JP7239403B2 - CONCENTRATION DETECTION DEVICE AND CONCENTRATION DETECTION PROGRAM FOR SAW SENSOR - Google Patents

Description

The present disclosure relates to technology for detecting antigen concentration in a specimen such as blood.

A surface acoustic wave sensor is used to detect the antigen concentration in a sample such as blood (see, for example, Patent Document 1). That is, the antigen concentration in the specimen is detected based on the change in the propagation phase of the surface acoustic wave before and after the specimen is dropped. A surface acoustic wave sensor comprises interdigitated electrodes and a detection area. Comb electrodes are formed on the piezoelectric substrate to transmit, receive or reflect surface acoustic waves. A detection region is formed on the piezoelectric substrate and onto which the sample is dropped.

JP 2017-009492 A

A prior art method for detecting antigen concentration is shown in FIG. First, a specimen containing an antigen is dropped onto a detection region on which an antibody for enzyme immunoassay is immobilized. Next, the antigen concentration in the specimen is detected based on the time change of the phase P(t) of the output signal of the antibody-fixed channel. The sampling frequency of the phase P(t) of the output signal of the antibody-fixed channel is arbitrary and is several Hz, for example. Also, the periods t ₀ to t ₁ , t ₁ to t ₂ , t ₂ to t ₃ and t ₃ to t ₄ may be different from the sampling interval of the phase P(t) of the output signal of the antibody-fixed channel. may well be equal.

Here, the phase P(t) of the output signal of the antibody-immobilized channel is the change in the propagation phase of the surface acoustic wave before and after the specimen is dropped. Then, prior to antigen concentration detection, for various known antigen concentrations in the sample, a period t ₁ to t ₂ after a short time from the dropping time t ₀ and a period t after a long time from the dropping time t ₀ After measuring the slope of the time change of the phase P(t) of the output signal of the antibody-immobilized channel from ₃ to _t4 , the phase slope/antigen concentration calibration curve is stored.

When the antigen concentration in the specimen is high, the slope of the time change of the phase P(t) of the output signal of the antibody-immobilized channel is negative after dropping time _t0 , and becomes negative after a short time after dropping time _t0 . The gradient is steep during the period t ₁ to t ₂ , and is almost 0 in a saturated state during the period t ₃ to t ₄ after a long time from the dropping time t ₀ . Therefore, in order to improve the detection accuracy of the antigen concentration in the sample, the time change of the phase P(t) of the output signal of the antibody-fixed channel during the period t ₁ to _{t 2 a} short time after the dropping time t 0 The antigen concentration in the sample is detected based on the slope and the phase slope/antigen concentration calibration curve.

When the antigen concentration in the specimen is low, the slope of the time change of the phase P(t) of the output signal of the antibody-immobilized channel is negative after the dropping time _t0 , and after a short time after the dropping time _t0 . The slope is gentle during the period t ₁ to t ₂ , and is still gentle during the period t ₃ to t ₄ after a long time from the dropping time t ₀ . Therefore, in order to improve the detection accuracy of the antigen concentration in the sample, the time change of the phase P(t) of the output signal of the antibody-fixed channel during the period t ₃ to t ₄ after a long time from the dropping time t ₀ The antigen concentration in the sample is detected based on the slope and the phase slope/antigen concentration calibration curve.

Here, the slope of the time change of the phase P(t) of the output signal of the antibody-immobilized channel is not significantly affected by the antigen-antibody reaction environment in the period t ₃ to t ₄ after a long time from the dropping time t ₀ , but the dropping During the period t ₁ to t ₂ , which is a short time after time t ₀ , the antigen-antibody reaction environment has a great influence. Here, the antigen-antibody reaction environment is a concept that includes antibody manufacturing lot and specimen dropping method. In other words, the detection accuracy of the antigen concentration in the specimen is not much affected by the antigen-antibody reaction environment at low concentrations, but is greatly affected by the antigen-antibody reaction environment at high concentrations. A plurality of phase slope/antigen concentration calibration curves are required for the period t ₁ to t ₂ after a short time from the dropping time t ₀ and the period t ₃ to t ₄ after a long time from the dropping time t ₀ . . Therefore, the production efficiency of surface acoustic wave sensors is reduced.

