Flow rate measurement apparatus, antigen concentration measurement apparatus, flow cell, flow rate measurement method, and antigen concentration measuring method

Abstract

A flow rate measurement apparatus includes a light oscillator; a thin metallic film which causes surface plasmon resonance by light output from the light oscillator; a focusing unit which fixes the thin metallic film and converts the output light of the light oscillator into incident light having a plurality of incident angles to focus the incident light at a location of a focal line in a straight line shape on the thin metallic film; a measurement part having antibody fixed areas to which an antibody is fixed and reference areas to which an antibody is not fixed, the antibody fixed areas and the reference areas being alternately arranged at a location along the focal line location on the thin metallic film; a light receiver which receives reflected light, at the focal line location, of the output light by surface plasmon resonance occurring at the focal line location, at each of the plurality of incident light angles; an SPR angle calculator which obtains a temporal change of an SPR angle in each of the antibody fixed areas and the reference areas in the measurement part; and a flow rate operation unit which calculates the flow rate of the sample flowing in the flow cell based on the temporal change of the SPR angle obtained by the SPR angle calculator.

1 . A flow rate measurement apparatus which measures a flow rate of a sample flowing in a long flow cell, different antibodies being arranged in a sample flow direction in the flow cell, the apparatus comprising:
 a light oscillator;   a thin metallic film which causes surface plasmon resonance by light output from the light oscillator;   a focusing unit which fixes the thin metallic film and converts the output light of the light oscillator into incident light having a plurality of incident angles to focus the incident light at a location of a focal line in a straight line shape on the thin metallic film;   a measurement part having antibody fixed areas to which an antibody is fixed and reference areas to which an antibody is not fixed, the antibody fixed areas and the reference areas being alternately arranged at a location along the focal line location on the thin metallic film;   a light receiver which receives reflected light, at the focal line location, of the output light by surface plasmon resonance occurring at the focal line location, at each of the plurality of incident light angles;   an SPR angle calculator which obtains a temporal change of an SPR angle in each of the antibody fixed areas and the reference areas in the measurement part; and   a flow rate operation unit which calculates the flow rate of the sample flowing in the flow cell based on the temporal change of the SPR angle obtained by the SPR angle calculator.   
     
     
         2 . The flow rate measurement apparatus according to  claim 1 , wherein the flow rate operation unit comprises:
 a waveform shifter which shifts one of adsorption curves indicating the temporal change of the SPR angles at two points in either the antibody fixed area or the reference area with respect to the other in a time direction;   a time difference detector which measures a shift time at which a difference between the SPR angles at the two points is smallest; and   a flow rate calculator which calculates the flow rate of the sample in the flow cell by dividing a location between the two points by the shift time.   
     
     
         3 . The flow rate measurement apparatus according to  claim 2 , further comprising a waveform differentiation operation unit which performs time differentiation on the adsorption curves for the two each points to obtain differential curves,
 wherein the waveform shifter shifts one of the differential curves for the two each points with respect to the other in a time direction, and the time difference detector measures a shift time at which a difference between the SPR angles in the differential curves for the two each points is smallest.   
     
     
         4 . The flow rate measurement apparatus according to  claim 2 , further comprising a waveform differentiation operation unit which performs time differentiation on differential data of the SPR angles,
 wherein the waveform shifter shifts one of the adsorption curves for the two each points with respect to each other in a time direction, and   each time the waveform shifter shifts the adsorption curve, the waveform differentiation operation unit performs the time differentiation on differential data of the adsorption curves for the two each points, and the time difference detector measures a shift time at which the time-differentiated differential data of SPR angles in the adsorption curves for the two each points is smallest.   
     
     
         5 . The flow rate measurement apparatus according to  claim 1 , wherein the flow rate operation unit calculates the flow rate of the sample flowing in the flow cell using Hough conversion. 
     
