JP6216627B2 - Sensing method - Google Patents

Description

  The present invention relates to a sensing method for sensing a sensing object using a piezoelectric vibrator.

  In the field of food hygiene, detection and quantification of relatively low molecular weight substances such as mold toxins and antibiotics in foods are performed. In the field of drug development, detection and quantification of low molecular weight substances may be performed. As a technique for performing such quantification, a sensing method using QCM (Quarts Crystal Microbalance) as disclosed in Patent Document 1 has been developed. In this sensing method, when the sensing object in the sample liquid is adsorbed to the adsorption layer formed on the surface of the crystal oscillator, the oscillation frequency of the crystal oscillator is determined according to the amount of adsorption of the sensing object due to the mass load effect. This is a technique that uses the fact that changes occur.

  By the way, in the sensing method by QCM, since the mass load effect is used, the larger the molecular weight of the sensing object, the larger the frequency variation with respect to the adsorption amount. That is, when the sensing object is a low-molecular substance, the frequency change amount with respect to the adsorption amount is small, so that the sensitivity is lowered. Therefore, if the concentration of the sensing object contained in the sample liquid is low, it becomes difficult to perform highly reliable measurement.

  To solve this problem, a method of increasing the amount of frequency change by adding a molecule larger than the low molecular weight substance that has the property of binding to the low molecular weight substance to be detected and binding to the low molecular weight substance, so-called increase. A technique called feeling is known. As a molecule used for the sensitization, for example, an antibody is often used in an apparatus for detecting an antigen. For the purpose of obtaining a greater sensitization effect, particles larger than the antibody should be used. There is. In this case, the particles used have a particle size of the order of nanometers or micrometers, and as the material, particles having a large mass such as colloidal gold, magnetic beads, and latex are mainly used.

  However, in the sensitization method using the above-described particles, when the particles become larger than a certain size, the particles attached to the surface of the quartz crystal vibrator vibrate at a unique frequency different from that of the quartz crystal. As described in Section 2, a phenomenon is known in which a sensitization effect due to addition of a desired mass cannot be obtained. For this reason, in spite of the addition of particles for the purpose of sensitization, the sensitivity sometimes decreases.

  Patent Document 2 discloses a method of sensitizing frequency amplitude by adding a molecule having a larger particle size than a measurement object and a crosslinkable compound. Patent Document 3 discloses a method of increasing the frequency amplitude by increasing the surface area of a thin film by attaching particles to the thin film adsorbing a measurement target and removing the particles. However, these methods are different in principle from the present invention.

JP 2007-178348 A JP 2006-275864 A JP 2007-147556 A

A sensitive new method for the determination of adhesive bonding between a particle and a substrate, J. Appl. Phys. 58 (7), 1, October 1985 Positive Frequency Shift Observed Upon Adsorbing Micron-Sized Solid Objects to a Quartz Crystal Microbalance from the Liquid Phase, Anal. Chem. 2010, 82, pp.2237-2242

  The present invention has been made under such circumstances, and an object of the present invention is to perform highly reliable measurement in sensing a sensing object in a sample liquid based on a change in frequency of a piezoelectric vibrator. It is in providing the sensing method which can do.

The sensing method of the present invention comprises:
In a method for sensing a sensing object in a sample liquid based on a change in frequency of a piezoelectric vibrator,
Supplying a sample liquid to an adsorption layer formed on the electrode surface on one side of the piezoelectric vibrator and adsorbing the sensing object;
Supplying a first liquid containing a group of first particles having a property of adsorbing to a portion corresponding to the sensing object after that to one surface side of the piezoelectric vibrator;
Subsequently, a second liquid containing a second group of particles having a particle size smaller than that of the first particles and adsorbing to a portion corresponding to the sensing object is supplied to one surface side of the piezoelectric vibrator. And packing the second particles between the first particles and the adsorption layer;
And oscillating the piezoelectric vibrator with an oscillation circuit, and measuring each oscillation frequency before and after supplying the first particles to one surface of the piezoelectric vibrator.