Therefore, in order to solve the above problems, the present disclosure uses a surface acoustic wave sensor and an enzyme immunoassay method to detect the antigen concentration in a specimen such as blood, and the detection accuracy of the antigen concentration in the specimen is is improved irrespective of the antigen-antibody reaction environment and antigen concentration, and the phase gradient/antigen concentration calibration curve is unified to improve the production efficiency of surface acoustic wave sensors.

As shown in FIG. 1, the temporal change in the phase of the output signal of the antibody-immobilized channel is not significantly affected by the antigen-antibody reaction environment for a long period of time after the dropping of the specimen. Therefore, in order to solve the above-mentioned problem, the antigen concentration in the sample is detected based on the overall image of the time change in the phase of the output signal of the antibody-fixed channel from the time of dropping the sample to the final time of the antigen-antibody reaction. I decided to Specifically, linear vectors approximating the temporal changes in the phase of the output signal of the antibody-fixed channel in each time domain divided from the time of dropping the specimen to the final time of the antigen-antibody reaction are added based on the slope of the vector. Therefore, we decided to detect the antigen concentration in the sample.

Specifically, the present disclosure provides a detection region on which an enzyme immunoassay antibody is immobilized, and a comb-shaped electrode that transmits and receives or transmits and reflects surface acoustic waves. an acquisition unit, a signal division unit that divides the time change of the phase of the output signal of the channel in all time domains into time changes of the phase of the output signal of the channel in a plurality of time domains, and the output signal of the channel. a linear approximation unit for approximating the time change of the phase of the channel output signal in each time domain to a linear vector in each time domain of the phase of the output signal of the channel; an addition calculation unit for adding vectors and calculating an addition vector in the entire time domain of the phase of the output signal of the channel; and a concentration detection unit for detecting the concentration of an antigen in a sample dropped onto a detection area.

In addition, the present disclosure includes a detection region on which an enzyme-linked immunosorbent assay antibody is immobilized, and a signal acquisition step of acquiring an output signal of a channel provided with interdigitated electrodes that transmit and receive or transmit and reflect surface acoustic waves. , a signal division step of dividing the time variation of the phase of the output signal of the channel in the entire time domain into the time variation of the phase of the output signal of the channel in a plurality of time domains; A linear approximation step of approximating the time change in each time domain to a linear vector in each time domain of the phase of the output signal of the channel, and adding the linear vectors in a plurality of time domains of the phase of the output signal of the channel. an addition calculation step of calculating an addition vector of the phase of the output signal of the channel over the entire time domain; A surface acoustic wave sensor concentration detection program for causing a computer to sequentially execute a concentration detection step of detecting the concentration of an antigen in a dropped sample.

According to these configurations, when detecting the antigen concentration in a specimen such as blood, the detection accuracy of the antigen concentration in the specimen can be improved regardless of the antigen-antibody reaction environment and the antigen concentration.

Further, according to the present disclosure, the concentration detection unit associates the gradient of the addition vector in the entire time domain of the phase of the output signal of the channel with the concentration of the antigen in the sample dropped into the detection region. The surface acoustic wave sensor concentration detection device is characterized by detecting the concentration of an antigen in a sample dropped onto the detection region based on a stored calibration curve.

According to this configuration, in detecting the antigen concentration in a specimen such as blood, it is possible to unify the phase gradient/antigen concentration calibration curve and improve the production efficiency of the surface acoustic wave sensor.

In this way, the present disclosure uses a surface acoustic wave sensor and an enzyme immunoassay method to detect the concentration of antigens in a specimen such as blood. The production efficiency of the surface acoustic wave sensor can be improved by improving it regardless of the antigen concentration and by unifying the phase gradient/antigen concentration calibration curve.

FIG. 1 shows a conventional method for detecting antigen concentration. It is a figure which shows the structure of the surface acoustic wave sensor of this indication. FIG. 4 is a diagram showing concentration detection procedures of the surface acoustic wave sensor of the present disclosure; FIG. 4 is a diagram showing a concentration detection method of the surface acoustic wave sensor of the present disclosure; It is a figure which shows the calibration curve preparation method of the surface acoustic wave sensor of this indication.

Embodiments of the present disclosure will be described with reference to the accompanying drawings. The embodiments described below are examples of implementing the present disclosure, and the present disclosure is not limited to the following embodiments.

