METHOD AND APPARATUS FOR DETECTING PARTICULAR PARTICULATE SUBSTANCE

Abstract: By use of excitation beam having a wavelength which is equal to, or close to, the sizes of particular parti­ culate substances as the object of analysis, the particu­ late substances such as an immunoreaction product between an antigen and an antibody or between the antigen and a material supporting thereon the antibody are detected selectively. Very small particles having specific sizes such as rheumatuid factors, cancer specific antigens can be detected highly sensitively.

Description: BACKGROUND OF THE INVENTION This invention relates to a method and apparatus for detecting a particulate substance in a liquid and more particularly to a detection method and apparatus of an immunoreaction product which may be suitable for detecting a trace amount of an immunoreaction product. Conventional methods and apparatuses for detecting very small particles in a liquid in accordance with photoacoustic spectroscopy are not equipped with a control mechanism which controls the wavelength of excitation light in such a manner as to bring it into conformity with the size of the very small particules as the object of measurement, as can be seen, for example, from Japanese Patent Laid-Open No. l0254l/l986. They do not either consider any mechanism which detects selectively particulate substaces having a specific size dependence of sensitivity of an photoacoustic spectrometry and determines the quantity of the particulate substances. Therefore, the prior art technique does not consider selective detection and determination of a reaction product such as an immunoreaction product in the system where an antibody, antibody support particles and reaction products coexist during non-reaction. The prior art technique described above does not take into consideration the particle size dependency of sensitivity of the photoacoustic spectrometry and cannot selectively detect and determine very small particles having specific sizes such a rheumatuid factors, cancer specific antigens, and the like. SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method and apparatus for detecting and determining very small particles having specific sizes in a liquid and more particularly, for determining selectively and highly sensitively immunoreaction products. The object described above can be accomplished in the following way. The sensitivity of an photoacoustic spectrometry to particulate substances as the object of analysis is increased by use of excitation beam having a wavelength which is in agreement with, or close to, the sizes of the particulate substances as the object of analysis by utilizing the size dependence of the sensitivity of photoacoustic spectroscopy which is found out by the present inventors, and the particulate substance are then detected and determined selectively. The object described above can be accomplished, too, by controlling the sizes of the resulting particulate substances so that they coincide with the wavelength of excitation beam of the photoacoustic spectroscope. Since the present invention can selectively detect particulate substance having a specific size in the liquid and can determine it highly sensitively, the invention provides the following effects. l) The invention can determined the amount of the immunoreaction product with a far higher level of sensitivity than conventional tubidimetry. 2) The present invention can determine a cancer or tumore maker antigen, whose determination has been difficult conventionally, and makes it possible to find out diseases such as cancer at an earlier stage. BRIEF DESCRIPTION OF THE DRAWINGS Fig. l is a block diagram showing the fundamental construction of a particulate substance determination apparatus in a liquid in accordance with the present invention; Fig. 2 is a diagram showing particle size dependence of sensitivity of a photoacoustic spectrometry; Fig. 3 is a structural view of one embodiment of the present invention; Fig. 4 is a structural view of a cell used in an embodiment of the invention; Fig. 5 is a structural view of a reactor and a sample introduction device used for the embodiment; Fig. 6 is a schematic view of polystyrene particles supporting thereon an antibody before and after reaction; and Fig. 7 is a diagram showing a calibration curve of a rheumatuid factor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the principle of the present invention will be described. Fig. l shows an example of the fundamental construction of a detector for an immunoreaction product in accordance with the present invention. The light beam ll from a light source l such as laser is converted to excitation light 3 having a wavelength which is equal to, or near, the size of a particulate substance as the object of analysis by a wavelength converter 2 consisting of a dye laser, a non-linear type optical element, spectrometer, and the like. The light beam is then incident to a cell 6. The particulate substance contained in a liquid sample inside the cell 