  The sensing method of the present invention uses a group of second particles smaller than the first particles in the sensing method for sensitization using the first particles having a molecular weight larger than that of the sensing object. A first particle group is fixed on the surface of the piezoelectric vibrator by filling a gap between the surface of the piezoelectric vibrator and the surface of the piezoelectric vibrator. Therefore, by suppressing the natural vibration of the first particles, the sensitization effect of the first particle group can be obtained with certainty, so that the sensing object can be sensed with high accuracy.

It is a block diagram of the sensing apparatus which concerns on the sensing method of this invention. It is a perspective view of the sensing sensor which comprises the said sensing device. It is a vertical side view of the sensing sensor. It is a top view of the crystal oscillator which comprises the said sensor. It is a circuit diagram of the sensing device. It is a schematic diagram of the surface of the crystal unit. It is a schematic diagram of the surface of the crystal unit. It is a schematic diagram of the surface of the crystal unit. It is a schematic diagram of the surface of the crystal unit. It is a schematic diagram of the surface of the crystal unit. It is the schematic diagram showing the change of the resonant frequency by the coupling | bonding of the particle | grain to the surface of a crystal oscillator. It is a schematic diagram of the surface of the crystal unit. 6 is a graph showing the effect of the sensing method according to the present invention. It is the graph which measured the effect of other sensing methods.

  FIG. 1 shows one embodiment of a sensing device for carrying out the sensing method of the present invention. The sensing device includes a sensor unit 2, a liquid supply system 1 for supplying a sample liquid, a first liquid, a second liquid, a third liquid and a buffer liquid to the sensor unit 2, as will be described later, and a sensor. A liquid discharge system 90 that stores liquid discharged from the unit 2, a crystal sensor 7 that is a piezoelectric sensor attached to the sensor unit 2, and an oscillation circuit unit 6 that is provided adjacent to the sensor unit 2. ing.

  The liquid supply system 1 includes a flow path switching mechanism 11, a sample liquid supply unit 12, a first liquid supply unit 13 that supplies a first liquid containing a first bead group 45, which will be described later, and a second liquid that will be described later. A second liquid supply unit 14 for supplying a second liquid containing a group of beads 47, a third liquid supply unit 15 for supplying a third liquid containing a biotinylated antibody 40 which is an intermediate substance described later, A buffer solution supply unit 17 for supplying a buffer solution is provided. The flow path switching mechanism 11 selects one of the sample liquid, the first liquid, the second liquid, the third liquid, and the buffer solution via the sample liquid supply path 16 and supplies the selected liquid into the sensor unit 2. It is configured. As the flow path switching mechanism 11, for example, an injection valve having six ports and an injection loop is used. In this case, the selected liquid is temporarily stored through one port in the injection loop, and then the liquid is sent from the other port.

  As shown in FIG. 2, the sensor unit 2 is configured by laminating a support body 21, a wiring substrate 3, a crystal resonator 4, a flow path forming member 5, and a cover portion 24 in this order from the lower side. The support 21 is formed with a support recess 22 for fitting and holding the crystal sensor 7 and the flow path forming member 5. Accordingly, the channel forming member 5 is pressed against the crystal sensor 7 with the crystal sensor 7 fitted in the support recess 22, so that the lower surface of the channel forming member 5 presses the crystal resonator 4 against the wiring substrate 3. And fix it. Furthermore, the support body 21 is covered with a cover body 24 from above.

  2 and 3, reference numeral 26 denotes a liquid supply pipe, and 27 denotes a liquid discharge pipe. The sample liquid supplied from the liquid supply system 1 to the reaction channel 52 via the liquid supply pipe 26 passes through the liquid discharge pipe 27. It is configured to be discharged into the discharge system 90. The liquid discharge system 90 includes a drainage reservoir.

