JP2017156159A - Sensing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique that heightens measurement accuracy when sensing a sensing object in a sample solution on the basis of a frequency change in a piezoelectric transducer.SOLUTION: A solution containing a biotin antibody 101 and a solution containing a sensitizing particle binding antibody 102 are previously mixed, with the mixture being a measurement solution. An attempt to have an antigen 100 captured by an antibody 111 fixed to the surface of an adsorption layer 47 results in that the antibody 111 can capture only an antigen 100 passing through near the surface of the adsorption layer 47 and a binding rate is low. On the contrary, when a solution containing the biotin antibody 101, the sample solution, and a solution containing the sensitizing particle binding antibody 102 are mixed, the antigen 100 and the antibody 111 portion of the biotin antibody 101 become easy to contact, so that it is possible to more reliably bind the antigen 100 with the biotin antibody 101.SELECTED DRAWING: Figure 10

Description

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

  In the clinical field, for example, a simple test method called POCT (Point of care testing) typified by self-monitoring of blood glucose level, influenza virus test, and the like is widespread, and is described in Patent Document 1 in this test. A method using such a QCM (Quartz Crystal Microbalance) is known. Briefly describing the QCM, the sensing object in the sample liquid is adsorbed on the surface of the crystal unit provided in the sensing device, and the oscillation frequency of the crystal unit is increased by the mass addition effect according to the amount of adsorption of the sensing object. Change. Based on the change in the oscillation frequency, detection or quantification of the sensing object in the sample liquid is performed.

When adsorbing a sensing object to a crystal resonator, an adsorption layer composed of, for example, an antibody is provided on the electrode surface, and a sample liquid containing the sensing object is flowed to the electrode surface, thereby becoming a sensing object by an antigen-antibody reaction. The antigen is adsorbed on the adsorption layer. However, since the antibody immobilized on the electrode surface and the antigen contained in the liquid flowing on the electrode surface have a low contact frequency, there is a problem that the adsorption efficiency is low.
Conventionally, it is necessary to provide an antibody corresponding to the antigen, and it is necessary to prepare a sensing sensor corresponding to the sensing object.

  Patent Document 2 describes a biosensor in which a biotinylated antibody is bound to avidin provided on the surface of a piezoelectric material, and an analyte is captured by the biotinylated antibody. However, the analyte is captured by immobilizing the biotinylated antibody to avidin and then flowing the sample solution containing the analyte, which does not solve the problem of the present invention.

JP 2007-147556 A Special table 2014-525568 gazette

  The present invention has been made under such circumstances, and an object of the present invention is to provide a technique for improving measurement accuracy when sensing a sensing object in a sample liquid based on a frequency change of a piezoelectric vibrator. It is in.

The sensing method of the present invention includes a sensing sensor in which an adsorption layer having an anchor material provided on the surface thereof is formed on an electrode surface of a piezoelectric vibrator, an oscillation circuit that oscillates the piezoelectric vibrator, and an oscillation frequency of the oscillation circuit And a measuring unit for measuring
Sensing is performed by mixing a liquid containing a target binding substance obtained by binding a target that specifically binds to the anchor substance with a substance having a property of adsorbing to a corresponding part of the sensing object and a sample liquid to be sensed. Obtaining a measurement liquid in which the target and the target binding substance are combined;
A blank corresponding to the amount of the target binding material adsorbed on the adsorption layer is measured by supplying a reference liquid comprising a liquid containing the target binding material to the sensing sensor to bind the target binding material to the adsorption layer and measuring the oscillation frequency. Determining the frequency;
Measurement liquid is supplied to the sensing sensor, the target binding substance and the sensing object are adsorbed on the adsorption layer, and the oscillation frequency is measured, which corresponds to the amount of the target binding substance and the sensing object adsorbed on the adsorption layer. Obtaining a detection frequency to perform,
And a step of detecting a sensing object adsorbed on the adsorption layer based on a difference value between the detection frequency and the blank frequency.

The present invention provides a measurement liquid in which a liquid containing a target binding substance and a sample liquid containing a sensing object are mixed to bind the sensing object and the target binding substance. Then, the measurement liquid is supplied to the sensing sensor in which the adsorption layer having the anchor material provided on the surface is formed on the electrode surface of the piezoelectric vibrator, and the target and the anchor material are combined, and the target binding material and the sensing object are obtained. The detection frequency is measured by adsorbing to the adsorption layer. In addition, the blank frequency is measured by adsorbing a target binding substance that is not bound to the sensing object to the adsorption layer. The sensing object is detected from the difference between the detection frequency and the blank frequency. Since the sensing object and the target binding substance are combined in advance by mixing the liquid and the liquid, the coupling efficiency is high, so that the efficiency of the sensing object to be adsorbed on the adsorption layer is increased and the measurement accuracy is increased.
In addition, by using a target binding substance corresponding to the sensing object, the sensing object and the target binding substance can be combined to adsorb the sensing object to the adsorption layer. It is possible to measure an object to be detected.