6 absorbs the excitation light and generates an photoacoustic signal. The resulting photoacoustic signal is detected by a detector such as a piezoelectric device that is fitted to the cell. The detected photoacoustic signal l3 is amplified by a lock-in amplifier 9 referring to a signal from an light modulator 4 such as an optical chopper being a reference signal l2. The magnitude of the photoacoustic signal measured by such an photoacoustic spectrometry is proportional to the quantity (concentration) of the particulate substance contained in the sample. Therefore, a calibration curve of the particulate substance in accordance with the magnitude of the photoacoustic signal can be obtained. Next, Fig. 2 shows particle size dependence of the photoacoustic spectroscope, that is, the slope of the calibration curve. The diagram shows the result of measurement of polystyrene particles. Measurement was performed with an intensity of excitation light of 0.8 W, a wavelength of 488 nm and modulation frequency of l8l Hz. As shown in Fig. 2, it was found for the first time that when the particle size coincides with the wavelength 488 nm (approx. 0.5 µm) of excitation light, the sensitivity of the photoacoustic spectrometry increases resonantly. This experimental result reveals that when the wavelength of excitation light 3 of the photoacoustic spectrometry shown in Fig. l is made to be equal to the particle size of the particulate substance as the object of analysis, particles having a specific size can be detected and determined selectively. Particularly in the case of detection of an immunoreaction product as the particulate substance, the reaction product can be measured selectively and highly sensitively even when unreacted antibodies or particles supporting the unreacted antibodies coexist, if the wavelength of excitation light is selected in such a manner that it is in agreement with the size of the reaction product but is out of agreement with the size of the antibody or that of the particle supporting the antibody. Particle size dependence of sensitivity,shown in Fig. 2, presumably results from the fact that dielectric loss due to particles increases resonantly at the wavelength which is in agreement with the particle size and near that wavelength in the same way as scattering of resonant light and heat resulting from the dielectric loss increases resonantly, too, so that the magnitude of the photoacoustic signal generated from the heat released to the medium increases resonantly. Hereinafter, one embodiment of the present invention will be explained with reference to Figs. 3 to 7. Fig. 3 shows the construction of a detector for an immunoreaction product in accordance with the present invention. An excitation beam source is an argon laser l7 having a l0 W output and laser beam 25 from the argon laser has emission lines of 488 nm, 5l4.5 nm and a line in the ultraviolet wavelength range. A pigment inside a dye laser l8 is excited (pumped) in accordance with the size of the reaction product as the object of analysis. When the size of the reaction product is from 0.6 to 0.7 µm , for example, the wavelength of the excitation beam 25 from the argon laser l7 is set to 5l4.5 nm while Rhodamine is used as the pigment of the dye laser l8. Part of the laser beam 27 whose wavelength is converted in accordance with the size of the object of analysis is branched by a half mirror 5, and is incident to a wavelength monitor 28 and its wavelength is confirmed by a wavelength monitor 28. The remaining laser light is subjected to light modulation to a periodical rectangular wave by an intensity modulator l9 consisting of a rotating blade type optical chopper and changes to excitation beam 26. This beam 26 is incident to a cell 23 and lets the sample inside the cell generate an photoacoustic signal. The photoacoustic signal generated in the cell 23 is detected by a piezoelectric element and is amplified by a lock-in amplifier 3l referring a signal 35 from a driver 29 of the light modulator l9. Part of the excitation beam 26 is branched by the half mirror 5 and its intensity is monitored by an optical intensity monitor 30. The lock-in amplifier 3l inputs information relating to the magnitude and phase of the photoacoustic signal while the wavelength monitor 28, the driver 29 of the light modulator and the optical intensity monitor 30 input information relating to the wavelength of the excitation beam, the modulation frequency and intensity, to a data processor 32. The data processor 32 displays the information of parameters relating to the measurement condition such as the excitation beam wavelength and the modulation frequency on a display 33 or processes the data of the measurement result and displays the calibration curve of the reaction product and the result of determination of an unknown sample on the display 33. The sample such as serum is placed in a reaction tube inside the reactor 2l and the antibody or particles supporting antibody is added by a reagent charger 22. The reaction