  The crystal sensor 7 is configured by providing a crystal resonator 4 that is a piezoelectric resonator on a wiring board 3. As shown in FIG. 4, the crystal resonator 4 is configured by providing excitation electrodes 42 and 43 on both surfaces of a disk-shaped crystal piece 41 that is a piezoelectric piece. In this example, as shown in FIG. 4B, the back side excitation electrode 43A and the second excitation electrode 43B are arranged so as to be spaced apart from each other and aligned in the X direction, and as shown in FIG. 4A. A common electrode 42 that is an excitation electrode for the two excitation electrodes 43A and 43B is disposed on the front surface side. Accordingly, the first excitation region 43A and the common electrode 42 form a first vibration region 4A, and the second excitation electrode 43B and the common electrode 42 form a second vibration region 4B.

  When the crystal sensor 7 is connected to the sensor unit 2, the first excitation electrode 43 </ b> A and the second excitation electrode 43 </ b> B are two oscillation circuits via the conductive paths 32 and 34 of the wiring board 3 as shown in FIG. 5. Connected to 6A and 6B, respectively. At this time, the common electrode 42 is connected to the ground side of the oscillation circuits 6A and 6B through the conductive path 33 of the wiring board 3. In the end region of the wiring board 3, connection terminals 35, 36, and 37 connected to the respective conductive paths 32, 33, and 34 are formed.

  An adsorption layer 46 made of an antibody 49 for adsorbing a sensing object, for example, an antigen, is formed on the first region corresponding to the first excitation electrode 43A in the common electrode 42 on the crystal unit 4. Yes. On the other hand, the second region corresponding to the second excitation electrode 43B in the common electrode 42 is exposed without the adsorption layer 46 being formed, and the temperature change of the oscillation frequency of the first region is caused during data processing. Plays a canceling role.

  Further, the oscillation circuit unit 6 is electrically connected to the connection terminal portion of the wiring board 3 as shown in FIG. 5 by being inserted into the sensor unit 2. A measurement circuit unit 81 and a data processing unit 82 are provided following the oscillation circuit unit 6. The measurement circuit unit 81 has a function of measuring the oscillation frequency. The data processing unit 82 is a part that stores time series data of measured frequencies and displays the time series data, and is composed of, for example, a personal computer.

  The oscillation frequencies of the first vibration area 4 </ b> A and the second vibration area 4 </ b> B of the crystal resonator 4 are taken into the measurement circuit unit 81. Then, the data processor 82 obtains time series data of the difference between the oscillation frequency in the first vibration region 4A and the oscillation frequency in the second vibration region 4B.

  A process for quantifying an antigen in a sample solution using the above-described sensing device will be described. First, a buffer solution is supplied from the buffer solution supply unit 17 to the reaction flow path 52 in the sensor unit 2, and the oscillation frequencies F4a and F4b of the first vibration region 4A and the second vibration region 4B are measured. Subsequently, the sample liquid is supplied into the sensor unit 2 from the sample liquid supply unit 12 of the liquid supply system 1, and the sample liquid flows into the reaction channel 52 from the liquid supply port 53. The sample solution that has flowed into the reaction channel 52 spreads inside the reaction channel 52 and reaches the surface of the crystal unit 4 on which the common electrode 42 is formed.

  Here, the antigen in the sample solution does not directly react with the crystal, and the electrode is directly exposed from the common electrode 42 of the second vibration region 4B, and the antigen does not react with the region. Therefore, when an antigen is supplied to the common electrode 42 in which the adsorption layer 46 is formed by the antibody 49 as shown in FIG. 6, the adsorption layer on the common electrode 42 in the first vibration region 4A as shown in FIG. The antigen reacts only with the antibody 49 constituting 46, and is bound on the adsorption layer 46. 7 to 10 and 12, reference numeral 71 denotes an antigen, and the binding density of the antigen 71 to the adsorption layer 46 is shown to be higher than the actual density.