It is a perspective view of the sensing device concerning an embodiment of the invention. It is a disassembled perspective view of a sensing sensor. It is the disassembled perspective view which showed the upper surface side of each part of a detection sensor. It is a top view of the surface side and the back surface side of a crystal oscillator. It is sectional drawing which shows the surface structure of an adsorption layer. It is the disassembled perspective view which showed the lower surface side of a part of sensing sensor. It is sectional drawing of a sensing sensor. It is a schematic block diagram of the main-body part of a sensing apparatus. It is a block diagram which shows the structure of the crystal oscillation circuit in a sensing apparatus. It is explanatory drawing which showed the measurement liquid typically. It is explanatory drawing which showed the reference liquid typically. It is a characteristic view which shows the time change of the frequency when a measurement liquid and a reference liquid are supplied. It is explanatory drawing which shows the surface of an adsorption film when a reference liquid is supplied. It is explanatory drawing which shows the surface of an adsorption film when a measurement liquid is supplied.

  A sensing device used in the sensing method according to the embodiment of the present invention will be described. This sensing device uses a microfluidic chip, and can detect the presence or absence of an antigen such as a virus in a sample liquid obtained from, for example, a wiping liquid of a human nasal cavity, and determine the presence or absence of a human virus infection. It is configured as follows. As shown in the external perspective view of FIG. 1, the sensing device includes a main body 12 and a sensing sensor 2. The main body 12 includes, for example, a plurality of insertion ports 17 for connecting the detection sensor 2, and the detection sensor 2 is detachably connected to the insertion port 17. On the upper surface of the main body 12, a display unit 16 configured by, for example, a liquid crystal display screen is provided. Displays the measurement results such as minutes or the presence or absence of virus detection.

  Next, the detection sensor 2 will be described. As shown in FIGS. 2 and 3, the detection sensor 2 includes a container 20 including an upper cover body 21 and a lower case 22. As shown in FIG. 3, a wiring board 3 having a shape extending in the length direction is provided above the lower case 22, and the difference between the main body portion 12 is provided at one end side in the length direction of the wiring board 3. An insertion portion 31 to be inserted into the insertion port 17 is formed. In the following description, the insertion portion 31 side of the sensor 2 is assumed to be the front.

A through hole 32 is formed at a position near the rear side of the wiring board 3. The wiring board 3 is plugged above the lower case 22, and the through hole 32 is blocked by the bottom surface of the lower case 22. The part 31 is disposed so as to protrude to the outside of the lower case 22. The lower portion of the through hole 32 is closed by the bottom surface of the lower case 22 to form a recess. Furthermore, two holes 33 penetrating in the thickness direction are formed side by side in the width direction of the wiring board 3 at the rear end of the wiring board 3.
Three wirings 25 to 27 extending in the length direction are provided on the surface side of the wiring board 3, and one ends of the wirings 25 to 27 are respectively connected to the terminal portions 252, 262, 272 is formed, and terminal portions 251, 261, and 271 are formed at the outer edge of the through hole 32 on the other end side, respectively.

4A and 4B, the crystal unit 4 includes, for example, an AT-cut disc-shaped crystal piece 41, and the surface side of the crystal piece 41 as shown in FIG. For example, excitation electrodes 42A and 42B formed of, for example, Au (gold) are provided so as to extend in parallel to each other. The excitation electrodes 42 </ b> A and 42 </ b> B are connected at one end in the length direction, and the extraction electrode 44 extends from the connected portion toward the peripheral edge of the crystal piece 41. The lead electrode 44 is routed around the side surface of the crystal piece 41, and a terminal portion 44a is formed at the peripheral portion of the back surface. As shown in FIG. 4B, the excitation electrodes 43A and 43B are provided on the back surface side of the crystal unit 4 so as to be opposed to the excitation electrodes 42A and 42B, for example, by Au. Lead electrodes 45 and 46 are led out from the excitation electrodes 43A and 43B toward the periphery of the crystal piece 41, and terminal portions 45a and 46a are formed at the periphery of the crystal piece 41, respectively.
A region sandwiched between the excitation electrode 42 </ b> A and the excitation electrode 43 </ b> A in the common electrode 42 of the crystal unit 4 is a vibration region 61. A region sandwiched between the excitation electrode 42 </ b> B and the excitation electrode 43 </ b> B becomes a second vibration region 62. These first and second vibration regions 61 and 62 are provided apart from each other and vibrate independently.