product formed under the predetermined reaction condition is moved from the reactor 2l to the cell 23 through a sample path 37. After measurement, the sample path 37 is washed by ultra-pure water from a ultra-pure water device 20 together with the cell 23 and the next measurement is prepared. The apparatus of the present invention measures automatically a plurality of samples and processes the data by repeating these processes. Fig. 4 is a detailed view of the cell 23 of the present apparatus. The inside of the cell is cylindrical. After the sample is packed into the cylinder, the excitation beam 26 is allowed to be incident along the center axis of the cylinder. The photoacoustic signal generated inside the cell is detected by the piezoelectric element 40 and sent as a piezoelectric signal from an electrode terminal 42 to the lock-in amplifier 3l. The piezoelectric element is disposed at the center of the cell 23 and its inner diameter is the same as that of the glass cylinder 4l of the cell, and the inside of the cell describes a smooth cylindrical shape. The sample flows into the cell through an inlet tube 37 and after measurement, is discharged to a drain through an outlet tube 38. Fig. 5 shows in detail the reactor 2l of the present apparatus and a sample introduction portion 43. The reactor 2l consists of a reaction tube 49 and a rotary holer 45. A sample such as serum is placed into the reaction tube 49 and the reagent such as the antibody is charged, too, through the reagent charger 22. A stirring device such as a stirrer may be disposed inside the reaction tube 49, whenever necessary. After the reaction, the sample is caused to flow into the sample introduction portion 43 from an extraction port at the lower part of the reaction tube 49 and sent into the cell. After the reaction, the reaction tube 49, the sample introduction portion 43, the sample flow path 37 and the cell 23 are washed by ultrapure water emitted from a spray 44. Fig. 6 is a schematic view of a material supporting the antibody to be used in the present invention and the reaction product after immunoreaction. The material supporting the antibody is preferably one that has a specific gravity near l, can be dispersed uniformly into the serum and exhibits a large exothermic quantity due to dielectric loss. From this aspect, polystyrene particles are particulate substance which is suitable for the apparatus of the present invention. The particle diameter of the polystyrene particles 46 supporting the antibody before reaction is set to be 0.2 µm. Since this embodiment is directed to detect a rheumatuid factor, the corresponding antibody is supported on the polystyrene particles 46. As a result of the immunoreaction under a suitable reaction condition, the polystyrene particles 46 agglutinate through the antigen and turn to the reaction product 47. The size of the reaction product is about 0.5 µm and it coexists with the unreacted polystyrene particles 48. Since the size of the unreacted particles 48 is 0.2 µm and that of the reaction product is 0.6 µm in this embodiment, the wavelength of the laser beam 25 from the argon laser l7 is set to be 5l4.4 nm so that the wavelength of the excitation beam becomes 0.6 µm, and hodamine is selected as the pigment of the dye laser l8. The modulation frequency is set to l8l Hz. Fig. 7 shows an example of the calibration curve of the rheumatuid factor obtained by the present apparatus. It can be understood from the result of measurement that the immunoreaction product can be determined in the concentration range of l0⁻³ U/ml (U: international unit). This result shows that the immunoreaction product can be measured in a concentration range which is incomparably lower than the lower determination limit obtained by ordinary turbiditmetry. In the conventional photoacoustic spectroscopes for liquid which cannot select the wavelength, the unreacted product and the reaction product cannot be distinguished and the total amount of the particulate substances in the sample are measured. Therefore, a calibration curve cannot be obtained. In accordance with the present apparatus, the wavelength can be scanned in the visible range so that the size of the reaction product in the sample can be known from the wavelength providing the peak of signal intensity by monitoring the signal intensity while scanning the wavelength. In this case, the excitation light intensity at each wavelength is monitored by the light intensity monitor and the signal magnitude is standardized. The wavelength that provides the peak is displayed on the display 33 after wavelength of scanning is completed. Therefore, the wavelength thus displayed can be used for the determination of the reaction product. The present invention can measure the phase of the photoacoustic signal by the lock-in amplifier 3l. The photoacoustic signal generated from the particulate substance in the liquid has a phase corresponding to the size of the particle. This phase depends on the heat radiation time from the particle to the