  After a predetermined time has elapsed since the sample solution was supplied, the third solution containing the biotinylated antibody 40 is supplied from the third solution supply unit 15 to the reaction channel 52 in the sensor unit 2. The biotinylated antibody 40 is bound to the antigen 71 as shown in FIG. The biotinylated antibody 40 is an intermediate substance formed by binding an antibody 40b that selectively binds to the antigen 71 and biotin 40a. Subsequently, the first liquid containing the first bead (particle) group 45 is supplied from the first liquid supply unit 13 to the reaction flow path 52 in the sensor unit 2, and the quartz crystal 4 A first group 45 of beads is supplied to the surface on which the common electrode 42 is formed. The first liquid in which the first bead group 45 is dispersed is, for example, a buffer solution, but may be pure water.

  As shown in FIG. 9, the first bead group 45 is treated with avidin particles 48 so that the surface binds to biotin 40 a on the biotinylated antibody 40. Since the avidin particle 48 and the biotinylated antibody 40 bound to the surface of the first group of beads 45 are bound 1: 1, the first bead 45 supplied on the surface of the crystal unit 4 is Then, the biotinylated antibody 40 overlaps and binds to the antigen 71 bound to the adsorption layer 46 described above. The size of the beads constituting the first bead group 45 is 200 nm to 3000 nm in diameter, and the material is, for example, magnetic beads, colloidal gold, latex, or the like.

  Subsequently, the second liquid containing the second bead (particle) group 47 is supplied from the second liquid supply unit 14 to the reaction flow path 52 in the sensor unit 2. The second bead group 47 supplied on the surface of the crystal unit 4 is treated with avidin particles 48 as in the first bead group 45 (not shown in FIG. 10). ). Then, as shown in FIG. 10, it enters the gap between the particles of the first bead group 45 bound to the biotinylated antibody 40 and the adsorption layer 46 to fill the gap. The size of the beads constituting the second group of beads 47 is 5 nm to 100 nm in diameter, and the material is, for example, magnetic beads, colloidal gold, latex, or the like. Then, after a predetermined time has elapsed, the buffer solution is supplied from the buffer solution supply unit 17 to the reaction channel 52 in the sensor unit 2 to block the adsorption layer 46.

  In this way, the sample solution passing through the reaction channel 52 is supplied to the surface of the common electrode 42 as well as the buffer solution and passes over the common electrode 42. Then, both the oscillation frequencies of the vibration regions 4A and 4B are equally reduced by the water pressure. When the sample liquid contains the antigen 71 that is a sensing object, simultaneously with this action, the antigen 71 is adsorbed to the adsorption film 46 on the first vibration region 4A. Further, by supplying the first bead group 45 to the surface of the common electrode 42, the first bead group 45 is overlapped and bound to the antigen 71, so that the oscillation frequency depends on the amount of adsorption due to the mass load effect. F1 decreases. On the other hand, since the antigen 71 is not adsorbed on the second vibration region 4B, the first bead group 45 is not bonded, and the oscillation frequency F2 is not lowered due to the mass load effect. As a result, the oscillation frequencies of the vibration regions 4A and 4B become F4a 'and F4b', and a change occurs between (F4a'-F4b ') and (F4a-F4b). The difference between (F4a′−F4b ′) and (F4a−F4b) is obtained, and a relational expression between the difference and the concentration of the sensing object in the sample liquid is obtained, and obtained by the relational expression and measurement. From the difference, the concentration of the sensing object in the sample solution is obtained.

  According to the above-described embodiment, in the sensing method in which sensitization is performed using the first bead group 45 having a molecular weight larger than that of the antigen 71 as the sensing object, the particles having a smaller particle size than the first bead group. The first bead group 45 is fixed on the surface of the crystal unit 4 by filling the gap between the first bead group 45 and the surface of the crystal unit 4. ing. Therefore, by suppressing the natural vibration of the particles of the first bead group 45, the sensitization effect of the first bead group 45 can be obtained with certainty, so that the antigen 71 can be sensed with high accuracy. There is an effect that can be done.

