WO2014132717A1 - Interaction analysis device - Google Patents

Abstract

Provided is an interaction analysis device which automates the immobilization of a sample to be measured on a measurement substrate and enables an operator to arbitrarily control the amount of the sample to be measured during observation. An interaction analysis device is characterized by comprising: a substrate; a solution sending mechanism for providing a sample to be measured and a reaction reagent to the substrate; an observation mechanism for observing the sample to be measured on the surface of the substrate; an analysis mechanism for analyzing the immobilized amount of the sample to be measured on the measurement substrate on the basis of the result of the observation by the observation mechanism; and a control mechanism for adjusting the immobilized amount of the sample to be measured on the substrate, and is characterized by immobilizing the sample to be measured on the surface of the substrate, measuring the immobilized amount of the sample to be measured immobilized on the surface of the substrate, and adjusting the immobilized amount of the sample to be measured on the substrate on the basis of the result of the measurement.

Description

Interaction analyzer

The present invention relates to an apparatus for analyzing an interaction between a substance and a living body. In particular, the present invention relates to a method and apparatus for measuring how a biological sample such as a cell shows a response to a stimulus.

As a method for screening effective drugs for drug discovery, a technique (cell-based assay) for determining whether cells are sensitive to drugs has become widespread. Conventionally, when the effect of a drug is simply evaluated, a method for determining whether or not the target molecule and the target drug are physically bound has been used. On the other hand, the effect of a drug can be more accurately evaluated by directly measuring the physiological response of the target cell of the drug. This cell-based assay technique is used not only for drug discovery but also in various fields. For example, as an allergy test, it is possible to determine whether a patient responds to a specific allergen, or to determine whether a substance may cause an allergic reaction. In addition, when administering a drug such as an anticancer drug, it is also applicable to personalized medicine in which the most effective drug is selected and administered.
A typical procedure for a cell-based assay is as follows. First, a cell to be a measurement sample is cultured or collected from a subject. Next, the cells are fixed on the surface of a substrate such as a sensor chip. Then, a reaction reagent is added, brought into contact with the measurement sample, observed and measured using means such as a fluorescence microscope, and the obtained data is analyzed to obtain the presence / absence and size of the cell.
As an observation means, in addition to fluorescence observation generally used in biology, a technique using surface plasmon resonance (SPR) has been used in recent years.

The principle of SPR will be described. When light is applied to a metal, the free electrons inside the metal collectively vibrate. This state is called plasmon. This phenomenon also occurs on the metal surface, and this is called surface plasmon. At this time, an electric field is generated on the metal surface by the vibration of electrons. Suppose that there is a glass substrate with a metal thin film deposited on the upper surface. When light is incident from the lower surface of the substrate at an angle that causes total reflection at the interface between the glass and the metal thin film, an evanescent wave is generated on the surface of the metal thin film. Resonance occurs when the wave number of the evanescent wave and the surface plasmon described above are aligned. This is called surface plasmon resonance: SPR. When SPR occurs, the energy of the incident light is lost by resonance, and the intensity of the reflected light decreases. The incident angle when SPR occurs is called the resonance angle, and its value depends on the dielectric constant of the metal thin film surface.

In Patent Document 1, a cell response to an external stimulus is observed using SPR. A gold thin film is formed on the surface, and a self-assembled monomolecular film having an amino group terminal is constructed on the substrate to obtain a sensor chip. The cell suspension is dropped onto the sensor chip to fix the cells on the surface. The substrate on which the cells are fixed is set in the apparatus and SPR observation is performed. The incident angle at this time is set to an angle at which resonance occurs due to the dielectric constant of the cell. For this reason, the intensity of the reflected light is reduced. When a stimulating substance is added in this state, the cell responds, for example, releases histamine. As a result, the dielectric constant of the cell changes, the resonance state collapses, and the intensity of reflected light increases. The change in the cell is observed by measuring the change in the signal intensity of the reflected light. A method of obtaining planar SPR information by using an image sensor such as a CMOS as in Patent Document 1 is referred to as SPR imaging (SPR imaging: SPRI).

However, the method of Patent Document 1 has a problem that the signal intensity of reflected light depends on the number of cells, so that quantitative evaluation is difficult and reproducibility for each measurement is low. Thus, as a technique for solving the above problem, Patent Document 2 provides a means for correcting the variation in the result due to the number of cells by calculating the number of cells from the SPR measurement result and converting it to the amount of change per unit cell. . First, an SPR substrate having a region to which cells can adhere and a region to which cells cannot adhere is prepared, and immersed and cultured in a cell culture medium. The substrate is washed and set in the apparatus, and SPR observation is performed while gradually changing the incident angle. Since the dielectric constant is different between the area where the cells are attached and the area where the cells are not attached, there is a difference in the resonance angle between the two. The number of cells is calculated by dividing the difference in angles by the fluctuation value of the resonance angle per cell obtained in advance. Thereafter, the incident angle is set to the resonance angle in the region where the cells are fixed, a stimulating substance is added, and the fluctuation amount of the resonance angle is measured. Finally, by dividing the fluctuation amount by the number of cells, a value normalized as the fluctuation amount per unit cell can be obtained.

Patent 3795312 JP2007-333612A

In Patent Document 2, the number of cells is calculated with a resonance angle fluctuation of 0.4 mdeg when one cell is fixed in an area of 1 mm ^ 2. In other words, in order to accurately calculate the number of cells, it is necessary to set the angular resolution of the rotation axis for setting the resonance angle to 0.4 mdeg or less. For this purpose, a highly accurate rotation stage is required, This leads to an increase in device cost. In practice, the resonance angle is obtained by curve fitting after creating a resonance curve using a rotary stage with low resolution, but this method inevitably causes an error in the calculation result. Moreover, since the value of the resonance angle fluctuation per cell differs depending on the size and type of the cell, the versatility is low.

