JPH0514219B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a photoacoustic analysis method and apparatus, and particularly to a photoacoustic analysis method and apparatus suitable for immunoassay.

[Conventional technology]

Conventional photoacoustic analyzers are disclosed in Japanese Patent Application Laid-Open No. 63-44149 or Analytical Chemistry, No. 53.
Volume, pages 539-540 (1981)
(Anal.Chem., 53.pp.539-540 (1981)), when subtracting the background of a sample, the sample and blank are sequentially packed into cells and the photoacoustic signal intensity of each is calculated. Alternatively, two cells were filled with a sample and a blank, and excitation light of the same wavelength and intensity was applied to each cell to find the difference. In addition, the background was kept constant, and quantitative analysis was performed without taking into account the difference between the sample and the blank.

[Problem to be solved by the invention]

In the above-mentioned prior art, when subtracting the background of a sample, a blank is always required or the background is assumed to be constant. On the other hand, since the sensitivity of photoacoustic spectroscopy is extremely high, if contaminants other than the target of analysis are mixed into the sample, high-level photoacoustic signals, that is, background, will be generated for the contaminants, so There is a problem in that photoacoustic signals from objects are buried in the background, making quantitative analysis difficult.

In particular, in immunoassays, impurities in serum samples vary from person to person, and even within the same person, they vary greatly depending on symptoms and complications. Therefore, it is not possible to perform quantitative analysis assuming that the background is constant, and it is necessary to subtract the background using a blank.

However, in applications such as immunoassays, it is not always possible to prepare blanks due to the relationship between sample and reagent amounts and constraints on analysis procedures, and in this case, the photoacoustic signal intensities of the sample and blank are measured separately. However, there was a problem in that it was not possible to subtract the background by taking the difference.

In addition, a method for correcting the background using only the sample without using a blank is to use excitation light with a wavelength that matches the absorption peak of the analyte and excitation light with a wavelength that matches the absorption peak of contaminants. A so-called two-wavelength method is considered, which determines the concentration of an analyte and a contaminant from a photoacoustic signal in light. However, in this case, since the measurement is performed using two excitation light wavelengths, the analysis time is long, and in the case of a photo-denatured sample, the state of the sample changes during the second measurement.

The purpose of the present invention is to provide a photoacoustic analysis method and apparatus that can eliminate the influence of the background and determine the concentration of an analyte from only the measurement results for a sample under these circumstances where it is difficult to separately prepare a blank. Our goal is to provide the following.

[Means to solve the problem]

In order to achieve the above object, the method of the present invention is a photoacoustic analysis method in which a sample is irradiated with excitation light and an analyte contained in the sample is quantified from the intensity of a photoacoustic signal generated by the photoacoustic effect. The present invention is characterized in that the intensity of a photoacoustic signal is measured for the sample at two or more different times, and the substance to be analyzed is quantified from the difference in the respective signal intensities.

Furthermore, the device of the present invention irradiates the sample with excitation light,
A photoacoustic analyzer for quantifying an analyte contained in the sample from the intensity of a photoacoustic signal generated by a photoacoustic effect, comprising a measuring means for measuring the photoacoustic signal of the sample at two or more different times; The method is characterized by comprising a processing means for quantifying the substance to be analyzed based on the difference between the measured values.

[Effect]

The principle of operation of the present invention will be explained below. There are various factors that determine the waveform, intensity, phase, etc. of a photoacoustic signal, such as the spatial distribution and wavelength of excitation light, the light intensity modulation frequency, and the material and shape of the cell.

Among these, the excitation wavelength λ can be increased as a parameter that particularly strongly reflects the characteristics of the sample.
Further, the parameter that determines the frequency of the generated photoacoustic signal is the light intensity modulation frequency ω. If the sample is in the process of reaction, the photoacoustic signal is measured at the measurement time t.
becomes a function of Other parameters can be kept constant as long as the cell or light source is not changed.

Therefore, the photoacoustic signal S is expressed as a function of the excitation light wavelength λ, the light intensity modulation frequency ω, and the measurement time t.
We will write it as ω, t). First, a case will be described in which the measurement time t is set to a constant time t ₀ and excitation light having two different wavelengths λ ₁ and λ ₂ and light intensity modulation frequencies ω ₁ and ω ₂ are simultaneously irradiated.