  In the embodiment described above, in order to solve the above-described problem that the sensitization effect is reduced when the particle exceeds a certain size in the sensitization method by particle bonding in the quartz crystal resonator, first, the sensing object. Are bonded to the adsorption layer 46 formed on the surface of the common electrode 42 on the crystal unit 4. Thereafter, the first bead group 45 having the property of binding to the sensing object is overlapped with the sensing object bound to the adsorption layer 46. Subsequently, when a second bead group 47 having a particle smaller than that of the first bead group 45 is supplied onto the crystal unit 4, the beads constituting the second bead group 47 are transferred to the first bead group 45. Since the particle diameter is about 1/10 to 1/100 as compared with the constituting beads, as described above with reference to FIG. 10, the second bead is formed in the gap between the first bead group 45 and the adsorption layer 46. A group 47 of beads enters. The second bead group 47 fills the gap, so that the natural vibration of the first bead group 45 is suppressed. In the present invention, the first bead group 45 is supplied onto the crystal resonator 4 prior to the second bead group 47, so that the amount of the antigen 71 and the first bead group 45 can contain the biotinylated antibody 40. Thus, a sensitizing effect can be obtained based on the correspondence relationship between the amount of the antigen 71 and the antigen 71. Further, since the second bead 47 is smaller in both volume and weight than the first bead 45, the sensitization effect by the first bead group 45 is not adversely affected.

  In this manner, the natural vibrations of the beads constituting the first bead group 45 can be suppressed by the second bead group 47. As a result, the mass loading effect of the first bead group 45 is exhibited, and the sensitization effect represented by the formula (1) described later can be efficiently obtained. Therefore, even when the molecular weight of the sensing object is small, it is possible to detect the sensing object with high sensitivity.

ここで、Δｍは負荷質量（ｇ）、Ｓは電極面積（ｃｍ^２）、ρは圧電振動子の密度（ｇ／ｃｍ^３）、μは圧電振動子の剪断応力（ｇ／ｃｍ・ｓｅｃ^２）、Ｎはオーバートーン次数、Ｆは公称周波数（Ｈｚ）、ΔＦは反応前後の圧電振動子における周波数変化（Ｈｚ）である。
しかし、圧電振動子に対して質量を負荷する物質がある程度の大きさを超えると、質量を負荷する物質が圧電振動子とは異なる固有の周波数にて振動するようになり、上述の（１）式の関係、即ち発振周波数の低下が成り立たなくなる現象が知られている。 Here, the effect of supplying the first bead group 45 and the second bead group 47 to the crystal unit 4 in the present embodiment will be described.
Regarding the relationship between the load mass and the frequency before and after the mass is applied to the surface of the piezoelectric vibrator, a relational expression (Sauerbrey's expression) represented by the following expression (1) is generally established. The sensitization effect described in the section of the background art uses the relational expression.

Here, Δm is the load mass (g), S is the electrode area (cm ² ), ρ is the density of the piezoelectric vibrator (g / cm ³ ), and μ is the shear stress of the piezoelectric vibrator (g / cm · sec ² ). , N is the overtone order, F is the nominal frequency (Hz), and ΔF is the frequency change (Hz) in the piezoelectric vibrator before and after the reaction.
However, when the mass loading material with respect to the piezoelectric vibrator exceeds a certain size, the mass loading material vibrates at a unique frequency different from that of the piezoelectric vibrator, and the above (1) It is known that there is a relationship between the equations, that is, a phenomenon in which a decrease in oscillation frequency does not hold.

This phenomenon will be described with reference to FIG.
Consider a state in which a sphere 83 is simply coupled to the crystal resonator 8 as shown in FIG. It is assumed that the mass of the crystal resonator 8 is M and the mass of the sphere 83 is m, and the vibrations of the crystal resonator 8 and the sphere 83 are spring vibrations having spring constants K and k, respectively. Since the crystal resonator 8 and the sphere 83 are coupled, resonance occurs in a state where two springs are coupled in series as shown in FIG.