On the surface of the excitation electrode 42A on the front side of the crystal unit 4, as shown in FIG. 5, an adsorption layer 47 provided with an avidin as an anchor material, for example, streptavidin 40, is provided. Further, the gold used for the excitation electrode 42A has a strong adsorptive property and adsorbs the sensing object contained in the sample liquid flowing on the surface or molecules dissolved in the sample liquid. The gap is filled with BSA (bovine serum albumin) 49. For this reason, when the sample liquid flows on the surface of the crystal unit 4, the sample liquid and the excitation electrode 42A are not touched.
Further, the excitation electrode 42B and the extraction electrode 44 formed on the surface side of the crystal unit 4 are a blocking layer 48 to which BSA 49 is applied in order to prevent adsorption of particles such as a sensing object to the electrode.
Returning to FIG. 3, in the crystal resonator 4, the excitation electrodes 43 </ b> A and 43 </ b> B on the back side face the through holes 32 of the wiring board 3, and the terminal portions 44 a, 45 a, and 46 a correspond to the wiring board 3. It arrange | positions so that it may overlap with the terminal parts 261, 251, 271, and it adhere | attaches with a conductive adhesive. Thereby, the crystal unit 4 is fixed to the wiring board 3 in a substantially horizontal state.

  On the rear side of the front surface side of the wiring substrate 3, a flow path forming member 5 is laminated so as to cover the upper side of the crystal resonator 4. The flow path forming member 5 is constituted by a plate-like member made of PDMS (polydimethylsiloxane), for example. At a position near the rear of the flow path forming member 5, a hole 58 for aligning the flow path forming member 5 is located at a position corresponding to the hole 33 formed in the wiring board 3. 5 is provided so as to penetrate through in the thickness direction.

  As shown in FIG. 6, a substantially circular recess 54 is formed on the lower surface side of the flow path forming member 5 so that the crystal resonator 4 is accommodated. Further, on the lower surface side of the flow path forming member 5, the respective wirings 25 to 27 formed on the wiring substrate 3 are accommodated, and grooves 253, 263, and 273 communicating with the depressions 54 are formed. The recess 54 is provided with an enclosing portion 51 that partitions and forms a sample solution supply channel 57 between the channel forming member 5 and the surface of the crystal unit 4 when the channel forming member 5 is pressed toward the wiring substrate 3. . The surrounding part 51 is configured by an annular projecting part whose outer edge is formed in an oval shape so that the length direction thereof faces in the front-rear direction of the detection sensor 2. The enclosure 51 is configured to protrude from the depression 54 to a thickness of 300 μm, and the area inside the enclosure 51 is a flat surface having the same height as the depression 54. The flow path forming member 5 is provided with through holes 52 and 53 that open to the front end and the rear end of the supply flow path 57 and penetrate the flow path forming member 5 in the thickness direction.

  The flow path forming member 5 is arranged so that the hole 58 is aligned with the hole 33 provided in the wiring board 3. At this time, the surrounding portion 51 is disposed on the upper surface of the crystal unit 4, and the lower surface side of the supply flow channel 57 is closed by the crystal unit 4. At this time, the excitation electrodes 42 </ b> A and 42 </ b> B are aligned in the center of the supply flow path 57. Therefore, as shown in FIG. 4A, the first and second vibration regions 61 and 62 are arranged so as to be lined up on the left and right in the direction from the through hole 52 toward the through hole 53. Further, the region surrounded by the enclosing portion 51 and the crystal unit 4 forms a supply flow channel 57 having a depth of 300 μm whose bottom surface is constituted by the crystal unit 4 and whose top surface and bottom surface extend in parallel.

Further, as shown in FIG. 3, the through holes 52 and 53 are provided with an inlet side capillary member 55 and an outlet side capillary member 56, which are each made of a porous member, in a detachable manner.
The inlet side capillary member 55 is, for example, a cylindrical member, and is configured of a chemical fiber bundle such as polyvinyl alcohol (PVA). The inlet-side capillary member 55 is disposed so as to close the through-hole 52, and is provided so that its upper end is exposed to the inlet 23 formed in the upper cover body 21 described later, and its lower end enters the supply channel 57. It has been. Similarly, the outlet side capillary member 56 is formed of a bundle of chemical fibers such as polyvinyl alcohol (PVA), and is formed into an L-shape that extends upward and then bends and extends horizontally. The outlet side capillary member 56 is disposed so as to block the through hole 53 and the lower end thereof enters the supply channel 57. Furthermore, the lower end of the outlet side capillary member 56 is inclined from the front side toward the rear side.