Since photoacoustic signals are essentially waves, the principle of superposition works. It is assumed that the wavelength λ ₁ of the first excitation light is adjusted to the optical absorption peak of the analyte, and the wavelength λ ₂ of the second excitation light is adjusted to the optical absorption peak of the contaminant.
The photoacoustic signals generated by each excitation light are caused by an analyte and a contaminant, respectively, but these are detected in an overlapping manner. Therefore, the detected photoacoustic signal S is as shown in the following equation.

S(t ₀ )=S(λ ₁ , ω ₁ , t ₀ ) +S(λ ₂ , ω ₂ , t ₀ )...(1) On the other hand, the detected photoacoustic signal is converted into excitation light by a lock-in amplifier or the like. The component synchronized with the intensity modulation period of is recovered from the noise field. Mathematically, a sine function synchronized with the signal to be recovered, that is, a convolution of the reference signal and the photoacoustic signal S, is taken, and is expressed as follows.

Sr(ω ₀ )=∫ei(ω ₀ t ₀ −θ) S(t ₀ )dt ₀ ……(2) Here, Sr(ω ₀ ) is the recovered signal and θ is the phase of the reference signal. . Since the photoacoustic signal S(t ₀ ) is a periodic function, it can be Fourier decomposed, and
From the orthogonal normality of the sine function, it can be easily understood that the signal Sr recovered from equation (2) is the first Fourier component of S(t ₀ ) synchronized with the reference signal.

Therefore, when recovering the signal from the object to be analyzed from the detected signal S(t ₀ ), if the frequency of the reference signal is ω ₁ , Sr(ω ₁ )=∫ei(ω ₁ t ₀ −θ) S(t ₀ )dt ₀ =∫ei(ω ₁ t ₀ −θ) S(λ ₁ , ω ₁ , t ₀ )+S(λ ₂ , ω ₂
, t ₀ )) dt ₀ = S^ ₁ (λ ₁ , ω ₁ )...(3), where S^ ₁ (λ ₁ , ω ₁ ) is S(λ ₁ , ω ₁ , t ₀ )
is the first Fourier component with respect to period ω 1 of ω ₁ . (3)
As is clear from the equation, ω ₂ is not synchronized with the period ω ₁
It can be seen that the component of the photoacoustic signal generated by the second excitation light having a frequency of is not recovered. Similarly, if the frequency of the reference signal is ω ₂ , the recovered signal will be S^ ₁ (λ ₂ , ω ₂ ), and only the second pumping light component will be recovered. Therefore, the first excitation light and the second excitation light
Even if the excitation lights are incident on the cell at the same time, signal components corresponding to both excitation lights can be recovered at the same time by inputting them to a signal processing device having two reference signals corresponding to each excitation light.

In particular, the background becomes a problem when the tail of the optical absorption spectrum of a contaminant overlaps the absorption peak of the analyte, as shown in FIG. In this case, the molar absorption coefficients at the absorption peak λ ₂ of the contaminant and the absorption peak λ ₁ of the analyte are each expressed as ε
(λ ₂ ) and ε(λ ₁ ), the absorbance α ₁ of impurities at λ ₁ is α ₁ =ε(λ ₁ )/ε(λ ₂ )α ₂ (4). Here, α ₁ is the absorbance of the contaminant at λ ₁ . Since the intensity of the photoacoustic signal is proportional to the absorbance, the amount of the photoacoustic signal intensity measured with the first excitation light that is derived from impurities is α ₁ /α ₂ ω ₂ /ω ₁ S from equation (4). ^ ₁ (λ ₂ , ω ₂ ) = ω (ε ₁ ) / ω (ε ₂ ) ω ₂ /ω ₁ S^ ₁ (λ ₂ , ω ₂ )
...(5) becomes. Here, the term ω ₂ /ω ₂ is a correction term for the photoacoustic signal intensity to be inversely proportional to the light intensity modulation frequency. Therefore, from the above, the photoacoustic signal strength S ₀ of only the separated object is calculated as follows from equation (5): S ₀ =S^ ₁ (λ ₁ , ω ₁ ) −ω ₂ ε(λ ₁ )/ω ₁ ε(λ ₂ )S^ ₁ (λ ₂ , ω ₂ )……(
6) ≡S^ ₁ (λ ₁ , ω ₁ )−kS^ ₁ (λ ₂ , ω ₂ ) ……(6′) can be obtained.

