the DNA probe E1 on the V-shaped cantilever (e1, e′1), based on the above correspondence,
ω _0-obs-e1i ≒ (k / m) ^1/2
ω _0-obs-e1f ≒ ([k + Δk] / [m + Δm]) ^1/2
Δω _0-obs-e1 = (ω _0-obs-e1i −ω _0-obs-e1f )
≒ [(k / m) ^1/2 -([k + Δk] / [m + Δm]) ^1/2 ]
This causes a change in the resonance frequency.

  For the target DNAs 1 to 6, the capacitance “resonance frequency after immersion treatment” measured after the hybridization reaction is measured according to the procedure described above, and the measurement result of the “initial resonance frequency” measured value in the initial state is The difference is calculated, and the presence / absence of changes in the mass m and the spring constant k of each V-shaped cantilever before and after the hybridization reaction is calculated.

  Table 2 summarizes the presence or absence of changes in the resonance frequency due to changes in the mass m and the spring constant k of each V-shaped cantilever before and after the hybridization reaction, with respect to the target DNAs 1 to 6.

  When there is a change in the resonance frequency of each V-shaped cantilever before and after the hybridization reaction, the DNA probe immobilized on the V-shaped cantilever and the target DNA form a hybrid, and the V-shaped cantilever It shows that adhesion has occurred. Therefore, whether each target DNA has four recognition sites of EcoRI and SacI, BamHI and XbaI, PstI and SphI, SphI and HindIII based on the measurement result of the presence or absence of each V-shaped cantilever resonance frequency change. It is possible to determine whether or not.

  Generally, as a multi-probe array type sensor, a pair of upper and lower V-shaped cantilevers are arranged in an array, and DNA probes having different base sequences are immobilized on each upper and lower pair of V-shaped cantilevers. By doing so, it is possible to determine whether or not n types of base sequences are included in the target DNA.

  According to the present invention, molecules or ionic species composed of one to several atoms, amino acids / peptides / proteins, antigenic molecules including these glycosides, conjugates with sugar chains, DNA and RNA A highly sensitive detection method and apparatus targeting nucleic acid molecules can be provided. Along with that, substances that exist minutely in water or in the atmosphere, such as gas sensors that detect rare gases or toxic gases in the atmosphere, or odor sensors (artificial olfaction) that detect odorous substances in the atmosphere, Useful resources such as uranium and rare metals, environmental hormones (endocrine disrupting chemicals) and environmental pollutants, detection devices such as bacteria, medical devices that detect some indicator substances and viruses in blood and breath, specific DNA for genetic diagnosis By applying the principle of the present invention to an array detector or the like, the detection sensitivity can be improved.