medium and according to the studies made by the present inventors, is a function of the square of the size. Therefore, if the polstyrene particles are 0.2 µm in diameter before the reaction and the reaction proceeds, the reaction product of 0.6 um in size increases. The present apparatus can monitor the progress of the reaction and the quantity of the reaction product or can detect and determine the reaction product by measuring the change of the phase. In this case, the modulation frequency of the excitation light intensity is set to about l0 KHz in order to increase the variation of the phase. Next, the case where two wavelengths, that is, the wavelength 0.6 µm which is in agreement with the size of the reaction product and the wavelength 0.2 µm which is in agreement with the size of the unreacted particles, are utilized as the wavelength of the excitation light, will be described. When measurement is made using the wavelength 0.6 µm, the apparatus has sensitivity of about l/l00 of the 0.6 µm paricles to the unreacted 0.2 µm particles. Therefore, when the amount of the unreacted particles becomes about l00 times that of the reaction product, the signal component from the unreacted particles, that is, the background, becomes substantially equal to the signal component from the reaction product and determination of the reaction product becomes difficult. Therefore, 0.2 µm particles are determined by the 0.6 µm excitation light exhibiting high sensitivity to the 0.2 µm particles and the data thereby obtained is reduced from the data obtained by using the 0.6 µm excitation light. In this manner, the background of the 0.2 µm particles can be reduced and the reaction product can be determined.

Claims: A method of detecting a particular particulate substance comprising the steps of: adjusting the wavelength of a light beam (11) to be substantially equal to the size of individual particles of the particular particulate substance as the object of analysis, radiating the light beam (11) onto said particulate substance, and measuring the magnitude of a photoacoustic signal (13) generated from said particulate substance. A method of detecting a particular particulate substance according to claim 1, further comprising the steps of: determining the concentration of said particulate substance by use of the relationship between the magnitude of the photoacoustic signal (13) and the concentration of said particulate substance. A method of detecting a particular particulate substance according to claim 2, wherein said particulate substance is an immunoreaction product between an antigen and an antibody or between the antigen and a material supporting thereon the antibody. A method of detecting a particular particulate substance according to claim 2 or 3, wherein the light beam (11) to be radiated to said particulate substance is a beam of continuous light within a wavelength range capable of embracing the particle size distribution of said particulate substance. A method of detecting a particular particulate substance according to claim 3, wherein said support material for said antibody is a material having a small light absorption coefficient for the wavelength of said light beam to be radiated. An apparatus for detecting a particular particulate substance comprising: a light source (1); a wavelength converter (2) for converting the wavelength of the light beam (11) provided from said light source (1) to a specific wavelength; a cell (6) into which said particulate substance as the object of analysis is charged and which receives the light beam (3) whose wavelength is converted, and outputs an photoacoustic signal (13), a data processor (32) for inputting said photoacoustic signal (13) and determining the concentration of said particulate substance by use of the relationship between the intensity of photoacoustic signals and the concentration of a particular particulate that is in advance stored therein; and a display (23) for displaying the output information of said data processor (32). An apparatus for detecting a particular particulate substance according to claim 6, wherein said particulate substance is an immunoreaction product between an antigen and an antibody or between said antigen and a material supporting thereon said antibody. An apparatus for detecting a particular particulate substance according to claim 6 or 7, wherein the light beam (11) to be radiated to said particulate substance is a beam of continuous light within a wavelength range capable embracing the particle size distribution of said particulate substance. An apparatus for detecting a particular particulate substance according to claim 7, wherein said support material for said antibody is a material having a small light absorption coefficient for the wavelength of said light beam to be radiated.

Inventors: FUJIMORI, HARUO, KITAMORI, TAKEHIKO, NOMURA, YASUSHI, SUZUKI, KAZUMICHI, YASUDA, KAZUO
Applicants: HITACHI, LTD.
Application year: 1987
Main classification: G01N15
IPC codes: G01N15, G01N33, G01N21, G01N29