As is clear from the above principle, according to the present invention, the background can be corrected without using a blank.

Next, by measurements at two different times,
A method for correcting the background will be explained. The components of the photoacoustic signal of the sample can be divided into a signal component S _S from the analyte and a component Sb derived from impurities, and S (λ, ω, t) = S ₂ (λ, ω, t) +Sb (λ, ω, t) ...(7) It can be written as. If the sample is in the process of a reaction and the analyte is a reaction product, the signal component attributable to the analyte changes from time to time, but the signal component attributable to contaminants remains constant. Therefore, if the measurement times are t ₁ and t ₂ , Sb (λ, ω, t ₁ ) = Sb (λ, ω, t ₂ ), so ΔS = (λ, ω, t ₂ )−S (λ, ω, t ₁ ) = S _S (λ, ω, t ₂ )−S _S (λ, ω, t ₁ ) ...(8). When time t ₁ is the reaction start time and t ₂ is the reaction end time, ΔS is a signal intensity proportional to the amount of reaction product.

Further, since the production rate of a reaction product can usually be expressed by a certain function, if the function is investigated in advance, the amount of the analyte can be determined from the photoacoustic signal intensity at a certain time during the reaction. For example, if the production rate of the reaction product is constant, if each sample is measured at constant t ₁ and t ₂ , a calibration curve can be obtained from ΔS and quantitative analysis can be performed. In this case, if the time required for the reaction to complete is _t3 , the final signal strength _S3 for the reaction product is _S3 = _t3 / _t2 - _t1ΔS (9). Therefore, by measuring the intensity of the photoacoustic signal at two different times in a sample in which a reaction is in progress, the background can be corrected without using a blank.

〔Example〕

A first embodiment of the present invention will be described below with reference to FIGS. 1 and 2.

This embodiment can be applied both to cases where excitation light having two different wavelengths and light intensity modulation frequencies are used and to cases where measurements are made at two different times.

The apparatus configuration of this embodiment is shown in FIG. The light source is
It is composed of two systems of dye lasers 1 and 2 that use an Ar laser as an excitation laser, and can emit wavelengths in the ultraviolet and visible regions. The laser beams emitted from each light source are intensity-modulated by rotating blade type optical intensity modulators 3 and 4, that is, optical choppers, into rectangular waves having different modulation frequencies, and become excitation lights 5 and 6.

The two excitation lights 5 and 6 follow the same optical path via the half mirror 7 and enter the cell 8. The cell 8 is a cylindrical photoacoustic cell, in which a cylindrical piezoelectric element having the same inner diameter is built into the center of a glass cylinder, and is used as a photoacoustic signal detector. The excitation lights 5 and 6 are incident along the central axis of the cylinder.

When the inside of the cylindrical photoacoustic cell 8 is filled with a liquid sample and the excitation lights 5 and 6 are incident, the photoacoustic signals generated in response to the two excitation lights overlap each other,
The acoustic wave is converted into an electrical signal 9 by the piezoelectric element. The photoacoustic signal converted into the electrical signal 9 is branched into two signals and input into two phase detection devices 10 and 11, which are signal processing devices.

The phase detection device 10 extracts only the photoacoustic signal 13 corresponding to the excitation light 5 from the detected photoacoustic signal 9 using the signal 12 synchronized with the modulation of the optical intensity modulator 3 as a reference signal, and extracts only the photoacoustic signal 13 corresponding to the excitation light 5 from the detected photoacoustic signal 9. Enter.

Similarly, the phase detection device 11 refers to the signal 15 from the optical intensity modulator 4, extracts only the photoacoustic signal 16 corresponding to the excitation light 6, and inputs it to the data processing device 14. The data processing device 14 creates a calibration curve for the analyte from the aforementioned equations (6) and (6') or equations (8) and (9) based on the photoacoustic signals 13 and 16. Quantitative analysis of unknown samples. Here, the proportionality constant k in equation (6') can also be determined experimentally in advance.

An example of application of the present invention to immunoassay will be described below.

In immunoassays using serum as a sample, bilirubin is included as a contaminant, but bilirubin is unstable to light and exhibits photobleaching. Therefore, the background due to bilirubin changes during the measurement, and the conventional photoacoustic immunoassay method using one excitation light cannot perform accurate measurements. Therefore, in this example, the background is corrected using two excitation lights without using a blank. In the figure, 17 is a mirror, and 18 is a beam stopper.