Further, in any of the methods described in Patent Document 1 and Patent Document 2, since the process of fixing cells to the substrate and the process of SPR observation are independent, information on how much cells are fixed on the substrate Becomes apparent only when the SPR observation is actually performed. When the SPR observation is started, if the number of cells on the substrate is out of the range suitable for the observation, the fixing process must be performed again, which places a heavy burden on the operator.

When cells are fixed on the surface of a substrate or the like, a method of controlling the amount of fixation by changing the contact time between the substrate and the cell suspension is common. However, the rate of attachment to the substrate varies depending on the cell type and environmental conditions. Furthermore, for example, when observing at a low magnification, there is no problem even if the number of cells is large to some extent, but when observing one cell at a high magnification, the number of cells optimal for observation is necessary so that the cells need to be separated from each other. Depends on the application. For this reason, it is extremely difficult to arbitrarily control the fixed amount of various types of cells using only the contact time parameter.

Therefore, the present invention provides an interaction analysis apparatus that automates fixing of a measurement sample to a measurement substrate and allows an operator to arbitrarily control the amount of the measurement sample at the time of observation.

In order to solve the above problems, the present invention adopts the configuration described in the claims.

One aspect includes a substrate, a liquid feeding means for providing a measurement sample and a reaction reagent to the substrate, an observation means for observing the measurement sample on the substrate surface, and based on the observation result, Analyzing means for analyzing information related to the amount of the measurement sample fixed on the substrate, and control means for adjusting the amount of the measurement sample fixed on the substrate based on the information; To do.

According to the present invention, it is possible to provide an interaction analysis device that is highly reliable in results and easy to use.

The apparatus configuration described in the first embodiment. 2 is a configuration around a flow cell described in the first embodiment. 3 is a flowchart of the operation described in the first embodiment. It is an analysis result of Example 1. The apparatus configuration described in the second embodiment. 2 is a configuration around a flow cell described in the second embodiment. The apparatus configuration described in the third embodiment. 4 is another apparatus configuration described in the third embodiment. The apparatus configuration described in the fourth embodiment. This is the apparatus configuration described in the fifth embodiment.

Hereinafter, the novel features and benefits of the present invention will be described with reference to the drawings. However, the drawings are for explanation only, and do not limit the scope of the present invention.

(Device configuration)
The apparatus configuration of the first embodiment is shown in FIG. The analysis apparatus 101 includes an irradiation unit 102, a detection unit 103, a flow cell 104, a prism 105, and a liquid feeding unit 106. The operation of the analysis apparatus 101 is controlled by the control PC 108. A monitor 109 is connected to the control PC 108 and displays analysis results and the like. In addition, the analysis device 101 is provided with a temperature control device 107, and by adjusting the temperature of the CO2 gas sent from the CO2 gas cylinder 110, the temperature inside the analysis device 101 or the entire temperature and CO2 concentration can be arbitrarily set. It can be set.

(Explanation of irradiation unit / detection unit structure)
The irradiation unit 102 and the detection unit 103 will be described. The irradiation unit 102 includes components such as a light source 111, an optical fiber 112, a condensing lens 113, and a polarizing element 114. The detection unit 103 includes components such as an imaging lens 115 and an image sensor 116. The light source 111 is an excitation light source for SPR observation. The excitation light 117 emitted from the optical fiber 112 is condensed by the condenser lens 113 and shaped into a parallel beam. The S-polarized light component is removed from the excitation light 117 that has become a parallel beam by the polarizing element 114, and only the P-polarized light component is obtained. The excitation light 117 that has become P-polarized light enters the prism 105, enters the flow cell 104 from the back side of the flow cell 104, and enters the measurement region 118 at an angle θ. Thereafter, the excitation light 117 is reflected at an angle θ to become reflected light 118, passes through the flow cell 104 and the prism 105 again, and is emitted out of the prism 105. The emitted reflected light is collected by the imaging lens 115 and imaged on the image sensor 116 as a two-dimensional image. The image sensor 116 acquires an image and transmits image data to the control PC 108. The control PC 108 includes an analysis unit 121 that analyzes the image data sent from the image sensor 116 and displays the result on the monitor 109. The irradiation unit 102 and the detection unit 103 are arranged symmetrically with respect to the center line of the prism. Note that the irradiation unit 102 and the detection unit 103 are arranged on a drive stage that can take an arbitrary angle with respect to the flow cell 104, but are omitted in FIG. Further, a diaphragm for changing the irradiation range of the excitation light is provided between the condenser lens 113 and the polarizing element 114, but this is omitted in FIG.

In this embodiment, an LED light source (wavelength 640 nm) is used as the light source 111, a quartz core fiber (core diameter 600 μm, NA 0.22) is used as the optical fiber 112, and a spherical achromatic lens (outer diameter 50 mm, focal length is used as the condenser lens 113. 25 mm), a polarizing filter as the polarizing element 114 (adaptive wavelength 400 to 700 nm), an objective telecentric lens (5 times magnification) as the imaging lens 115, and a 1/2 type CMOS camera (effective pixel number 1280 ×) as the image sensor 116 1024 (1.3 million pixels) and pixel size 5.2 μm) were used. The spot diameter of the excitation light 117 after passing through the condensing lens 113 is φ10 mm. In this embodiment, an LED having a wavelength of 640 nm is used as the light source 111, but an apparatus that oscillates electromagnetic waves having different wavelengths may be used. The range of wavelengths that can be selected is, for example, 300 nm to 300 μm. In addition to the LED, a solid-state laser, a gas laser, a semiconductor laser, or the like can be used. Further, a desired wavelength component may be extracted from a single light source 111 that oscillates a plurality of wavelengths by a band-pass filter or the like. The light source 111 is, for example, a xenon lamp. In this embodiment, a commercially available polarizing filter is used as the polarizing element 114, but components having the same function such as a polarizing beam splitter and a Glan-Thompson prism can be used.