  Non-Patent Document 1 discloses that the angular frequency of the resonance is higher than the angular frequency of the crystal resonator 8, and therefore the relationship represented by the above-described equation (1) does not hold. Therefore, in the present embodiment, the natural vibration of the sphere 83 is suppressed by filling the gap between the sphere 83 and the crystal resonator 8 with particles smaller than the sphere 83, and therefore only the vibration of the crystal resonator 8 alone is suppressed. Like to get.

  In this embodiment, the relational expression between the difference between (F4a′−F4b ′) and (F4a−F4b) acquired in advance and the concentration of the sensing object in the sample liquid, and the difference obtained by the measurement Based on the above, the concentration of the sensing object in the sample liquid has been obtained, but instead of the quantification, it is detected that there is a difference between (F4a′−F4b ′) and (F4a−F4b). Thus, the presence or absence of the antigen may be determined.

  In the embodiment described above, the frequencies F4a and F4b of the first vibration region 4A and the second vibration region 4B are measured before supplying the sample solution, and (F4a′−F4b ′) and (F4a−F4b) are measured. ), The antigen 71 as the sensing object is quantified. However, the timing of measuring the oscillation frequencies F4a and F4b is before the first bead group 45 is supplied. The sample liquid may be supplied before the third liquid is supplied or after the third liquid is supplied.

  Further, in the embodiment described above, instead of supplying the third liquid containing the biotinylated antibody 40 from the third liquid supply unit 15, as shown in FIG. The surfaces of the 45 and second bead groups 47 may be treated with avidin particles 48 and biotinylated antibodies 40 that bind to the avidin particles 48. Then, the same result can be obtained by supplying the first bead group 45 and the second bead group 47 to the crystal unit 4 in this order. That is, each biotinylated antibody 40 of the first bead group 45 and the second bead group 47 is captured by the antigen 71 on the adsorption film 46.

  Further, in the embodiment described above, the crystal resonator 4 in which the vibration region is divided into 4A and 4B is used in order to cancel the frequency change due to the temperature characteristic of the crystal resonator 4. Instead of the crystal resonator 4, a crystal resonator having one vibration region, that is, one corresponding electrode on each side of the crystal resonator may be used. In this case, an adsorption layer that adsorbs the sensing object is provided on one surface side of the crystal unit, and the difference in the oscillation frequency of the crystal unit before and after the supply of the first bead group 45 is measured.

A test for measuring the effect of the present invention was performed using the sensing device according to the embodiment of the present invention described above. Biotin-conjugated bovine serum albumin (BSA) was used as the sensing object. Biotin-bound BSA has the property of binding in a small amount to the surface of a gold electrode.
(Evaluation test)
A sample solution containing 100 μg / mL of biotin-conjugated BSA is supplied to the sensor unit, and after a predetermined time, a buffer solution consisting of BSA containing avidin-conjugated magnetic beads having a diameter of 1 μm is supplied to the sensor unit, and the frequency before the beads are supplied. Got the change. Then, after a certain period of time, a buffer solution made of BSA containing avidin-bound magnetic beads having a diameter of 50 nm was supplied to the sensor unit 2 to obtain further frequency changes.

(Reference test)
Using the same sensing device as in the evaluation test, a biotin-conjugated BSA sample solution having the same concentration is supplied to the sensor unit, and after a predetermined time, a buffer solution made of BSA containing avidin-conjugated magnetic beads having a diameter of 50 nm is supplied to the sensor unit. The change in the frequency before the bead supply was obtained. Further, after a predetermined time, a buffer solution made of BSA containing avidin-bound magnetic beads having a diameter of 1 μm was supplied to the sensor unit, and further changes in frequency were obtained.

