JPH02122243A - Method and device for acoustooptical analysis - Google Patents

Abstract

PURPOSE:To eliminate the influence of a background from only a result of measurement against a sample and to derive the concentration of an object to be analyzed by irradiating a sample simultaneously by two or more excitation light beams having each different optical wavelength and optical intensity modulation frequency. CONSTITUTION:When the inside of an acoustooptical cell 8 is filled with a liquid sample and excitation light beams 5, 6 are made incident, acoustooptical signals generated in accordance with two excitation light beams are superposed to each other, and converted as the acoustooptical wave to an electric signal 9 by a piezo-electric element. The acoustooptical signals converted to this signal 9 are allowed to branch to two signals, and inputted to a phase detecting device 10 and 11. Subsequently, in the device 10, by using a signal 12 synchronizing with the modulation of an optical intensity modulator 3 as a reference signal, only an acoustooptical signal 13 corresponding to the excitation light beam 5 is extracted from the detected signal 9, and inputted to a data processor 14. In the same manner, in the device 11, by referring to a signal 15 from an optical intensity modulator 4, only an acoustooptical signal 16 corresponding to the excitation light beam 6 is extracted and inputted to the device 14. In the device 14, based on the signals 13, 16, the analytical cure of an object to be analyzed is generated, and an unknown sample is brought to quantitative analysis.

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, Vol. 53, No. 5.
pp. 39-540 (1981) (Anal, Ch.
em,,53. pp. 539-540 (1981
)), the excitation light incident on the cell was limited to one wavelength and light intensity modulation frequency.

Therefore, when subtracting the background of a sample, it is necessary to fill the sample and blank into cells sequentially and measure the photoacoustic signal intensities of each and find the difference, or alternatively, fill two cells with the sample and blank and use the same wavelength , excitation light with the same light intensity modulation frequency was incident on each cell and the difference was taken. In addition, the background was kept constant, and the quantitative analysis was carried out focusing on the difference between the sample and the blank.

[Problem to be solved by the invention]

In the above conventional techniques, 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, a high level of photoacoustic signal, that is, a background, will be generated for the contaminants. There is a problem in that photoacoustic signals from objects are buried deep in the bank crown, making quantitative analysis difficult.

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

However, in applications such as immunoassays, it is not always possible to prepare a blank due to the relationship between the sample amount and reagent amount and the limitations of the analytical procedure. There was a problem in that it was not possible to subtract the background by measuring and taking the difference.

In addition, as a method for correcting Basok Graland using only a sample without using a blank, 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 is used. A so-called two-wavelength method is considered, which determines the concentration of the analyte and impurities from the photoacoustic signal. However, in this case, since 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 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 second object, the method of the present invention is a photoacoustic method in which a sample is irradiated with excitation light and the analyte contained in the sample is quantified from the intensity of the photoacoustic signal generated by the photoacoustic effect. In the analysis method, the sample is simultaneously irradiated with two or more excitation lights having different light wavelengths and light intensity modulation frequencies, photoacoustic signals synchronized with the light intensity modulation of each excitation light are detected, and these signals are detected. The method is characterized in that the substance to be analyzed is quantified from the difference in intensity, and the intensity of the photoacoustic signal at two or more different times with respect to the sample is measured,
This method is characterized in that the substance to be analyzed is quantified from the difference in signal strength.

Further, the apparatus of the present invention is a photoacoustic analyzer that irradiates a sample with excitation light and quantifies an analyte contained in the sample from the intensity of a photoacoustic signal generated by a photoacoustic effect. A means for generating two or more excitation lights having different light wavelengths and light intensity modulation frequencies, and a detection means for inputting the excitation light into the sample and detecting a photoacoustic signal synchronized with each light intensity modulation. The method further comprises a measuring means for measuring the photoacoustic signal of the sample at two or more different times, and a processing means for quantifying the substance to be analyzed based on the difference between the measured values. This is a characteristic feature.