α-fetoprotein (hereinafter abbreviated as AFP) was measured using the apparatus according to the above example.

(1) Preparation of reagent for AFP measurement Anti-human AFP antibody in 0.1M Bricine buffer (PH
6.5) Add 1 ml of white polystyrene latex (solid content concentration 10%) with an average particle size of 0.22 μm to 10 ml, leave it at room temperature and stir for 3 hours, and then
Centrifugation was performed at 1200 rpm for 40 minutes under cooling at ℃.
The obtained precipitate was mixed with 0.5% bovine serum albumin,
An anti-AFP antibody sensitized latex reagent having a concentration of 1% of the sensitized latex particles was prepared by suspending the sensitized latex particles in 10 mM phosphate buffer (PH6.5) containing 0.1 M NaCl and 2 mM MgCl ₂ .

(2) Sample measurement Human AFP and bilirubin as sample solutions
Bovine serum albumin solution 10μ supplemented with 10mg/dl
Add 50μ of the anti-AFP antibody sensitized latex reagent prepared in (1) and 10mM phosphate buffer (PH6.5).
200μ was added and antigen-antibody reaction was performed at 37°C for 10 minutes. This reaction solution is filled into the photoacoustic cell 8,
Photoacoustic signals were measured. The reaction solution in the photoacoustic cell 8 is simultaneously irradiated with excitation light of 33Hz, 465nm, and 181Hz, 520nm to generate a photoacoustic signal S^ ₁ , respectively.
(465 nm, 33 Hz) and S^ ₁ (520 nm, 181 Hz) were measured.

S^ ₁ (465 nm, 33 Hz) mainly includes signals derived from impurity components contained in the sample, but also includes signals derived from reaction products. However, the latter is very small and can be ignored in measurement.

S^ ₁ (520 nm, 181 Hz) includes signals derived from antigen-antibody reaction products and signals derived from reaction products and impurities. Although the latter is a small amount compared to the former, it cannot be ignored because the former, which is the measurement target, is weak.

In advance, we measure the photoacoustic signal of bilirubin, which is a problem as a contaminant component when performing visible light photometry using a human serum sample, and calculate the relationship of equation (6) for the above wavelength. The value of is 1/100
It was hot. That is, the measurement target signal S ₀ is determined as follows: S ₀ =S^ ₁ (520 nm, 181 Hz) - 1/100 S^ ₁ (465 nm, 33 Hz). The calibration curve for AFP quantification obtained as described above is shown in Figure 2.

Note that the simultaneous reproducibility of the 5.0 fg/ml AFP sample was good with n=30 CV=2.3%.

In this way, according to this embodiment, the background can be corrected without using a blank.
It has the following effects.

(1) Analyte content can be accurately quantified even in samples with varying backgrounds.

(2) Blank cells and samples become unnecessary.

(3) When applied to immunoassays, individual differences in contaminants,
Differences between cases and changes in symptoms do not impede quantitative analysis.

(4) Uncertainty in quantitative analysis due to reagent purity can be resolved.

(5) Even if the contaminants contain photoreactive chemicals, spectroscopic analysis becomes possible.

Next, a second embodiment of the present invention will be described with reference to FIGS. 4 and 5.

FIG. 4 shows the apparatus configuration of this embodiment. A laser is used as the light source 1. For example, if a dye laser using an Ar laser as an excitation laser is used, it is possible to emit wavelengths in the ultraviolet and visible regions. The laser beam from the light source 1 is intensity-modulated into a rectangular wave by a rotating blade type optical intensity modulator 3, that is, an optical chopper.
The cell 8 is irradiated.

Cell 8 is a cylindrical photoacoustic cell, in which a cylindrical piezoelectric element with the same inner diameter is installed in the center of the glass cylinder.
This is used as a photoacoustic signal detector. Excitation light 5 is incident along the central axis of the cylinder.

When the inside of this cell 8 is filled with a liquid sample and excitation light 5 is incident, a photoacoustic wave is generated according to the amount of light absorption of the sample at the wavelength of the excitation light, and is converted into an electric signal 9 by a piezoelectric element. This electrical signal 9 is input to a phase detection device 10 which is a signal processing device.

The phase detection device 10 extracts a signal component synchronized with the modulation of the optical intensity modulator 3 from the detected photoacoustic signal 9 using the signal 12 synchronized with the modulation of the optical intensity modulator 3 as a reference signal. This signal strength is the aforementioned S(t) and is sent to the data acquisition device 20.