In this embodiment, an objective telecentric lens is used as the imaging lens, but a commercially available machine vision lens, zoom lens, or the like can be used. By changing the type of imaging lens, observation at low to high magnification is possible according to the application.

(Structure explanation of liquid feeding part)
Next, the liquid feeding unit 106 will be described. The liquid feeding unit 106 includes reagent reservoirs 123 to 125, liquid feeding tubes 126 to 127, switching valves 128 to 129, a liquid feeding pump 130, and a waste liquid tank 131. A liquid supply tube is connected to each of the reagent reservoirs 123 to 125 and is integrated into the liquid supply tube 126 via the switching valves 128 to 129. The liquid feeding tube 126 is connected to the opening (for introduction) of the flow cell 104. The flow cell 104 is provided with another opening (for discharge), and another liquid supply tube 127 is connected thereto. The end portion of the liquid feeding tube 127 is connected to the waste liquid tank 131. Further, a liquid feed pump 130 is attached to the liquid feed tube 127 and moves the liquid from the reagent reservoirs 123 to 125 to the flow cell 104. The kind of reagent, the amount of liquid, the liquid supply speed, the liquid supply timing, etc. sent from the reservoirs 123 to 125 to the flow cell 104 are determined by the liquid supply system control unit 120 of the control PC 108 to the switching valves 128 to 129 and the liquid supply pump 130. This is done by issuing an operation instruction to the user.

In this embodiment, the reservoir 123 is filled with a measurement sample, the reservoir 124 is filled with a reaction reagent, and the reservoir 125 is filled with a buffer. In this embodiment, TYGON R-3603 (inner diameter 0.8 mm, manufactured by Saint-Gobain) is used as the liquid feeding tubes 126 to 127, a three-way solenoid valve is used as the switching valves 128 to 129, and a peristaltic pump (maximum) is used as the liquid feeding pump 130. A pressure of 0.1 MPa was used. The individual parts are not limited to this, and the number of parts can be reduced by using, for example, a 6-way switching valve as the switching valve. Further, as the liquid feeding pump 130, another liquid feeding means such as a syringe pump can be selected.

(Description of flow cell structure)
The structure of the mechanism that holds the flow cell 104 and the prism 105 is shown in FIG. The flow cell 104 includes a substrate 201 and a flow path component 202. The flow cell 104 and the prism 105 are held by a holder 203. The substrate 201 is produced by forming a gold thin film on an observation surface (front surface / upper side in FIG. 2) for fixing a measurement sample of a glass substrate, like a general SPR substrate. A groove 204 as a flow path pattern is formed on the surface of the flow path component 202. By functioning the gold thin film surface of the substrate 201 and the groove side plane of the flow path component 202, the flow cell 104 functions. The holder 203 is provided with openings 209 to 210 through which the liquid feeding tubes 126 to 127 are passed. The liquid feeding tubes 126 to 127 pass through the holder openings 209 to 210 and are connected to the openings 207 to 208 on the flow path component 202, respectively. In addition, the holder 203 is provided with an opening 212 through which the temperature-controlled CO2 gas pipe 211 is passed. Polydimethylsiloxane (PDMS) used as the flow path component 202 has a high gas exchange function, and the temperature and CO2 concentration on the surface of the substrate 201 can be kept constant. In this embodiment, the temperature is adjusted to 37 ° C. and the CO 2 concentration is 5% so that the cells are most active. The holder 203 is attached with a linear guide for visual field movement and focus adjustment, but is omitted in FIG.

The material of the substrate 201 used in this example is S-LAL10 manufactured by Ohara, and the shape is a plate shape of 20 × 20 mm in length and width and 1.0 mm in thickness. The thickness of the gold thin film formed on the surface is 50 nm. In addition, 1 nm-thick chromium exists as an adhesive layer between the glass substrate and the gold thin film. In addition, the material of the flow path component 202 used in this example is PDMS, and the shape is a plate shape having a length and width of 18 mm and a thickness of 5 mm. The size of the groove 204 is 2 mm in width, 1 mm in depth, and 10 mm in length. There are vertical flow paths 205 to 206 extending from both ends of the groove 204 to the surface opposite to the groove 204, and openings 207 to 208 are provided at the respective ends. The material of the prism 105 used in this example is S-LAL10 manufactured by Ohara, which is the same as that of the substrate 201, and the shape is a triangular prism shape having a height of 20 mm with an equilateral triangle having a side of 20 mm as the bottom. The substrate 201 and the prism 105 side surface (square surface) are in optical contact with matching oil (not shown). In this embodiment, Cargill standard refraction liquid (refractive index 1.72) is used as matching oil.

The surface of the substrate 201 may be processed to promote the fixation of the measurement sample. For example, it may be treated with poly L-lysine or various antibodies according to the measurement sample. In addition, it is possible to easily hold the measurement sample by providing minute unevenness on the surface of the substrate 201.