[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 light wavelength λ can be mentioned as a parameter that particularly strongly reflects the characteristics of the sample, and the light intensity modulation frequency ω is a parameter that determines the frequency of the generated photoacoustic signal. If the sample is undergoing reaction, the photoacoustic signal will be a function of the measurement time t. 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 S(λ) as a function of the excitation light wavelength λ, the light intensity modulation frequency ω, and the measurement time t.

I will write it as ω+1). First, the measurement time t is set to a constant time t. binding, two different wavelengths λ3. λ2 and light intensity modulation frequency ω3. A case where excitation light of ω2 is simultaneously irradiated will be explained.

Since photoacoustic signals are essentially waves, the principle of superposition works. It is assumed that the wavelength λ of the first excitation light is matched to the light absorption peak of the analyte, and the second excitation light wavelength λ2 is matched to the light 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.

5(to)”5(zi, ωx, to)+S(λz+C
112, jo)"・(1) On the other hand, the detected photoacoustic signal uses a lock-in amplifier or the like to recover the component synchronized with the intensity modulation period of the excitation light from the noise field. Mathematically, the signal to be recovered is A sine function synchronized with , that is, a convolution of the reference signal and the photoacoustic signal S, is taken,
It is expressed as follows.

Here, Sr(ω.) is the recovered signal and O is the phase of the reference signal. Since the photoacoustic signal 5(tO) is a periodic function, it can be subjected to Fourier decomposition, and due to the orthogonal normality of the sine function, the signal Sr recovered from equation (2) is 5(tO) synchronized with the reference signal. It can be easily understood that this is the first Fourier component of .

Therefore, when recovering the signal from the analyte from the detected signal 5(to), if the frequency of the reference signal is ω, then from equation (2) S r(C1)=fei(""-I )S(t,)d t
.

=S□(λ1.ω,) ・・・・・・
...(3), where s i, (λ□, ω1) is S(λ1 g C1) 1. f, is the first Fourier component with respect to the period ω of O). As is clear from equation (3), it can be seen that the component of the photoacoustic signal generated by the second excitation light having the frequency C2 that is not synchronized with the period ω1 is not recovered. Similarly, if the frequency of the reference signal is C2, the recovered signal will be S□(λ2.ω2), and only the second excitation light component will be recovered. Therefore, even if the first excitation light and the second excitation light enter the cell at the same time, if they are input to a signal processing device that has two reference signals corresponding to each excitation light, the signal components corresponding to both excitation lights can be processed. can be recovered at the same time.

In particular, pack ground 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, if the molar extinction coefficients at the absorption peak λ2 of the contaminant and the absorption peak λ of the analyte are ε(λ2) and ε(λ,), respectively, then the absorbance α□ of the contaminant at λ is as follows. Here, α is the absorbance of contaminants at λ1. Since the intensity of the photoacoustic signal is proportional to the absorbance, the portion of the photoacoustic signal intensity measured with the first excitation light that is derived from contaminants is determined by equation (4). Here, the term C2/ω□ 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 intensity S of only the aromatic object. As is clear from the above principle from equation (5),
According to the present invention, back ground can be corrected without using a blank.

Next, a method of correcting the background by measuring at two different times will be described. The components of the photoacoustic signal of the sample can be divided into a signal component Ss from the analyte and a component Sb derived from impurities, and S (λ, ω, t) = Ss (λ, ω, t) + Sb ( λ,
It can be written as (,1,t)−(7). 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 concubine times are t□ and t2, then sb(λ, ω
, 1;□)=sb(λ, ω, t2), so ΔS-(λ, ω, tZ) S(λ, ω, t□)=Ss
(λ+ω+t2) ss(λ+ω+t+) ”'(
It becomes U. When time t1 is the reaction start time and t2 is the reaction end time, ΔS is a signal intensity proportional to the amount of reaction product.