It is a figure which shows typically the operation principle utilized in the detection method of the target substance using the cantilever sensor concerning this invention. It is a figure which shows typically the operation principle utilized in the detection method of the target substance using the cantilever sensor concerning this invention. It is a figure which shows one form of the cantilever sensor utilized in this invention, and shows typically an example of a structure of the chip | tip with one cantilever. FIG. 2 is an example of a cantilever resonance frequency measuring device used in the present invention, and is a diagram schematically showing an example of a device configuration to which an optical lever type displacement amount detecting device for measuring a deflection amount of a cantilever tip is applied. It is. It is a figure which shows typically the relationship between the vibration frequency and amplitude in the vicinity of the resonance frequency of a cantilever when forcedly vibrating a cantilever. It is a figure which shows one form of the means to introduce | transduce a bending to a cantilever used in this invention, and shows typically an example of the method of applying external force using the cantilever probe for probe microscopes. It is a figure which shows one form of the cantilever sensor utilized in this invention, and shows typically an example of a structure of a chip | tip with a set of 2 cantilevers. It is a figure which shows typically an example of the apparatus for contacting with a target material in the state which introduce | transduced the bending to the cantilever utilized in this invention. It is a figure which shows one form of the cantilever sensor utilized in this invention, and shows typically an example of a structure of a chip | tip with two sets of V-shaped cantilever. Utilizing means for introducing a bend into a pair of V-shaped cantilevers used in the present invention, and means for detecting a change in the amount of bend at the tip of the V-shaped cantilever via a change in capacitance, It is a figure which shows typically an example of the means to measure the resonant frequency of a cantilever.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Oscillator which vibrates cantilever 11 Chip which fixes cantilever 12 Target substance recognition substance 13 Cantilever 14 External force 15 which deflects cantilever Target substance 16 Repulsive force between target substances 31 Chip with cantilever 32 Hole which cantilever is accommodated 33 Cantilever body 41 Cantilever tip fixing stage 42 of displacement amount measuring device 42 Cantilever tip fixing clamp 43 of displacement amount measuring device 43 Laser light source 44 Laser beam 45 Lens 46, 46 ', 46''Laser beam 47 reflected by tip of cantilever 47 Laser beam detector 48 PC for device control with AD converter 49 High-frequency oscillator 51 Tip portion of cantilever for probe microscope 52 Cantilever body for probe microscope 53 Can for probe microscope Chiller probe 54 Spacers 63, 63 ′ for adjusting the deflection amount of the cantilever Cantilever main body 71 Calcium carbonate aqueous solution 72 containing PPA Paddle 73 for generating water flow channel 74 for aqueous solution Cantilever main body 81 deflected by water flow Tip with cantilever array 82 Slit 83, 83 'V-shaped cantilever body 84-1-4 conducting wire 85-1-4 electrode 91 power supply device 92 capacitance meter 93, 93' V-shaped bent inward by electromagnetic force Cantilever body 94, 94 'V-shaped cantilever body bent outward by electromagnetic force

Claims (5)

A method for detecting a target substance using a cantilever sensor,
(A) A step of fixing a target substance recognition substance that can be combined with the target substance on the surface of the cantilever constituting the cantilever sensor;
(C) A step of applying an external force to the cantilever so as to make at least one surface convex and hold it in a bent state;
(D) The step of bringing the cantilever held in a bent state into contact with a sample to enable binding of the target material to the target material recognition material;
(E) removing the external force applied to the cantilever;
(F) measuring the resonance frequency of the cantilever after making contact with the sample for the purpose of binding to the target substance;
(G) calculating the difference between the resonance frequency of the cantilever after contact with the sample and the initial resonance frequency of the cantilever before contact with the sample, measured in the step (F);
A method for detecting a target substance, comprising: To measure the initial resonant frequency of the cantilever before contact with the sample,
Between the step (A) and the step (C),
(B) measuring the initial resonant frequency of the cantilever;
The detection method according to claim 1, wherein: Between the step (D) and the step (E),
(C ′) An external force in a direction opposite to the external force applied in the step (C) is applied to the cantilever, and the cantilever is held in a state bent in the opposite direction to the direction of the bending in the step (C). The process of
(D ′) a step of bringing the cantilever held in a state of bending in the opposite direction into contact with a sample and allowing the target substance to bind to the target substance recognition substance;
The detection method according to claim 1, further comprising: A detection device that detects a target substance using a cantilever sensor,
(A) a cantilever sensor including a cantilever having a target substance recognition substance fixed on the surface;
(C) means for applying an external force to the cantilever so that at least one surface is convex and held in a bent state;
(D) means for bringing the cantilever held in the bent state into contact with the sample in a state enabling the binding of the target material to the target material recognition material;
(E) means for removing external force applied to the cantilever;
(F) means for measuring the resonant frequency of the cantilever after contact with the sample for the purpose of binding to the target substance;
(H) A target material fixed on the surface of the cantilever based on a resonance frequency difference calculated from the measured resonance frequency of the cantilever after contact with the sample and the initial resonance frequency of the cantilever before contact with the sample. A target substance detection apparatus comprising means for calculating the presence or absence of a target substance bound to a recognition substance or the amount of the bound target substance. (F) In the means for measuring the resonance frequency of the cantilever after contact with the sample, a vibrator for forcibly vibrating the cantilever used for measurement of the resonance frequency is:
5. The detection device according to claim 4, wherein the detection device is incorporated into the (a) cantilever sensor, or is made to contact the (a) cantilever sensor during measurement.

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