In this embodiment, the PDMS flow path 202 is attached to the substrate 201. However, a structure in which a flow path pattern is formed on a thin sheet of PDMS, for example, may be sandwiched between the substrate 201 and a member such as a cover glass. .

A plurality of the grooves 204 of the flow cell 104 may be provided. The number and width of the grooves 204 and the interval between the grooves 204 are not limited, but the measurement region 118 covers part or all of the plurality of grooves 204, and the grooves 204 can be separated on the image sensor 116. It is desirable that the image is formed under various conditions.

In this embodiment, S-LAL10 is used as the material of the prism 105 and the substrate 201. However, any material can be used as long as it is optically transparent, such as glass, sapphire, quartz, acrylic resin, and the like. It can be used. The prism 105 and the substrate 201 are preferably made of the same material in order to prevent reflection at the interface when light is moved between them, but a combination of different materials is used as long as it is not totally reflected at the interface. It does not matter. The shape of the prism 105 is not limited to a triangular prism, and may be another shape such as a trapezoid or a quadrangle. For example, when a semi-cylindrical prism is used, the excitation light 117 always enters the prism 105 regardless of the incident angle, and the loss due to reflection can be minimized.

In this embodiment, the substrate 201 on which a gold thin film is formed is used. However, the gold thin film is directly formed on the surface of the prism 105, and the analysis may be performed there. By integrating the substrate 201 and the prism 105, light reflection loss at the interface between the two can be reduced. In addition, the number of parts is reduced and no matching oil is required, so that the operability is improved.

In this embodiment, the temperature of the substrate 201 is controlled by introducing warm air into the holder 203, but the temperature of the entire apparatus 101 may be controlled. Further, for example, a temperature control component such as a Peltier element may be brought into contact with the side surface of the prism 105 directly or indirectly. The temperature may be controlled by forming a conductive thin film such as ITO on the side surface of the prism 105 and energizing it.

In this embodiment, the CO2 concentration in the holder 203 is controlled, but the CO2 concentration of the entire apparatus 101 may be controlled.

(Flow chart of device operation)
A series of observation processes according to this example will be described with reference to measurement of the response function of RBL-2H3 cells, a cell line derived from rat basophils, in response to stimulation of DNP-HSA antigen (Sigma-Aldrich).

RBL-2H3 cells are activated when the IgE receptor on the surface binds to an antigen via IgE bound to the receptor. Then, granules containing intracellular histamine and proteolytic enzymes are released (degranulated). This is said to play an important role in the development of type I allergy. This response phenomenon of RBL-2H3 cells to antigen stimulation is widely used as a method for evaluating allergic reactions. For example, foods, drugs, and environmental substances (pollen, house dust, etc.) can be used as antigens (reaction reagents), and the antigenicity of substances (whether they cause allergic reactions) can be evaluated. Or you may evaluate the antiallergic effect of the substance added besides an antigen. That is, using a combination of a cell and an antigen whose response is already known, such as RBL-2H3 cells sensitized with an anti-DNP-IgE antibody (as described below), DNP-HSA antigen, What is necessary is just to measure whether a response is suppressed or enhanced by adding a test substance further here.

FIG. 3 is a flowchart of observation according to the present invention. Hereinafter, the cell function observation method according to the present invention will be described with reference to FIGS.

・ Prepare measurement samples and reaction reagents before starting the process. First, RBL-2H3 cells were cultured (5% CO2, 37 ° C.) in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% fetal calf serum (FCS), 100 unit / mL penicillin, and 100 μg / mL streptomycin. Next, the cells are cultured for 24 hours (5% CO 2, 37 ° C.) in a culture medium supplemented with 50 ng / mL mouse monoclonal anti-DNP-IgE antibody (Sigma-Aldrich) in a cell culture dish (HydroCell, Cellseed). Sensitized the cells. Thereafter, the sensitized cells were treated with a buffer (Siramian buffer containing 5.6 mM glucose, 1 mM CaCl2, 0.1% BSA (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl, 25 mM piperazine-N, N′-bis (2- resuspended in ethanolsulfonic acid (PIPES), 40 mM NaOH, pH 7.2) to a concentration of 1 × 10 6 / mL, and used as a measurement sample for measurement. The measurement sample was pre-heated (5% CO 2, 37 ° C.) for 10 minutes before being set in the apparatus. As the reaction reagent, DNP-HSA antigen prepared with the above-mentioned buffer to a concentration of 50 ng / mL was used.

Various reagents adjusted by the above procedure and the flow cell 104 were set in the apparatus 101. The inside of the substrate holder 203 is previously controlled at a temperature of 37 ° C. and a CO 2 concentration of 5%.

In step P1, the incident angle is adjusted. The liquid feeding system control unit 120 operates the pump 130 and the valves 128 to 129 to send the buffer to the flow cell 104, then stops the pump 130 and fills the flow cell 104 with the buffer. In this example, liquid feeding was performed at a liquid feeding amount of 500 μL and a liquid feeding speed of 1.6 μL / sec (100 μL / min). After filling the buffer, the optical system control unit 122 operates the irradiation unit 102 and the detection unit 103 to perform SPR observation while continuously changing the incident angle of the excitation light 117. In this example, images were acquired while continuously changing the incident angle range of 45 to 70 ° at intervals of 0.5 °. The exposure time of the image sensor 116 is 0.1 second. The image data is sequentially transmitted to the control PC 108, and the analysis unit 121 analyzes the signal intensity of the reflected light 119 from the transmitted image. In this embodiment, as the intensity of the reflected light 119, a value obtained by averaging the sum of the signal intensities in the region where the buffer exists by the number of pixels is used. The analysis unit 121 creates a plot with the incident angle of the excitation light 117 as the X-axis and the signal intensity of the reflected light 119 as the Y-axis, and sets the point showing the minimum value as the incident angle in response observation described later. In this example, the incident angle was 57.5 °.