Further, when the production rate of the reaction product is constant, if the measurement is performed at a constant time t2 for each sample, 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 signal strength S3 for the final reaction product is as follows. Therefore, in a sample during a reaction, by measuring the intensity of the photoacoustic signal at two different times, the background can be corrected without using a blank.

〔Example〕

An 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 composed of two systems of dye lasers 1 and 2 using an Ar laser as an excitation laser, and is capable of oscillating 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, ie, optical choppers, into rectangular waves having different modulation frequencies, and become excitation lights 5 and 6.

The two excitation lights 5 and 6 travel along the same optical path via a half mirror 7 and enter a 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 a liquid sample is filled inside a cylindrical photoacoustic cell 8 and excitation lights 5 and 6 are incident, the photoacoustic signals generated in response to the two excitation lights overlap with each other and form one acoustic wave on the piezoelectric element. is converted into an electrical signal 9 by. This electric signal 9
The converted photoacoustic signal is branched into two signals and input to 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,
Data processing device]-4.

Similarly, the phase detection device 11 refers to the signal 15 from the optical intensity modulator 4 to generate a photoacoustic signal corresponding to the excitation light 6.
6 is extracted and input to the data processing device 14. In the data processing device 14, based on the photoacoustic signals 13 and 16, the above-mentioned equations (6) and (6'') or (8) and (
9) From the formula, create a calibration curve for the analyte and quantitatively analyze the unknown sample. Here, the proportionality constant of equation (6') can 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 fluctuates during measurement, making accurate measurement impossible with the conventional photoacoustic immunoassay method using one excitation light. 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-described example.

(1) Preparation of reagent for AFP measurement Anti-human AFP antibody in 0.1M pridine buffer (pH 6,
5) Add 1 mQ of white polystyrene latex (solid content concentration 10%) with an average particle size of Q and 22 pm to 10 mR, stir at room temperature for 3 hours, and then cool to 4°C under cooling at 2 to 4°C.
0 minutes, 12. Centrifugation was performed at OOOrpm. The obtained precipitate was mixed with 0.5% bovine serum albumin, 0.1M NaCQ,
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 (pH 6.5) containing 2 mM M g CQ2.

(2) Measurement of sample To 10μQ of bovine serum albumin solution to which human AFP and bilirubin were added 10μΩ/semi, anti-AFP antibody sensitized latex reagent prepared in (1), 50μΩ, 1
200 μQ of 0 mM phosphate buffer (pH 6,5) was added, and anti-antigen reaction was carried out at 37° C. for 10 minutes. This reaction solution was filled into a photoacoustic cell 8, and a photoacoustic signal was measured. The reaction solution in the photoacoustic cell 8 was exposed to 33 Hz, 465 nm and 1.
The respective photoacoustic signals S, (465 nm, 33 Hz) and S, (520 nm, 181 Hz) were measured by simultaneously irradiating with 818 z 520 nm excitation light.

Sl (465 nm, 33 Hz) mainly contains signals derived from contaminant components contained in the sample, but also contains signals derived from reaction products. However, the latter is very small and can be ignored in measurement.

S, (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.

If we first measure the photoacoustic signal of bilirubin, which is a problem as a contaminant when performing visible light photometry using a human serum sample, and find the relationship of equation (6) for the above wavelength, we get (6
') The value of k in formula was 1/100. That is, the measurement target signal S. is determined. A obtained as above
A calibration curve for FP quantification is shown in Figure 2.

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

As described above, according to this embodiment, the background can be corrected without using a blank, so the following effects can be achieved.

(1) Even in samples with varying backgrounds, the amount to be analyzed can be quantified accurately.

(2) Blank cells and samples are no longer needed.

(3) Individual differences in contaminants when applied to immunoassays.

Differences between cases and differences due to changes in symptoms do not impede quantitative analysis.