FIG. 2 shows the configuration of the surface acoustic wave sensor of the present disclosure. A surface acoustic wave sensor S is composed of an antibody immobilized channel 1 , a reference channel 2 and a concentration detector 3 . The antibody-immobilized channel 1 is composed of a detection region 11 and interdigitated electrodes 12,12. The reference channel 2 consists of a detection region 21 and comb electrodes 22 , 22 . The concentration detection device 3 includes a signal acquisition unit 31, a signal division unit 32, a linear approximation unit 33, an addition calculation unit 34, a concentration detection unit 35, and a calibration curve storage unit 36. ) is realized by installing the concentration detection program shown in FIG.

The detection region 11 is immobilized with an enzyme-linked immunosorbent assay antibody. The comb electrodes 12, 12 transmit and receive or transmit and reflect surface acoustic waves. A blocking membrane that does not react with antigens in the specimen is immobilized on the detection region 21 for reference. The comb electrodes 22, 22 transmit and receive or transmit and reflect surface acoustic waves for reference. The signal acquisition section 31, the signal division section 32, the linear approximation section 33, the addition calculation section 34, the concentration detection section 35, and the calibration curve storage section 36 will be described with reference to FIGS.

FIG. 3 shows the concentration detection procedure of the surface acoustic wave sensor of the present disclosure. FIG. 4 shows the concentration detection method of the surface acoustic wave sensor of the present disclosure. FIG. 5 shows a calibration curve creation method for the surface acoustic wave sensor of the present disclosure. Note that the sampling frequency of the phase P(t) of the output signal of the antibody-fixed channel 1 is arbitrary and is, for example, several Hz. Also, the time interval between adjacent black circles and black triangles shown in FIG. First, a specimen containing an antigen is dropped onto the detection regions 11 and 21 . Here, it is desirable to prevent insufficient drip wetting, ie, entrapment of air bubbles.

Next, as shown in the upper part of FIG. 4, the signal acquisition unit 31 acquires the output signal of the antibody immobilization channel 1 (step S1). Here, the output signal of the antibody-immobilized channel 1 is the change in the propagation phase of the surface acoustic wave before and after dropping the sample. By using the output signal of the reference channel 2 as a reference, the effect of the viscoelasticity of the sample is removed, and only the effect of the antibody on the detection area 11 and the antigen in the sample bound thereto is extracted. . The upper part of FIG. 4 shows the phase P(t) of the output signal of the antibody-immobilized channel 1 when there is noise.

As shown in FIG. 1, the time change of the phase P(t) of the output signal of the antibody-immobilized channel 1 is not significantly affected by the antigen-antibody reaction environment for a long time after the sample dropping time. Here, the antigen-antibody reaction environment is a concept that includes antibody manufacturing lot and specimen dropping method. Therefore, the antigen concentration in the sample is detected based on the overall image of the time change of the phase P(t) of the output signal of the antibody-fixing channel 1 from the time of dropping the sample to the final time of the antigen-antibody reaction.

In the present embodiment, linear vectors approximating the temporal changes in the phase P(t) of the output signal of the antibody-fixed channel 1 in each time domain obtained by dividing the time from the dropping time of the sample to the end time of the antigen-antibody reaction are added. The antigen concentration in the sample is detected based on the slope of the vector (steps S2 to S5). Steps S2 to S5 will be described below.

First, as shown in the upper part of FIG. 4, the signal dividing unit 32 divides the time variation of the phase P(t) of the output signal of the antibody-fixed channel 1 over the entire time region t ₀ to t ₅ into the antibody-fixed channel 1 phase P(t) of the output signal in a plurality of time regions t ₀ to t ₁ , t ₁ to t ₂ , t ₂ to t ₃ , t ₃ to t ₄ , t ₄ to t ₅ . (Step S2). Here, the time _t0 is the dropping time of the sample, and the time _t5 is the end time of the antigen-antibody reaction. The time widths of the respective time regions t ₀ to t ₁ , t ₁ to t ₂ , t ₂ to t ₃ , t ₃ to t ₄ , and t ₄ to t ₅ are such that the linear approximation in step S3 is highly accurate. is desirably long, and it is desirably short enough for the linear approximation in step S3 to hold.