     
         6 . The flow rate measurement apparatus according to  claim 1 , wherein the antibody, and a sample detection substance having a refractive index varying by reacting with a substance other than a substance expected to react with the antibody among substances contained in the sample are fixed on the thin metallic film, and the apparatus further comprises:
 a reaction-result derivation unit which obtains a resonant angle that is an incident angle at which reflectivity is smallest based on a correlation between the incident angle of the incident light and reflectivity of the reflected light in the antibody, and obtains a reaction amount between the antibody and the sample detection substance from the resonant angle;   a refractive-index derivation unit which obtains a resonant angle based on a correlation between the incident angle and reflectivity in the sample detection substance, and obtains refractive indices of the sample detection substance and the substance contained in the reacting sample from the resonant angle; and   a reaction-result correction unit which obtains an amount of error for the flow rate of the sample from a rate of change of the refractive index obtained by the refractive-index derivation unit and obtains a correction amount for the reaction amount obtained by the reaction-result derivation unit from an amount of error of the flow rate to correct the reaction amount based on the correction amount.   
     
     
         7 . The flow rate measurement apparatus according to  claim 6 , further comprising:
 a measurement-initiation-signal output unit which outputs a measurement initiation signal when the resonant angle is obtained from the correlation between then incident angle and the reflectivity obtained by the image processor and the sample beginning to flow on the flow cell is detected from a change of the resonant angle; and   an image acquisition period controller which iteratively outputs an image acquisition timing signal to instruct the light receiver to acquire an image, making the period of the image acquisition timing signal shorter than the normal period between an output time of the measurement initiation signal and a predetermined time, and returning the period of the image acquisition timing signal to the normal period after the predetermined time lapses.   
     
     
         8 . The flow rate measurement apparatus according to  claim 6  or  7 , further comprising a storage unit in which the ideal value of the rate of change of the refractive index, a relationship between the amount of error of the rate of change and the amount of error for the flow rate of the sample, and a relationship between the amount of error of the flow rate and the correction amount are registered in advance,
 wherein the reaction-result correction unit obtains an amount of error between the rate of change of the refractive index obtained by the refractive-index derivation unit and the ideal value of the rate of change registered in the storage unit, acquires the amount of error of the flow rate corresponding to the amount of error of the rate of change from the storage unit, and acquires the correction amount corresponding to the amount of error of the flow rate from the storage unit.   
     
     
         9 . An antigen concentration measurement apparatus which measures a concentration of an antigen contained in a sample, different antibodies being arranged in a sample flow direction in a long flow cell, the apparatus comprising:
 a light oscillator;   a thin metallic film which causes surface plasmon resonance by light output from the light oscillator, the antibody and a sample detection substance having a refractive index varying by reacting with a substance other than antigen expected to react with the antibody among substances contained in the sample being fixed on the thin metallic film;   a focusing unit which fixes the thin metallic film and converts the output light of the light oscillator into incident light having a plurality of incident angles to focus the incident light at a location of a focal line in a straight line shape on the thin metallic film;   a measurement part having antibody fixed areas to which an antibody is fixed and reference areas to which an antibody is not fixed, the antibody fixed areas and the reference areas being alternately arranged at a location along the focal line location on the thin metallic film;   a light receiver which receives reflected light, at the focal line location, of the output light by surface plasmon resonance occurring at the focal line location, at each of the plurality of incident light angles;   an SPR angle calculator which obtains a temporal change of an SPR angle in each of the antibody fixed areas and the reference areas in the measurement part;   a flow rate operation unit which calculates a flow rate of the sample flowing in the flow cell based on the temporal change of the SPR angle obtained by the SPR angle calculator;   a reaction-result derivation unit which obtains a resonant angle that is an incident angle at which reflectivity is smallest based on a correlation between the incident angle of the incident light and reflectivity of the reflected light in the antibody, and obtains a reaction amount between the antibody and the sample detection substance from the resonant angle;   a refractive-index derivation unit which obtains a resonant angle based on a correlation between the incident angle and reflectivity in the sample detection substance, and obtains refractive indices of the sample detection substance and the substance contained in the reacting sample from the resonant angle;   a reaction-result correction unit which obtains an amount of error for the flow rate of the sample from a rate of change of the refractive index obtained by the refractive-index derivation unit and obtains a correction amount for the reaction amount obtained by the reaction-result derivation unit from an amount of error of the flow rate to correct the reaction amount based on the correction amount; and   an antigen concentration calculation unit which calculates a concentration of the antigen contained in the sample based on the correction result from the reaction result correction unit.   
     