  The other end side of the outlet side capillary member 56 is inserted into one end side of a waste liquid channel 59 formed of a glass tube. On the other end side of the waste liquid channel 59, for example, a capillary sheet 71 that sucks the liquid flowing out from the waste liquid channel 59 and an absorption member 72 that absorbs the liquid sucked by the capillary sheet 71 are absorbed. Part 7 is connected. The lower case 22 is provided with a case body 73 for housing the waste liquid absorbing portion 7 and preventing liquid leakage from the absorbing member 72. Note that reference numeral 75 in FIG. 3 denotes a support member that supports the waste liquid channel 59.

  As shown in FIG. 6, a pressing portion 90 for pressing the flow path forming member 5 against the wiring board 3 is provided on the back surface side of the upper cover body 21. The pressing portion 90 is configured, for example, in a substantially box shape, and when the upper cover body 21 is fitted into the lower case 22 and locked together, the lower surface presses the entire upper surface of the flow path forming member 5 vertically downward. Then, the enclosure 51 is brought into close contact with the crystal unit 4. The pressing portion 90 is provided with a through hole 91 that penetrates the injection port 23 at a position corresponding to the through hole 52.

Further, the pressing portion 90 is formed with a notch 92 for securing an installation area for the waste liquid channel 59 and the outlet side capillary member 56 from the position corresponding to the through hole 53 toward the rear side. Further, the pressing portion 90 includes a fixing column 93 that is inserted into holes 58 and 33 provided in the flow path forming member 5 and the wiring board 3, respectively, and restricts the displacement of the flow path forming member 5 and the wiring board 3. Is provided.
As shown in FIGS. 2 and 3, the lower case body 22 is connected to the wiring substrate 3 excluding the insertion portion 31, the crystal resonator 4, the inlet side capillary member 55, and the outlet side capillary member 56. The forming member 5, the waste liquid channel 59, and the waste liquid absorption part 7 are accommodated, and the upper cover body 21 is provided so as to cover the lower case body 22 from above.
In this sensor 2, as shown in FIG. 7, the processing liquid supplied to the inlet 23 is the inlet 23 → the inlet side capillary member 55 → the supply channel 57 → the outlet side capillary member 56 → the waste liquid channel 59 → the waste liquid. It flows by a capillary phenomenon through a series of channels that follow the absorber 7.

  Next, the overall configuration of the sensing device will be described. As shown in FIG. 8, the main body unit 12 includes an oscillation circuit unit 6 and a data processing unit 66. When the insertion part 31 of the sensing sensor 2 is inserted into the insertion port 17 formed in the main body part 12, the terminal parts 252, 262 and 272 formed in the insertion part 31 are connected to the main body part 12. Are electrically connected to connection terminal portions (not shown) formed to correspond to the terminal portions 252, 262, and 272. A switch 60 is provided at the subsequent stage of the insertion port 17 so that the detection sensor 2 electrically connected to the oscillation circuit unit 6 can be switched.

  Here, an example in which one sensing sensor 2A is connected to the oscillation circuit unit 6 will be described. As schematically shown in FIG. 9, the oscillation circuit unit 6 includes a first oscillation circuit 63 and a second oscillation circuit 64 configured by, for example, a Colpitts circuit, and the first oscillation circuit 63 includes a crystal oscillation. The second oscillation circuit 64 is a region sandwiched between the excitation electrode 42B and the excitation electrode 43B. The second oscillation circuit 64 is a region sandwiched between the excitation electrode 42B and the excitation electrode 43B. Each of the vibration regions 62 is oscillated. The excitation electrode 42A and the excitation electrode 43A are connected so as to have a ground potential during oscillation.

  The output sides of the first and second oscillation circuits 63 and 64 are connected to the switch unit 65, and a data processing unit 66 is provided at the subsequent stage of the switch unit 65. The data processing unit 66 performs digital processing of the frequency signal that is the input signal, and the time-series data of the oscillation frequency “f1” output from the first oscillation circuit 63 and the oscillation output from the second oscillation circuit 64. Time-series data of frequency “f2” is acquired.

  In the sensing device of the present invention, channel 1 connecting data processing unit 66 and first oscillation circuit 63 and channel 2 connecting data processing unit 66 and second oscillation circuit 64 are provided by switch unit 65. By performing intermittent oscillation that is alternately switched, interference between the two vibration regions 61 and 62 of the sensor 2 can be avoided and a stable frequency signal can be acquired. These frequency signals are time-divided, for example, and taken into the data processing unit 66. The data processing unit 66 calculates the frequency signal as, for example, a digital value, performs arithmetic processing based on the time-division data of the calculated digital value, and displays, for example, a calculation result such as the presence or absence of an antigen on the display unit 16. .