Next, as shown in the upper part of FIG. 4, the linear approximation unit 33 calculates the phase P(t) of the output signal of the antibody-immobilized channel 1 in each time region t ₀ to t ₁ , t ₁ to t ₂ , t ₂ ∼ t ₃ , t ₃ - t ₄ , t ₄ - t ₅ , the phase P(t) of the output signal of the antibody-fixed channel 1 is measured in each time region t ₀ - t ₁ , t ₁ - t ₂ , Linear vectors V ₀ , V ₁ , V ₂ , V _{3 and V 4 are approximated at t 2 to t 3 , t 3} to t ₄ and t ₄ to t ₅ (step S3). Here, the slopes of the linear vectors V ₀ , V ₁ , V ₂ , V ₃ , V ₄ in principle decrease in this order. However, the slopes of the linear vectors V ₁ and V ₂ happen to be almost the same due to the influence of noise. The end point of linear vector V _n (n=0 to 3) does not necessarily coincide with the starting point of linear vector V _n+1 (n=0 to 3).

Next, as shown in the lower part of FIG. 4, the addition calculator 34 calculates a plurality of time regions t ₀ to t ₁ , t ₁ to t ₂ , t Add _the linear vectors V ₀ , V _{1 ,} V ₂ , V ₃ , V ₄ at 2 to t 3 , t ₃ to t 4 , t ₄ to t ₅ to obtain the phase _P (t ) in the entire time region t ₀ to t ₅ (step _S4 ). Here, after aligning the end point of the linear vector V _n (n=0 to 3) with the starting point of the linear vector V _n+1 (n=0 to 3), the linear vectors V ₀ , V ₁ , V ₂ , Add V ₃ and V ₄ . The lower part of FIG. 4 shows the phase P(t) of the output signal of the antibody-immobilized channel 1 when there is no noise. It can be seen that the start point and end point of the addition vector _VS approximately coincide with the start point and end point of the time change of the phase P(t) of the output signal of the antibody-fixed channel 1 in the absence of noise.

Next, as shown in the lower part of FIG. 4, the concentration detection unit 35 detects the slope of the addition vector V _S in the entire time region t ₀ to t ₅ of the phase P(t) of the output signal of the antibody-fixed channel 1. Based on this, the concentration of the antigen in the specimen dropped onto the detection area 11 is detected (step S5). Here, the calibration curve storage unit 36 stores the slope of the addition vector V _S in the entire time region t ₀ to t ₅ of the phase P(t) of the output signal of the antibody-fixed channel 1 and the sample dripped onto the detection region 11. A calibration curve that associates the concentration of the antigen in the medium with the standard curve is stored. Then, the concentration detection unit 35 detects the concentration of the antigen in the sample dropped onto the detection area 11 based on the calibration curve stored in the calibration curve storage unit 36 .

The calibration curve stored in the calibration curve storage unit 36 is created as follows. _{First ,} as _shown in the _{upper part of} FIG _. Gradients g 0 , g _{1 ,} g of addition vectors V _S0 , V _S1 , V _S2 , V _S3 , V _S4 , V _S5 , V _S6 in the entire time domain t ₀ to t ₅ of the phase P(t) of the output signal ₂ , _g3 , _g4 , _g5 , _g6 are measured. where c ₀ =0<c ₁ <c ₂ <c ₃ <c ₄ <c ₅ <c ₆ and g ₀ <g ₁ <g ₂ <g ₃ <g ₄ <g ₅ <g ₆ . Next _, as _shown in the lower part _{of FIG .} 5 _, the addition vector gradients g ₀ , _{g 1 ,} g ₂ , g ₃ , g ₄ , g ₅ , g ₆ are plotted. Here, the scale of the antigen concentration and the slope of the addition vector is assumed to be a logarithmic scale. The area between the plots above is then interpolated, such as with a sigmoidal curve. Then, based on the calibration curve stored in the calibration curve storage unit 36, the concentration detection unit 35 can detect that the antigen concentration is _cD when the slope of the addition vector is _gC .

Therefore, when detecting the antigen concentration in a specimen such as blood, the detection accuracy of the antigen concentration in the specimen is improved regardless of the antigen-antibody reaction environment and antigen concentration, and the phase slope/antigen concentration calibration curve is unified. The production efficiency of the surface acoustic wave sensor S can be improved.

By the way, in detecting the antigen concentration in the specimen based on the overall image of the time change of the phase P(t) of the output signal of the antibody-fixing channel 1 from the time of dropping the specimen to the final time of the antigen-antibody reaction, Not only the embodiments but also the following modified examples are applicable.