In step P2, a target value for the amount of fixed cells at the time of observation is set. The target value of the cell fixation amount is directly input by the operator. Alternatively, it is automatically set by the operator selecting a condition (for example, “high magnification observation”) recorded in the apparatus 101 in advance.

When observing cells in units of one, it is desirable that a certain amount of cells exist in the observation field and the cells are fixed without overlapping. In this example, the average cell density was adopted as an index of the amount of fixed cells. Below, the calculation method for setting the target value of average cell density is demonstrated.

Assuming that the cells are randomly fixed on the substrate, when the average cell density δ, the probability B (x) that x cells exist within the area s is expressed by the following equation (1) according to the Poisson distribution. It can be expressed as

B (x) = (sδ) x / x! exp (-sδ) ... Formula (1)

The area s is a range of radius r around a certain cell (s = πr ^ 2). The probability B (0) that another cell does not exist within this range can be expressed by the following equation (2).

B (0) = exp (−sδ)
= Exp (-πr ^ 2δ) Equation (2)
In the case where the cells are circular, if the area s is the area of the cells, it can be said that B (0) is “probability of cells not overlapping each other”. In this case, r is equal to the center-to-center distance (cell diameter) of each cell when two cells contact each other. The relationship between the average cell density δ and B (0) is given by the following equation (3).

δ = − {logB (0)} / (πr ^ 2) (3)
For example, using a cell having a diameter of 10 μm, the average cell density δ satisfying the condition that 50% or more cells do not overlap can be obtained by solving Equation (3) for δ (Equation (4)).

δ ≦ − {log 0.5} / (πr ^ 2)
≦ 2206 / mm ^ 2 Formula (4)
By setting the target value of the average cell density to 2206 cells / mm ^ 2 or less, the number of cells that do not overlap in the visual field becomes a majority. That is, the amount of work for selecting cells without overlapping can be reduced, and the efficiency of analysis is improved. A preferable average cell density δ is 335 cells / mm 2 or less, and at this time, the condition that 90% of cells exist without overlapping is satisfied. A more preferable average cell density δ is 163 cells / mm ^ 2, which satisfies the condition that 95% of cells exist without overlapping. In the above conditions, the number of non-overlapping cells in the visual field is 427 and 219, respectively.

On the other hand, if the target value of the average cell density is set to 335 cells / mm ^ 2 or more, the ratio of cells with overlapping exceeds 10%, so it takes time to select cells without overlapping. It is possible to observe. That is, an analysis result with little variation can be obtained by increasing the absolute number of cells to be observed. A preferable average cell density is 1135 cells / mm 2 or more, and the number of non-overlapping cells in the field of view is 1126. A more preferable average cell density is 2206 cells / mm 2 or more, and the number of non-overlapping cells in the field of view is 1563. When the average cell density exceeds 3182 cells / mm ^ 2, the number of non-overlapping cells in the field of view decreases, and a large number of overlapping cells occupy. For this reason, it is preferable that the average cell density is 3182 cells / mm ^ 2 or more in applications such as mainly observing overlapping cells.

By the above calculation method, the operator can set an optimum average cell density according to the purpose of observation. For example, the operator may input a desired ratio of non-overlapping cells (for example, 90%) from a text box or the like, calculate a suitable average cell density, and set it as a target value. In this example, the set value of the average cell density was set to 100 cells / mm 2 as a condition that a sufficient number of cells exist in the visual field and most of the cells do not overlap.

In step P3, cells are fixed on the surface of the substrate 201. The liquid supply system control unit 120 operates the pump 130 and the valves 128 to 129 to send the measurement sample to the flow cell 104, stops the pump 130, and fills the flow cell 104 with the measurement sample. In this example, liquid feeding was performed at a liquid feeding amount of 500 μL and a liquid feeding speed of 1.6 μL / sec (100 μL / min).

In step P4, the number of cells attached to the surface of the substrate 201 is monitored. The optical system control unit 122 operates the irradiation unit 102 and the detection unit 103 to acquire an image after a lapse of a certain time from the start of this process, and sends the image data to the control PC 108. In this embodiment, the setting is made so that an image is acquired 10 minutes after the start of the process. The analysis unit 121 analyzes the image and calculates the amount of fixed cells at that time. In this example, first cut out the image data of the region where the measurement sample exists, binarize the cut out image, obtain the number of cells by performing particle number analysis, find the area from the number of pixels of the cut out image data, Cell density was calculated by dividing cell number by area. In addition, it is possible to calculate by various methods such as a method of calculating from the area (counting the number of pixels other than the background and dividing by the number of pixels corresponding to one cell obtained in advance). It is. Only the number of cells may be used as a fixed amount without calculating the density. Moreover, you may use the distance between cells as a fixed quantity parameter | index. For example, the distance between cells, for example, the distance between the centers or the distance between the contours of the cells is calculated from the acquired image, and the maximum value, the minimum value, the average value, the median value, etc. It may be controlled as an index.