  Next, the operation of the embodiment of the present invention will be described. First, as shown in FIG. 10, a solution containing biotinylated antibody 101 in which biotin 112, which is an avidin target, is bound to antibody 111, a sample solution containing antigen 100 as a sensing target, sensitizing particles 113, and antibody A measurement liquid prepared by mixing a liquid containing the sensitized particle-bound antibody 102 with 111 bound thereto is prepared. At this time, mixing is performed so that the number of moles of biotinylated antibody 101 and the number of moles of sensitized particle-bound antibody 102 are more than the number of moles of antigen 100.

  As a result, the sensing object contained in the sample solution, for example, the antigen 100 and the site of the antibody 111 of the biotinylated antibody 101 are bound at a ratio of 1: 1 by the antigen-antibody reaction. Further, the antigen 111 binds to the antigen 100 at a ratio of 1: 1 by the antibody-antibody reaction in the sensitized particle-bound antibody 102. Therefore, a complex 110 of the antigen 100, the biotinylated antibody 101, and the sensitized particle-bound antibody 102 is obtained. At this time, since the number of moles of the biotinylated antibody 101 and the number of moles of the sensitized particle-bound antibody 102 are larger than the number of moles of the antigen 100, assuming that the antigen 100 substantially comprises the complex 110, In FIG. 4, the biotinylated antibody 101 and the sensitized particle-bound antibody 102 are separately present together with the complex 110.

  In addition, the sample solution containing the antigen 100 as the sensing object is removed from the measurement solution, and a reference solution is prepared by mixing the sample solution with the same amount of the reference measurement sensing solution as the sensing object, for example, physiological saline. To do. As shown in FIG. 11, since the reference solution does not contain the antigen 100, the biotinylated antibody 101 and the sensitized particle-bound antibody 102 are contained as individual molecules.

  Subsequently, the reference liquid and the measurement liquid are measured by the sensing device. First, the sensing sensors 2A and 2B are connected to the insertion port 17. In the following description, they are referred to as a first detection sensor 2A and a second detection sensor 2B. Further, it is assumed that the first detection sensor 2A and the second detection sensor 2B are configured in the same manner, and there is no difference in adsorption efficiency in the adsorption layer 47.

Next, the user supplies, for example, a buffer solution such as physiological saline to the inlets 23 of the first sensor 2A and the second sensor 2B with a dropper. As a result, the buffer solution fills the supply channel 57, and the surface of the crystal unit 4 on the side of the supply channel 57 changes to a liquid phase.
Subsequently, the reference liquid and the measurement liquid are supplied to the detection sensors 2A and 2B, respectively, and the oscillation frequency is measured. FIG. 12 shows a difference value (f1-f2) between the oscillation frequency f1 output from the first oscillation circuit 63 and the oscillation frequency f2 output from the second oscillation circuit 64 when the measurement liquid and the reference liquid are measured. It is a characteristic view which shows the time change of. At time 0, the supply flow path 57 is filled with the buffer solution.

  First, the switch 60 is switched so as to connect the oscillation circuit section 6 and the first sensing sensor 2A. Then, the oscillation of the first oscillation circuit 63 and the second oscillation circuit 64 is started. For example, in the data processing unit 66, the oscillation frequency f1 of the first oscillation circuit 63 and the oscillation frequency f2 of the second oscillation circuit 64 are A difference value (f1-f2) is calculated. At time 0, the supply flow path 57 is filled with the buffer solution, and the excitation electrodes 42A and 42B are equally pressured by the buffer solution. Therefore, since there is no difference between the oscillation frequency f1 of the first oscillation circuit 63 and the oscillation frequency f2 of the second oscillation circuit 64, the difference value (f1-f2) is zero.

  Next, at time 0 in FIG. 12, the reference liquid is injected into the injection port 23 of the first sensing sensor 2A. As a result, the reference liquid flows into the supply channel 57 in the first sensing sensor 2A, and the inside of the supply channel 57 is gradually replaced from the buffer solution to the reference solution. Therefore, the excitation electrodes 42A and 42B on the surface of the crystal unit 4 are in contact with the reference liquid. As shown in FIG. 5, the surface of the excitation electrode 42 </ b> A is configured such that the streptavidin 40 is exposed in the BSA 49. The surface of the excitation electrode 42 </ b> B and the surface of the extraction electrode 44 are a blocking layer 48 of BSA 49.