The data acquisition device 20 records the signal strength S(t) that changes over time, and records the reaction start time of the analyte.
The signal intensities S(t ₁ ) and S(t ₂ ) at time t ₁ and time t ₂ optimal for quantifying the analyte are extracted and transmitted to the data processing control device 21 .

The data processing control device 21 calculates the concentration of the analyte from the above-mentioned equation (8) and a pre-examined function.

An example of application of the present invention to immunoassay will be described below.

α-fetoprotein (hereinafter abbreviated as AFP) was measured using the apparatus according to the above example.

Sample solution human AFP and bilirubin 10
10μ bovine serum albumin solution supplemented with mg/dl
Add 50μ of anti-AFP antibody sensitized latex reagent prepared as described above and 10mM phosphate buffer (PH6.5).
A solution containing 200μ was immediately filled into the photoacoustic cell 8, and changes in the photoacoustic signal over time were measured. As the excitation light 5, Ar laser light having a wavelength of 520 nm, which is the absorption wavelength of the reaction product of the antigen-antibody reaction, which is the object to be analyzed, was used, and the intensity was about 1 W. The modulation frequency was 181Hz.

FIG. 5 shows the temporal change in the photoacoustic signal measured as described above. At time t ₁ immediately after mixing, the reaction has not yet started. Therefore, the photoacoustic signal S(t ₁ ) at this time is a signal caused only by contaminants. After that, as time passes, the reaction progresses and absorption by the reaction products increases,
Therefore, the photoacoustic signal strength increases. Thereafter, when the reaction ends, the photoacoustic signal becomes constant. During this reaction, the contaminant concentration does not change, so at a given time
The difference between the photoacoustic signals S(t ₂ ) and S(t ₁ ) at t ₂ is
As shown in equation (8), this is caused only by the reaction product. Therefore, by measuring the intensity of the photoacoustic signal at two different times in a sample in which a reaction is in progress, it is possible to correct the background without using a blank.

In addition, in this example, two excitation wavelengths were irradiated at two modulation frequencies using the apparatus shown in FIG.
It is also possible to measure the intensity of the photoacoustic signal at two times. For example, when serum is used as a sample, it contains bilirubin as a contaminant, but bilirubin is unstable to light and exhibits photobleaching. It is also possible that it will change. Even in this case, as mentioned above, by irradiating two excitation wavelengths at two modulation frequencies and measuring the intensity of the photoacoustic signal at each of the analyte and the contaminant, the background fluctuation can be corrected.
Here, if the intensity of the photoacoustic signal is further measured at two times for each of the analyte and the contaminant, there is an effect that the accuracy of the correction can be improved.

〔Effect of the invention〕

As described above, according to the present invention, the concentration of the substance to be analyzed can be determined in a short time by eliminating the influence of the background from only the measurement results for the sample without separately preparing a blank.

[Brief explanation of drawings]

FIG. 1 is a device configuration diagram showing an embodiment of the present invention;
Fig. 2 is a graph showing the calibration curve of alphafuetoprotein when using the device of this example, Fig. 3 is a graph showing the photoacoustic spectrum when the target substance and impurities are mixed, Fig. 4 5 is a diagram showing the configuration of an apparatus according to another second embodiment of the present invention, and FIG. 5 is a graph showing changes over time in the alpha afeto protein production reaction when the apparatus of the second embodiment is used. 1, 2... light source, 3, 4... light intensity modulator,
5, 6...excitation light, 7...half mirror, 8...
Photoacoustic cell, 9...Photoacoustic signal, 10, 11...
Phase detection device, 12, 15...Reference signal, 16...
...Photoacoustic signal, 13...Photoacoustic signal by excitation light 5, 14, 21...Data processing control device, 16...
...Photoacoustic signal by excitation light 6, 17...Mirror,
18...beam stopper, 20...data acquisition device.