     
         10 . A flow cell for surface plasmon resonance measurement having a thin metallic film on which an antibody is fixed in a portion thereof,
 wherein the antibody and a sample detection substance having a refractive index varying by reacting with a substance other than antigen expected to react with the antibody among substances contained in the sample flowing in the flow cell are fixed on the thin metallic film.   
     
     
         11 . A flow rate measurement method of measuring a flow rate of a sample flowing in a long flow cell using a flow rate measurement apparatus comprising: a light oscillator, a thin metallic film which causes surface plasmon resonance by light output from the light oscillator; a focusing unit which fixes the thin metallic film and converts the output light of the light oscillator into incident light having a plurality of incident angles to focus the incident light at a location of a focal line in a straight line shape on the thin metallic film; a measurement part having antibody fixed areas to which an antibody is fixed and reference areas to which an antibody is not fixed, the antibody fixed areas and the reference areas being alternately arranged in the flow cell and the flow cell being formed at a location along the focal line location on the thin metallic film; and a light receiver which receives reflected light, at the focal line location, of the output light by surface plasmon resonance occurring at the focal line location, at each of the plurality of incident light angles, the method comprising:
 an SPR angle calculation process of obtaining a temporal change of an SPR angle in each of the antibody fixed areas and the reference areas in the measurement part; and   a flow rate operation process of calculating the flow rate of the sample flowing in the flow cell based on the temporal change of the SPR angle obtained by the SPR angle calculator.   
     
     
         12 . A flow rate measurement method of measuring a flow rate of a sample flowing in a long flow cell using a flow rate measurement apparatus comprising: a light oscillator; a thin metallic film which causes surface plasmon resonance by light output from the light oscillator, the antibody and a sample detection substance having a refractive index varying by reacting with a substance other than an antigen expected to react with the antibody among substances contained in the sample being fixed on the thin metallic film; a focusing unit which fixes the thin metallic film and converts the output light of the light oscillator into incident light having a plurality of incident angles to focus the incident light at a location of a focal line in a straight line shape on the thin metallic film; a measurement part having antibody fixed areas to which an antibody is fixed and reference areas to which an antibody is not fixed, the antibody fixed areas and the reference areas being alternately arranged in the flow cell and the flow cell being formed at a location along the focal line location on the thin metallic film; and a light receiver which receives reflected light, at the focal line location, of the output light by surface plasmon resonance occurring at the focal line location, at each of the plurality of incident light angles, an antigen concentration measurement method comprising:
 an SPR angle calculation process of obtaining a temporal change of an SPR angle in each of the antibody fixed areas and the reference areas in the measurement part;   a flow rate operation process of calculating a flow rate of the sample flowing in the flow cell based on the temporal change of the SPR angle obtained in the SPR angle calculation process;   a reaction-result derivation process of obtaining a resonant angle that is an incident angle at which reflectivity is smallest based on a correlation between the incident angle of the incident light and reflectivity of the reflected light in the antibody, and obtaining a reaction amount between the antibody and the sample detection substance from the resonant angle;   a refractive-index derivation process of obtaining a resonant angle based on a correlation between the incident angle and reflectivity in the sample detection substance, and obtaining refractive indices of the sample detection substance and the substance contained in the reacting sample from the resonant angle;   a reaction-result correction process of obtaining an amount of error for the flow rate of the sample from a rate of change of the refractive index obtained in the refractive-index derivation process and obtaining a correction amount for the reaction amount obtained in the reaction-result derivation process from an amount of error of the flow rate to correct the reaction amount based on the correction amount; and   an antigen concentration calculation process of calculating a concentration of the antigen contained in the sample based on the correction result in the reaction result correction process.