  As described above, the reference solution contains the biotinylated antibody 101 and the sensitized particle-bound antibody 102 as individual molecules. Biotin 112 has a property of binding to streptavidin 40 (biotin: streptavidin = 1: 4). In addition, the portion covered with BSA 49 has a property that molecules such as biotinylated antibody 101 and sensitized particle-bound antibody 102 are difficult to adsorb. Therefore, as shown in FIG. 13, the biotin 112 site in the biotinylated antibody 101 contained in the reference solution is bound 1: 4 to the streptavidin 40 exposed in the adsorption layer 47. Further, the sensitized particle-bound antibody 102 remains in the reference solution because there is no site to be adsorbed to the streptavidin 40. Furthermore, since the surface of the excitation electrode 42B is covered with the blocking layer 48, neither the biotinylated antibody 101 nor the sensitized particle-bound antibody 102 is adsorbed. In order to avoid complication, the streptavidin 40 and the biotin 112 are described as being bound at 1: 1 in FIGS. 13 and 14, but in reality, the binding site of the biotin 112 is bound to the streptavidin 40. There are four places, and four biotins 112 bind to one streptavidin 40.

  Therefore, as the buffer solution is replaced with the reference solution, the biotinylated antibody 101 adheres to the excitation electrode 42A and the weight increases. As a result, the oscillation frequency f1 of the first oscillation circuit 63 decreases. On the other hand, since neither the biotinylated antibody 101 nor the sensitized particle-bound antibody 102 is adsorbed to the excitation electrode 42B, the oscillation frequency f2 of the second oscillation circuit 64 does not change. Therefore, the difference value (f1-f2) between the oscillation frequency f1 of the first oscillation circuit 63 and the oscillation frequency f2 of the second oscillation circuit 64 is reduced by an amount corresponding to the weight to which the biotinylated antibody 101 is attached. . That is, the absolute value increases. The absolute value of the difference value (f1-f2) when this reference liquid is measured using the first sensing sensor 2A is the blank frequency Fr. For example, the blank frequency Fr calculated by the data processing unit 66 is displayed on the display unit 16.

Subsequently, the switch 60 is switched to connect the oscillation circuit unit 6 and the second sensing sensor 2B. Then, the measurement liquid is injected into the injection port 23 of the second detection sensor 2B.
At the time 0, the second sensing sensor 2B is filled with the buffer solution 57, and the excitation electrodes 42A and 42B are equally pressured by the buffer solution, so that the oscillation frequency of the first oscillation circuit 63 is increased. There is no difference between f1 and the oscillation frequency f2 of the second oscillation circuit 64, and the difference value (f1-f2) is zero.

  When the measurement liquid is injected into the injection port 23 in the second sensor 2B, the supply channel 57 is gradually replaced with the measurement solution from the buffer solution, and the supply channel 57 is eventually filled with the measurement solution, and the excitation electrode 42A. 42B comes into contact with the measurement liquid. As described above, biotin 112 is bound to streptavidin 40 by (biotin: streptavidin = 1: 4). In the measurement liquid, a complex in which the biotinylated antibody 101, the antigen 100, and the sensitized particle-bound antibody 102 are bound to each other and an excess amount of the biotinylated antibody 101 are present. Therefore, as shown in FIG. 14, the streptavidin 40 exposed to the adsorption layer 47 in the excitation electrode 42 </ b> A includes the complex 110 of the biotinylated antibody 101, the antigen 100 and the sensitized particle-bound antibody 102, and an excess amount of biotin. Antibody 101 will be bound.

  Since complex 110 corresponds to the number of moles of antigen 100, the excess number of moles of biotinylated antibody 101 is the difference between the number of moles of biotinylated antibody 101 mixed with the measurement solution and the number of moles of antigen 100. It is a value. Therefore, the ratio of the complex 110 to the total number of the biotinylated antibody 101 and the complex adsorbed on the adsorption layer 47 is (number of moles of antigen 100) / (number of moles of biotinylated antibody 101 mixed in the measurement solution). Further, since the surface of the excitation electrode 42B is covered with the blocking layer 48, none of the complex 110, the biotinylated antibody 101, and the sensitized particle-bound antibody 102 is adsorbed.

As a result, as the supply flow path 57 of the second sensor 2B is replaced from the buffer solution to the measurement solution, the biotinylated antibody 101 and the complex 110 adhere to the excitation electrode 42A and the weight increases. As a result, the oscillation frequency f1 of the first oscillation circuit 63 decreases. On the other hand, since neither the biotinylated antibody 101 nor the sensitized particle-bound antibody 102 is adsorbed to the excitation electrode 42B, the oscillation frequency f2 of the second oscillation circuit 64 does not change.
Therefore, the difference value (f1-f2) between the oscillation frequency f1 of the first oscillation circuit 63 and the oscillation frequency f2 of the second oscillation circuit 64 is reduced by an amount corresponding to the weight of the biotinylated antibody 101 and the complex 110 attached. Will do. The absolute value of the difference value (f1-f2) when this measurement liquid is measured using the second sensor 2B is the detection frequency Fa. For example, the detection frequency Fa calculated by the data processing unit 66 is displayed on the display unit 16.