In step P5, the amount of fixed cells is determined. The analysis unit 121 compares the fixed amount calculated in the process P4 with the target value set in the process P2, and instructs one or both of the liquid feeding system control unit 120 and the optical system control unit 122 according to the result. put out. As a comparison method between the fixed amount and the target value, the magnitude of the value, whether the difference between the two is larger than the reference value, whether the ratio between the two is larger than the reference value, or the like may be used. Alternatively, not only a fixed amount at one point in time but also a change with time may be used. For example, a differential value at a certain time point, an average increase / decrease amount or increase / decrease rate in a certain time zone, an integrated value, an integrated value, or the like may be used. Parameters such as a reference value used for comparison may be set in advance at the time of manufacture, adjustment, and setting of the apparatus. In the course of the analysis flow, for example, at the time of setting a fixed amount target value in the process P2, a GUI is used. Alternatively, an operator may input from the command line or read from a setting file or the like. The analysis unit 121 uses these comparison methods to determine whether the fixed amount needs to be adjusted or whether the fixed amount needs to be adjusted, so that it is possible to proceed to the next step P6.

For example, if the fixed amount has not reached the target value set in step P2, it is necessary to adjust the fixed amount. Therefore, the analysis unit 121 performs necessary processing of the liquid feeding system control unit 120 and the optical system control unit 122. Either or both are instructed, and the process proceeds to step P4 again. As the content of the instruction, for example, the progress of the fixation reaction may be promoted by simply issuing an instruction to wait for a certain period of time. Moreover, you may instruct | indicate the change of conditions, temperature, liquid stirring, flow velocity, and other physical conditions which accelerate | stimulate a fixed reaction. Or you may progress to the fixing reaction of process P3. Alternatively, these instructions may be combined or appropriately selected from these.

For example, when the fixed amount has reached the target value set in step P2, the adjustment to the fixed amount is unnecessary, and thus the process proceeds to step 6. However, processing for stopping the fixed reaction is performed as necessary. Specifically, the liquid supply system control unit 120 operates the pump 130 and the valves 128 to 129 to send a buffer to the flow cell 104, and replaces the measurement sample in the flow cell 104 with the buffer. In this example, liquid feeding was performed at a liquid feeding amount of 500 μL and 1.6 μL / second (100 μL / min). The fixation reaction termination process is not limited to this method, and various methods can be used. A drug that inhibits or stops the fixation reaction may be added. Other physical conditions such as temperature and pH may be changed.

In step P6, response observation is performed. The optical system control unit 122 operates the irradiation unit 102 and the detection unit 103 to continuously acquire images at a constant time interval (time-lapse shooting). In this embodiment, the exposure time is 0.1 seconds and the shooting interval is 10 seconds. The captured image data is sequentially transmitted to the control PC 108, and the analysis unit 121 analyzes the signal intensity of the reflected light 119 from the transmitted image. In this embodiment, the signal intensity of the reflected light 119 is analyzed using a value obtained by averaging the signal intensity of all the pixels in the region where the buffer exists. For example, all the pixels in all the regions including the flow path component are used. May be calculated. Further, the analysis may be limited to one or more arbitrary regions. The analysis unit 121 creates a plot with the elapsed time as the X axis and the reflected light signal intensity as the Y axis, and displays the plot on the monitor 109 in real time.

¡After a certain period of time has elapsed since the start of time-lapse photography, a reaction reagent containing a stimulating substance is brought into contact with the cells. After the liquid supply system control unit 120 operates the pump 130 and the valves 128 to 129 to send the reaction reagent to the flow cell 104, the pump 130 is stopped and the flow cell 104 is filled with the reaction reagent. In this example, after the elapse of 60 seconds from the start, the reaction reagent was fed at a feeding rate of 500 μL and a feeding rate of 1.6 μL / sec (100 μL / min).

タ イ ム Time lapse photography ends after a certain period of time has elapsed since the reagent was charged. The optical system control unit 122 operates the irradiating unit 102 and the detecting unit 103 to stop the operations of the irradiating unit 102 and the detecting unit 103, and displays on the monitor 109 that the measurement is completed to notify the operator. In this example, time-lapse imaging was completed after 1800 seconds had elapsed since the reaction reagent was charged.

Finally, the liquid supply system control unit 120 operates the pump 130 and the valves 128 to 129 to send the buffer to the flow cell 104, and after washing the liquid supply tubes 126 to 127 and the flow cell 104 with the buffer, the pump 130 is stopped. . In this example, liquid feeding was performed at a liquid feeding amount of 4000 μL and a liquid feeding speed of 3.2 μL / sec (200 μL / min). A solution containing trypsin may be sent before washing with a buffer. By adding the above process, cells attached to the surface of the substrate 201 drop off, and the surface of the substrate 201 returns to the state before the measurement, so that the measurement can be repeated without replacing the substrate 201.

Timeout error determination may be included in or before or after the process P5. The timing of this determination may be, for example, before or after the comparison between the fixed amount and the target value, or the processing for adjusting the fixed reaction is performed by either or both of the liquid feeding system control unit 120 and the optical system control unit 122. It may be before or after instructing the process, or before or after instructing either or both of the liquid feeding system control unit 120 and the optical system control unit 122 to stop the fixation reaction. The determination criterion for the timeout error may be when a predetermined time is exceeded or when a predetermined number of times is exceeded. Here, the number of times such as comparison between a fixed amount and a target value, calculation of a fixed amount, execution or instruction of fixed amount adjustment may be used. The time may be the elapsed time calculated from the start time of the fixation reaction of the measurement sample, the preparation of the apparatus, the installation of the measurement sample, the reaction reagent, etc. or the preparation time. Moreover, you may combine the some criteria mentioned here. In addition, the operator may select which of these determination criteria is adopted or not to perform the determination, and the timing of selection may be before or after the analysis flow starts (before the end). As operations after the time-out error is determined, the analysis flow may be temporarily stopped or canceled, a special fixed amount adjustment for time-out error, forcibly shifting to the process P6, etc. The operation content necessary for them is appropriately instructed to one or both of the liquid feeding system control unit 120 and the optical system control unit 122. When the analysis flow is temporarily stopped, for example, the analysis unit 121 notifies the worker to that effect via the monitor 108, voice, or the like, prompts the worker to instruct the next operation, and sets the instruction means as GUI, CUI, It may be provided via gestures, voices and the like. Operation options may include resumption of flow, suspension of flow, special fixed amount adjustment for time-out error, forced transition to step P6, and the like. The apparatus 101 may have a specification that allows the measurement sample, the reaction reagent, various setting values, and apparatus settings to be changed before restarting when the operator chooses to restart the flow. When the cancellation of the flow is selected, the analysis unit 121 may instruct the liquid supply system control unit 120 and the optical system control unit 122 or both to finish the process as necessary.