Claims (1)

[Scope of Claims] 1. A photoacoustic analysis method for quantifying an analyte contained in a sample, including the step of irradiating the sample with at least two or more excitation lights having different light wavelengths and different light intensity modulation frequencies. , a step of quantifying the substance to be analyzed using photoacoustics based on the difference in signal intensity of the detected photoacoustic signals. 2. The step of irradiating with two or more excitation lights,
2. The photoacoustic analysis method according to claim 1, wherein the two or more excitation lights are irradiated substantially simultaneously. 3. The photoacoustic analysis method according to claim 1, wherein the wavelength of one of the at least two or more excitation lights substantially matches the optical absorption wavelength of the immune product. 4. The wavelength of one of the at least two or more excitation lights substantially matches the light absorption wavelength of the immune product, and the wavelength of at least the other one substantially matches the light absorption wavelength of the contaminant or unreacted substance. 2. The photoacoustic analysis method according to claim 1, wherein 5. A photoacoustic analysis method for quantifying an analyte contained in a sample, comprising: irradiating the sample with excitation light having a single wavelength of light modulated at a single light intensity modulation frequency; a step of detecting frequency-synchronized photoacoustic signals on the same sample at two or more different times, and utilizing the photoacoustic effect to detect the substance to be analyzed based on the difference in signal strength of the two or more detected photoacoustic signals. 1. A photoacoustic analysis method comprising: 6. The photoacoustic analysis method according to claim 3, wherein the substance to be analyzed is a reaction product, and the two or more different times are two or more different times in a reaction process of the reaction product. 7. In the photoacoustic analysis method according to claim 1,
a step of detecting a photoacoustic signal synchronized with the light intensity modulation frequency on the same sample at two or more different times; 1. A photoacoustic analysis method, comprising: a step of quantifying using acoustic effects. 8. In a photoacoustic analyzer for quantifying an analyte contained in a sample, at least two means for irradiating the sample with excitation light, each having a different light wavelength, and excitation light emitted from the irradiation means. at least two or more light intensity modulation means for modulating light to different light intensity modulation frequencies; and irradiating the sample with at least two or more excitation lights having the light wavelength and the light intensity modulation frequency. means for detecting a photoacoustic signal synchronized with each of the light intensity modulation frequencies, and quantifying the substance to be analyzed using the photoacoustic effect based on a difference in signal intensity of the detected photoacoustic signals. A photoacoustic analysis device characterized by comprising means for. 9. The photoacoustic analysis device according to claim 8, wherein the means for irradiating the two or more excitation lights irradiates the two or more excitation lights substantially simultaneously. 10. The photoacoustic analysis device according to claim 8, wherein the wavelength of one of the at least two or more excitation lights substantially matches the optical absorption wavelength of the immune product. 11 The wavelength of one of the at least two or more excitation lights substantially matches the light absorption wavelength of the immune product, and at least the other one substantially matches the light absorption wavelength of the contaminant or unreacted substance. 9. The photoacoustic analysis device according to claim 8, wherein the photoacoustic analysis device has the same characteristics. 12. The method according to claim 8, further comprising a reaction container in which the sample is subjected to an immune reaction, and a transport means for holding the sample after the immune reaction and transporting the sample to a cell into which the excitation light is incident. Photoacoustic analyzer. 13. The photoacoustic analyzer according to claim 8, wherein the cell that holds the sample and receives the excitation light also has the function of causing an immune reaction. 14 In a photoacoustic analyzer for quantifying an analyte contained in a sample, a means for irradiating the sample with a single light wavelength, and a single light intensity modulation for excitation light irradiated from the irradiation means. a light intensity modulation means for irradiating the sample with excitation light having the light wavelength and the light intensity modulation frequency; and a means for irradiating the sample with excitation light having the light intensity modulation frequency; and means for quantifying the substance to be analyzed using a photoacoustic effect based on the difference in signal strength of the two or more detected photoacoustic signals. Analysis equipment. 15. The photoacoustic analysis device according to claim 14, wherein the substance to be analyzed is a reaction product, and the two or more different times are two or more different times in a reaction process of the reaction product. 16. The photoacoustic analysis according to claim 14, comprising a reaction container in which the sample undergoes an immune reaction, and a transport means for holding the sample after the immune reaction and sending the excitation light to a cell where it is incident. Device. 17. The photoacoustic analysis device according to claim 14, wherein the cell that holds the sample and receives the excitation light also has the function of causing an immune reaction. 18. The photoacoustic analysis method according to claim 8, comprising means for detecting a photoacoustic signal synchronized with the light intensity modulation frequency at two or more times, and a difference in signal strength between the two or more detected photoacoustic signals. A photoacoustic analysis method, comprising means for quantifying the substance to be analyzed based on the photoacoustic effect.