Subsequently, in the data processing unit 66, the detection liquid Fa, which is an absolute value of the difference value (f1-f2) when the measurement liquid is measured using the second detection sensor 2B, and the reference liquid are used as the first detection sensor. A frequency change amount F that is a difference value between the blank frequency Fr that is an absolute value of the difference value (f1-f2) when measured using 2A is calculated.
The frequency change amount F will be described. As described above, the blank frequency Fr corresponds to the weight of the biotinylated antibody 101 adsorbed on the adsorption layer 47 in the first sensing sensor 2A. The detection frequency Fa corresponds to the weight of the biotinylated antibody 101 and the complex 110 adsorbed on the adsorption layer 47 in the second sensor 2B.

  The biotinylated antibody 101 and the complex 110 are adsorbed to the excitation electrode 42A of the second sensing sensor 2B. The concentration of the biotinylated antibody 101 contained in the measurement solution and the biotinylated antibody 101 contained in the reference solution are absorbed. The concentration of is the same. Therefore, in the adsorbate adsorbed on the adsorption layer 47 of the first detection sensor 2A, the weight of the biotinylated antibody 101 minutes is the amount of the biotinylated antibody 101 minutes in the adsorbate adsorbed on the adsorption layer 47 of the second detection sensor 2B. Is equal to the weight of

  Therefore, the difference value (Fa−Fr) between the detection frequency Fa and the blank frequency Fr is the sensitized particle-bound antibody and the antigen 100 constituting the adsorbate complex 110 in the adsorption layer 47 of the second sensor 2B. This corresponds to the weight of 102. The antigen 100 constituting the complex 110 and the sensitized particle-bound antibody 102 are bound at 1: 1 as shown in FIG. Therefore, the difference value (Fa−Fr) is proportional to the number of antigens 100 constituting the adsorbate complex 110. The ratio of the complex 110 to the total number of the biotinylated antibody 101 and the complex adsorbed on the adsorption layer 47 is (number of moles of antigen 100) / (number of moles of biotinylated antibody 101 mixed in the measurement solution). Therefore, the number of antigens 100 contained in the adsorbate is determined by the concentration of antigen 100 in the measurement solution. Therefore, the difference value (Fa−Fr), which is the frequency change amount, indicates a value corresponding to the amount of the antigen 100 contained in the sample solution, that is, the sensing object.

  In the above-described embodiment, a liquid containing the biotinylated antibody 101, a sample liquid, and a liquid containing the sensitized particle-bound antibody 102 are mixed in advance to obtain a measurement liquid. When trying to capture the antigen 100 with the antibody 111 immobilized on the surface of the adsorption layer 47, the antibody 111 can only capture the antigen 100 passing near the surface of the adsorption layer 47, so the binding rate is low and the capture rate is low. End up. Therefore, a sample with a low concentration of the antigen 100 may not be sufficiently adsorbed on the adsorption film. On the other hand, the antigen 100 and the site of the antibody 111 of the biotinylated antibody 101 are brought into contact with each other by mixing the liquid containing the biotinylated antibody 101, the sample liquid, and the liquid containing the sensitized particle-bound antibody 102. Therefore, the antigen 100 can be more reliably bound to the biotinylated antibody 101.

Thereafter, the site of biotin 112 in the complex 110 of the biotinylated antibody 101, the antigen 100 and the sensitizing particle-bound antibody 102 is bound to the streptavidin 40 fixed to the excitation electrode 42A. Since it has a high affinity with 112, it is easier to bind than an antigen-antibody reaction. Therefore, even when a liquid containing biotin 112 is passed through the surface of the immobilized streptavidin 40, it can be efficiently bound. Therefore, the antigen 100 can be efficiently adsorbed on the adsorption layer 47. Therefore, even if the concentration of the antigen 100 is low, the antigen 100 can be reliably adsorbed on the adsorption film 47 and the measurement accuracy is improved.
The sensitized particle-bound antibody 102 specifically binds to the antigen 100 and increases its weight, and detects a change in the frequency of the crystal unit 4 when the antigen 100 is adsorbed to the adsorption layer 47. This is to make it easier. Therefore, the measurement liquid need not be mixed with a liquid containing the sensitizing particles 113.

  Further, according to the present invention, a substance that specifically binds to the sensing object is selected according to the sensing object to be sensed, and a target substance, for example, biotin 112 is bound to the substance to form a biotinylated substance. The solution containing the biotinylated substance and the sample solution containing the sensing object may be mixed. Therefore, a plurality of types of sensing objects can be measured without changing the type of the adsorption layer 47 of the sensing sensor. In addition, since a substance that specifically binds to a sensing target only needs to be biotinylated, for example, a nucleic acid, a sugar, or the like may be biotinylated.