The result of the evaluation test is shown in FIG. 13, and the result of the reference test is shown in FIG. 14 as a graph.
In the evaluation test, the frequency decreased after supplying 1 μm beads (a ₁ ), and then the frequency decreased greatly by supplying 50 nm beads (a ₂ ), and the effect of the above-described formula (1) was obtained. It was shown that On the other hand, in the reference test, when 50 nm beads were supplied, the frequency decreased (b ₁ ), but when 1 μm beads were supplied thereafter, the frequency increased (b ₂ ). This means that even if a small (50 nm) bead is supplied before supplying a large (1 μm) bead, the sensitizing effect cannot be obtained. It is speculated that this happened. Therefore, it can be said that the sensitization method using beads of the present invention can significantly suppress the coupling resonance between the crystal resonator and the beads and realize sensitization by a desired mass load effect.

DESCRIPTION OF SYMBOLS 1 Liquid supply system 2 Sensor unit 3 Wiring board 4 Crystal oscillator 40 Biotinylated antibody 42 Common electrode 45 First bead group 46 Adsorption layer 47 Second bead group 48 Avidin particle 49 Antibody 5 Flow path forming member 71 Antigen

Claims (9)

In a method for sensing a sensing object in a sample liquid based on a change in frequency of a piezoelectric vibrator,
Supplying a sample liquid to an adsorption layer formed on the electrode surface on one side of the piezoelectric vibrator and adsorbing the sensing object;
Supplying a first liquid containing a group of first particles having a property of adsorbing to a portion corresponding to the sensing object after that to one surface side of the piezoelectric vibrator;
Subsequently, a second liquid containing a second group of particles having a particle size smaller than that of the first particles and adsorbing to a portion corresponding to the sensing object is supplied to one surface side of the piezoelectric vibrator. And packing the second particles between the first particles and the adsorption layer;
And a step of oscillating the piezoelectric vibrator with an oscillation circuit and measuring each oscillation frequency before and after supplying the first particles to one surface of the piezoelectric vibrator. After performing the step of supplying the sample liquid, before the step of supplying the first liquid to one surface side of the piezoelectric vibrator, a third liquid containing an intermediate substance adsorbed on the sensing object is added to the third liquid. The process of supplying to one side of the piezoelectric vibrator is performed,
The sensing method according to claim 1, wherein the first particles and the second particles have a property of being adsorbed by the intermediate substance.   The first particle is a group of adsorbing particles having a base constituting the particle main body and a property of adhering to the outer peripheral surface of the base and adsorbing to a site corresponding to the sensing object and having a smaller particle size than the base. The sensing method according to claim 1, further comprising:   4. The sensing method according to claim 3, wherein the adsorption particles provided in the first particles are avidin particles.   The second particle is a group of adsorbent particles having a base constituting the particle main body and a property of adhering to the outer peripheral surface of the base and adsorbing to a site corresponding to the sensing object and having a smaller particle size than the base. The sensing method according to any one of claims 1 to 4, further comprising:   6. The sensing method according to claim 5, wherein the adsorption particles provided in the second particles are avidin particles.   After performing the step of supplying the sample liquid, before the step of supplying the first liquid to the one surface side of the piezoelectric vibrator, biotin, which is an intermediate substance adsorbed on the sensing object, is bound. The sensing method according to claim 4 or 6, wherein a step of supplying a third liquid containing bovine serum albumin to one surface side of the piezoelectric vibrator is performed. The first particles and the second particles are a base constituting the particle main body, adsorbing particles attached to the outer peripheral surface of the base and having a smaller particle diameter than the base, and an intermediate substance attached to the adsorbing particles. And comprising
The sensing method according to claim 1, wherein the intermediate substance has a property of being adsorbed by the sensing object.   9. The sensing method according to claim 8, wherein the adsorption particles provided in the first particles and the second particles are avidin particles, and the intermediate substance is a biotinylated antibody.

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