Note that the target value for the process P2 may be set before the process P5. The setting of the target value in the process P2 is preferably after the device preparation in the process P1 and before the start of the fixed reaction in the process P3. In this case, for example, after preparing the measurement sample, reaction reagents, and apparatus at the start of the experiment, a fixed amount target value is set as the experimental condition, and the results obtained by performing the analysis are added. As a new experimental condition, a fixed amount of target value is corrected and analyzed, it is possible to efficiently proceed with the experiment because it is not necessary to redo the device preparation. Alternatively, the target value for the process P2 may be set before preparing the apparatus for the process P1, and in this case, after setting a certain fixed amount of target value, the apparatus is prepared and the analysis proceeds. It is not necessary to set the target value again in an experiment in which the setting is changed based on the result, or various reagents are exchanged, re-prepared, and analysis is performed again, so that the experiment can be carried out efficiently. Become. In such cases, when the measurement sample or reaction reagent is inferior from the experimental results, the deviation (error) between the actual fixed amount and the target value is larger than expected, a timeout error, etc. In the case where the target fixed amount is not obtained in (1), the time required to reach the desired fixed amount is longer than expected and the measurement sample or the analysis result is adversely affected.

FIG. 4 shows the results of the antigen response reaction of RBL-2H3 cells measured using the above means. 60 seconds after the start of the measurement, the reaction reagent was started to be fed, and after about 50 seconds, an increase in the SPR reflected light intensity accompanying the cell response could be confirmed.

FIG. 5 shows an example in which a pipetter mechanism 504 is used for the liquid feeding unit 106 and a well substrate 601 is used instead of the flow cell 104. Other configurations are the same as those of the first embodiment. Here, the pipetter mechanism is a mechanism capable of discharging a specified volume of liquid. A method of discharging the liquid sucked from a separate liquid reservoir before discharging, a method of discharging the liquid directly in a reservoir directly connected to the pipetter mechanism, and discharging intermittently as necessary may be used.

(Description of structure)
The liquid feeding unit 106 of this embodiment includes reagent reservoirs 501 to 503 and a pipetter mechanism 504. The pipetter mechanism 504 includes a nozzle 505, a robot arm 506, a pipe 507, a syringe pump 508, a liquid feed pump 509, a switching valve 510, a water tank 511, a cleaning tank 512, and a waste liquid tank 513.

The nozzle 505 is held by the robot arm 506, and can be moved to the well substrate 601, the reagent reservoirs 501 to 503, the cleaning tank 512, the waste liquid tank 513, and the like as necessary. The nozzle 505 is connected to a syringe pump 508 via a pipe 507, and can suck or discharge a specified volume of liquid from the reagent reservoirs 501 to 503, the well substrate 601 and the like. Unnecessary liquid sucked from the reagent reservoirs 501 to 503 and the well substrate 601 is discharged to the waste liquid tank 503. A liquid feed pump 509 is connected to the syringe pump 508 and can fill the water in the water tank 511. In addition, after moving the nozzle 505 to the waste liquid tank 513, water is discharged from the liquid feed pump 509 via the syringe pump 508 and the nozzle 505, thereby cleaning the inside of the flow path and the inside of the nozzle 505 to prevent contamination and suction. Carryover of discharged liquid can be prevented and reduced. Furthermore, after moving the nozzle 505 to the cleaning tank 512, the switching valve 510 is switched to discharge water to the cleaning tank 510, and water is applied to the nozzle 505, so that the outside of the nozzle 505 can also be cleaned. Contamination and carry-over of sucked / discharged liquid can be prevented and reduced. These operations are performed by sending operation instructions to the robot arm 506, the syringe pump 508, the liquid feeding pump 509, and the switching valve 510 from the liquid feeding system control unit, which is omitted in the drawing.