  Further, in the above-described embodiment, the two detection sensors 2A and 2B are used, and the reference liquid and the measurement liquid are measured by the respective detection sensors 2A and 2B. You may make it measure a measurement liquid. For example, after a reference solution is passed through one sensing sensor 2 and a blank frequency is measured, a phosphate buffer solution is injected from the injection port 23 to wash away the surface of the adsorption layer 47. An example in which a measurement liquid is then injected from the inlet 23 and the detection frequency is measured is given. Further, it may be a flow type sensing device provided with a liquid feeding means for allowing the sample liquid to flow through the supply flow path 57 of the sensing sensor 2.

In order to confirm the effect of the sensing method according to the embodiment of the present invention, the following test was performed.
Using the sensing device described in the embodiment as an example, the measurement liquid and the reference liquid are injected into the first and second detection sensors 2A and 2B according to the procedure described in the embodiment, and the blank frequency Fr, detection is performed. The frequency Fa and the frequency change amount (Fa−Fr) were calculated. As the sample solution, a sample solution containing 20 ng / ml antigen was used.
As a comparative example, except that the biotinylated antibody 101 was bound to the streptavidin 40 provided on the surface of the excitation electrode 42A to form an adsorption layer, and the liquid containing the biotinylated antibody 101 was removed from the reference solution and the measurement solution. The blank frequency Fr, the detection frequency Fa, and the frequency change amount (Fa−Fr) were calculated in the same manner as in the example.

  Table 1 shows this result. The frequency difference (blank frequency) between the first vibration region 61 and the second vibration region 62 in each of the reference liquids of the example and the comparative example, and the first vibration region 61 in the measurement liquid. Difference between the frequency difference between the first and second vibration regions 62 (detection frequency), the detection frequency when measuring the sample liquid, the blank frequency when measuring the reference liquid, and the frequency when measuring the constant liquid (frequency) Change amount).

  In the comparative example, the blank frequency Fr was −0.8 Hz, the detection frequency Fa was 32.8 Hz, and the difference value (Fa−Fr) was 33.6 Hz. In the example, the blank frequency Fr was 222.0 Hz, the detection frequency Fa was 300.8 Hz, and the difference value (Fa−Fr) was 78.8 Hz. Therefore, it can be said that by mixing the sample solution containing the antigen 100 and the solution containing the biotinylated antibody 101 and supplying the mixture to the sensing sensor 2, the frequency change amount is increased by about 2.3 times and the detection sensitivity is improved. Therefore, it can be said that a great effect can be obtained by using the sensing method according to the embodiment of the present invention.

2 Sensing sensor 4 Crystal resonator 12 Main body 40 Streptavidin 42A, 42B, 43A, 43B
Excitation electrode 47 Adsorption layer 48 Blocking layer 57 Supply flow path 64 First oscillation circuit 65 Second oscillation circuit 100 Antigen 101 Biotinylated antibody 102 Sensitizing particle-bound antibody 110 Complex 112 Biotin

Claims (4)

A sensing sensor in which an adsorption layer provided with an anchor material on the surface is formed on the electrode surface of the piezoelectric vibrator, an oscillation circuit that oscillates the piezoelectric vibrator, a measurement unit that measures the oscillation frequency of the oscillation circuit, Use
Sensing is performed by mixing a liquid containing a target binding substance obtained by binding a target that specifically binds to the anchor substance with a substance having a property of adsorbing to a corresponding part of the sensing object and a sample liquid to be sensed. Obtaining a measurement liquid in which the target and the target binding substance are combined;
A blank corresponding to the amount of the target binding material adsorbed on the adsorption layer is measured by supplying a reference liquid comprising a liquid containing the target binding material to the sensing sensor to bind the target binding material to the adsorption layer and measuring the oscillation frequency. Determining the frequency;
Measurement liquid is supplied to the sensing sensor, the target binding substance and the sensing object are adsorbed on the adsorption layer, and the oscillation frequency is measured, which corresponds to the amount of the target binding substance and the sensing object adsorbed on the adsorption layer. Obtaining a detection frequency to perform,
Detecting a sensing object adsorbed on the adsorption layer based on a difference value between the detection frequency and the blank frequency.   The sensing method according to claim 1, wherein the anchor substance is avidin and the target is biotin.   The sensing method according to claim 1, wherein a sensing sensor that supplies the reference liquid is different from a sensing sensor that supplies the measurement liquid.   4. The measurement liquid and the reference liquid each include particles having a particle size larger than that of the sensing object and having a property of adsorbing to a site corresponding to the sensing object. The sensing method according to claim 1.

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