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

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

〔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 diagram showing the configuration of an apparatus showing an embodiment of the present invention, Fig. 2 is a graph showing a calibration curve of alpha-fetoprotein when using the apparatus of this embodiment, and Fig. 3 is a graph showing analyte substances and contaminants. It is a graph which shows the photoacoustic spectrum when . 1.2... Light source, 3, 4... Light intensity modulator, 5, 6 Excitation light, 7... Half mirror 18... Photoacoustic cell, 9
...Photoacoustic signal, 10.11...Phase detection device, 12
．． 15. Reference signal, 5. Beam stopper, 16 Photoacoustic signal, 13. Photoacoustic signal by excitation light 5, 14
...Data processing device, 16.. Photoacoustic signal by excitation light 6, 17.. Mirror, 18.. Beam stopper.

Claims (1)

[Scope of Claims] 1. In 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 a photoacoustic effect, Two or more excitation lights with different light wavelengths and light intensity modulation frequencies are irradiated simultaneously, a photoacoustic signal synchronized with the light intensity modulation of each excitation light is detected, and the target substance to be analyzed is detected from the difference in signal strength. A photoacoustic analysis method characterized by quantifying. 2. In a photoacoustic analysis method in which a sample is irradiated with excitation light and an analyte substance contained in the sample is determined from the intensity of a photoacoustic signal generated by the photoacoustic effect, two or more different substances are used for the sample. A photoacoustic analysis method characterized by measuring the intensity of a photoacoustic signal at a time of , and quantifying the substance to be analyzed from the difference between the respective signal intensities. 3. In a photoacoustic analyzer that irradiates a sample with excitation light and quantifies the target substance contained in the sample from the intensity of a photoacoustic signal generated by the photoacoustic effect, different wavelengths of light irradiated onto the sample and and a detection means for making the excitation light incident on the sample and detecting a photoacoustic signal synchronized with each light intensity modulation. Photoacoustic analysis device. 4. In a photoacoustic analyzer that irradiates a sample with excitation light and quantifies the target substance contained in the sample from the intensity of the photoacoustic signal generated by the photoacoustic effect, two or more different photoacoustic signals of the sample are used. A photoacoustic analysis device comprising: a measuring means for measuring at a time of , and a processing means for quantifying a substance to be analyzed based on a difference between the measured values. 5. The photoacoustic analysis method according to claim 1, wherein the wavelength of one of the excitation lights matches the optical absorption wavelength of the immune reaction product. 6. The method according to claim 1, wherein the wavelength of one of the excitation lights matches the light absorption wavelength of the immunoreaction product, and at least one wavelength of the other excitation light matches the wavelength of the light absorbed by the immune reaction product, and the wavelength of at least one of the other excitation lights matches the wavelength of the light absorbed by the immunoreaction product. A photoacoustic analysis method characterized by matching the optical absorption wavelength of. 7. In the method according to claim 2, the sample is a product of an immune reaction, and the amount of the immune reaction product produced is determined from the difference in photoacoustic signals at two or more different times in the course of the immune reaction. A photoacoustic analysis method characterized by: 8. The apparatus according to claim 3, 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 analysis device. 9. The photoacoustic analyzer according to claim 3, wherein the cell that holds the sample and receives the excitation light also has the function of causing an immune reaction. 10. The photoacoustic analysis device according to claim 4, wherein the amount of the immune reaction product produced is determined from the difference between photoacoustic signals at two or more different times during the course of the immune reaction of the sample. 11. The photoacoustic analyzer according to claim 8, wherein the wavelength of one of the excitation lights incident on the cell matches the optical absorption wavelength of the immune reaction product. 12. The device according to claim 8, wherein the wavelength of one of the excitation lights incident on the cell matches the light absorption wavelength of the immunoreaction product, and at least one wavelength of the other excitation light matches the wavelength of light absorption of the immunoreaction product. Alternatively, a photoacoustic analyzer characterized in that the wavelength matches the light absorption wavelength of an unreacted substance.