As a first modified example, noise is removed by low-pass filter processing with respect to the time change in the phase P(t) of the output signal of the antibody-fixed channel 1 from the time when the sample is dropped to the end time of the antigen-antibody reaction. , it is conceivable to detect the antigen concentration in the sample. Here, it is desirable to set the cutoff frequency after evaluating the noise frequency.

As a second modification, noise is removed by curve fitting with respect to the time change of the phase P(t) of the output signal of the antibody-immobilized channel 1 from the time of dropping the sample to the final time of the antigen-antibody reaction. , to detect the antigen concentration in the specimen. Here, it is desirable to appropriately set a fitting curve (for example, a decay exponential function, etc.).

As a third modification, the theoretical curve (upper solid line (see ). However, when the noise component is large, it may become difficult to compare with the theoretical curve.

As a fourth modification, the slope of a straight line that simply connects the start and end points with respect to the time change of the phase P(t) of the output signal of the antibody-fixed channel 1 from the time of dropping the specimen to the final time of the antigen-antibody reaction Based on this, it is conceivable to detect the antigen concentration in the sample. However, when the noise component is large, there is a possibility that the slope of the straight line that simply connects the start and end points will be inaccurate.

On the other hand, in the present embodiment, only time domain division and linear straight line approximation should be performed. Therefore, unlike the first modification, it is not necessary to set the cutoff frequency, and unlike the second modification, it is not necessary to set the fitting curve. And unlike the third and fourth modifications, even when the noise component is large, the linear straight line approximation is easy and highly accurate.

The concentration detection device and the concentration detection program of the surface acoustic wave sensor of the present disclosure can be applied to the use of detecting the antigen concentration in a sample such as blood using the enzyme immunoassay method.

S: Surface acoustic wave sensors _V0 , _V1 , _V2 , _V3 , _V4 : Linear vectors _Vs , _Vs0 , Vs1, _Vs2 , _Vs3 , _Vs4 , _Vs5 , _Vs6 : Addition vector ₁ : Antibody-immobilized channel 2: Reference channel 3: Concentration detection device 11, 21: Detection regions 12, 22: Comb electrode 31: Signal acquisition unit 32: Signal division unit 33: Linear approximation unit 34: Addition calculation unit 35: Concentration detection unit 36: calibration curve storage unit

Claims (3)

a signal acquisition unit for acquiring an output signal of a channel comprising a detection region on which an enzyme immunoassay antibody is immobilized, and a comb-like electrode for transmitting and receiving or transmitting and reflecting a surface acoustic wave;
a signal division unit that divides the time change of the phase of the output signal of the channel in the entire time domain into time changes of the phase of the output signal of the channel in a plurality of time domains;
a linear approximation unit for approximating a time change in each time domain of the phase of the output signal of the channel to a linear vector in each time domain of the phase of the output signal of the channel;
an addition calculation unit that adds linear vectors of the phases of the output signals of the channels in a plurality of time domains and calculates an addition vector of the phases of the output signals of the channels in the entire time domain;
a concentration detection unit that detects the concentration of the antigen in the sample dropped into the detection region based on the gradient of the addition vector in the entire time domain of the phase of the output signal of the channel;
A concentration detection device for a surface acoustic wave sensor, comprising: The concentration detection unit stores a calibration curve in which the slope of the addition vector in the entire time domain of the phase of the output signal of the channel is associated with the concentration of the antigen in the sample dropped into the detection region. 2. The concentration detection device for a surface acoustic wave sensor according to claim 1, wherein the concentration of the antigen in the sample dropped onto the detection area is detected based on: A signal acquisition step of acquiring an output signal of a channel comprising a detection region on which an enzyme immunoassay antibody is immobilized and a comb-shaped electrode that transmits and receives or transmits and reflects a surface acoustic wave;
a signal division step of dividing the time variation of the phase of the output signal of the channel in the entire time domain into the time variation of the phase of the output signal of the channel in a plurality of time domains;
a linear approximation step of approximating a time change in each time domain of the phase of the output signal of the channel to a linear vector in each time domain of the phase of the output signal of the channel;
an addition calculation step of adding linear vectors of the phases of the output signals of the channels in a plurality of time domains and calculating an addition vector of the phases of the output signals of the channels in the entire time domain;
a concentration detection step of detecting the concentration of the antigen in the sample dropped into the detection area based on the slope of the addition vector in the entire time domain of the phase of the output signal of the channel;
concentration detection program for a surface acoustic wave sensor for causing a computer to sequentially execute .

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