A configuration diagram of the well substrate 601 is shown in FIG. The well substrate 601 includes a well component 602, a substrate 201, a prism 105, and a holder 603 for holding them together. Regarding the substrate 201 and the prism 105, the same configuration as that of the first embodiment can be basically used. Well part 602 includes one or more liquid holding sites 604. By installing the well component 602 on the substrate 601, the liquid holding site 604 and the substrate 201 form one or more wells 605. The holder 603 is provided with an opening 606 through which the nozzle 505 passes. A septa 607 is installed in the opening 606. The nozzle 505 passes through the septa 607 of the opening 606 and reaches the well 605 on the well substrate 601. The holder 603 is provided with an opening 608 for passing the temperature-controlled CO2 gas pipe 211. Thereby, the temperature and CO2 concentration on the surface of the well substrate 601 can be kept constant. In this embodiment, the temperature is adjusted to 37 ° C. and the CO 2 concentration is 5% so that the cells are most active. The holder 603 is provided with a linear motion guide for moving the visual field and adjusting the focus, but is omitted in FIG.
When there are a plurality of wells 605 in one well substrate 601, there is an effect that an interaction analysis under a plurality of conditions can be performed by setting up the well substrate 601 once. In particular, when a plurality of wells 605 can be observed in all time periods or temporarily in parallel by an optical system including the irradiation unit 102 and the detection unit 103, a plurality of conditions can be analyzed in parallel. Each well 605 has a function of holding liquid in or on the well 605 and preventing the liquid between the wells 605 from being mixed or moved unintentionally. Each well 605 has at least one opening through which the liquid to be held can be introduced. As the well component 602, various structures having such a function may be used. The amount of liquid that can be held in each well 605 can be appropriately changed depending on the application, but is preferably 1 nL to 1 mL, more preferably 10 nL to 100 μL, and more preferably 100 nL to 10 μL.

As another form of the well part 602, a plate-like member having a through hole as the liquid holding site 604 and a wall surface surrounding the liquid as the liquid holding site 604 (for example, the bottom surface of the multi-well plate is cut out) Water repellent to the liquid held on the surface of the substrate 201 or the sheet-like member provided with a through hole as the liquid holding site 604 Or a surface treatment exhibiting hydrophilicity (and functional groups introduced into the surface by the treatment, arrangement of molecules / atoms, etc.) having a pattern that functions as the liquid holding site 604 can be used. As the pattern, for example, water-repellent surface treatment is performed except for each liquid holding site 604, and each liquid holding site 604 is untreated, or each liquid is held on the surface of a relatively water-repellent substrate 201. A pattern in which a hydrophilic surface treatment is applied only to the outer periphery of the site 604 can be used. As another example of the well component 602, a flow cell-like member having an opening provided at the end or in the middle of the flow path may be used. In this case, the liquid may be introduced by dropping the liquid into the opening or inserting the nozzle 505 into the opening. Since the liquid in each well 605 is semi-confined inside the well component 602 and the surface area that directly touches the atmosphere is reduced, the liquid is less likely to evaporate, and more accurate analysis can be easily realized.

When the pipetter mechanism 504 as described above is used, only the nozzle 505 or the pipe 504 in the vicinity of the nozzle 505 is in contact with different measurement samples and reaction reagents in common, so that the effect of preventing and reducing carryover is obtained. is there.

In addition, as described in the first embodiment, various methods can be used for the method of controlling the material, temperature, and CO2 concentration of the prism 105 and the substrate 201.

(Overview of operation)
An actual analysis example when the combination of the pipetter mechanism 504 and the well substrate 601 is used will be described. Here, the analysis target is the same as in Example 1, except that RBL-2H3 cells, which are a rat basophil-derived cell line, are used as measurement samples, and DNP-HSA antigen is used as a reaction reagent to measure the response function to cell antigen stimulation. went. The reagent reservoir 501 was filled with a measurement sample, the reagent reservoir 502 was filled with a reaction reagent, and the reagent reservoir 503 was filled with a buffer. As the well component 602, a plate-like PDMS (longitudinal and lateral 19 × 19 mm, thickness 5 mm) with a through hole (φ4 mm) is used, and the upper surface (the thin gold film) of the substrate 201 provided with the thin gold film as in the first embodiment. The holder 603 was held so as to be in close contact with the existing surface) side.

In addition, since the same flowchart as the first embodiment can be used for the basic operation flowchart, only the parts different from the first embodiment will be described with reference to FIG. All operations are performed in accordance with instructions from the liquid feeding system control unit 120 and the optical system control unit 122 in the control PC 108 described in the first embodiment.

In step P1, the well 605 is filled with a buffer, and the incident angle is adjusted. The nozzle 505 moves to the reagent reservoir 503, sucks the buffer, moves to the well substrate 601, and discharges the buffer to the well 605. The amount of buffer used in this step is 10 μL. After discharging the buffer, the nozzle 505 moves to the waste liquid tank 513 and discharges a small amount of water to clean the inside. Thereafter, the nozzle 505 moves to the cleaning tank 512 and cleans the outside. After the above operation, the incident angle is adjusted by the method shown in the first embodiment. In the case where a plurality of wells 605 are present on the well substrate 601, the incident angle can be adjusted by filling the buffer only at one specific location and measuring the SPR reflected light intensity in that region. After the adjustment of the incident angle is completed, the nozzle 505 moves to the well substrate 601, sucks the buffer, and discharges it to the waste liquid tank 513. Thereafter, the cleaning operation is performed in the same procedure as before. In step P3, the measurement sample is immobilized. The reaction is performed by discharging the measurement sample to the well 605 and waiting as it is in the same procedure as in the process P1. The liquid volume of the measurement sample used in this step is 10 μL. While waiting, cells sink to the bottom of the well 605 by gravity. The gold thin film is exposed at the bottom of the well, and the cells adhere to the surface of the gold thin film due to their natural properties. In addition, after the measurement sample is aspirated and discharged into the well 605, another drug that initiates or accelerates the fixation reaction may be aspirated from another reservoir (not shown) and added to the same well 605 for discharge.

In the fixed amount determination in step P5, when the fixed amount is less than the target value, 5 μL of the measurement sample is sucked from the well 605 and discarded in the waste liquid tank 513, and then 5 μL of the measurement sample is sucked from the reagent reservoir 501. The liquid was discharged into the well 605.

When the fixed amount was not less than the target value, first, 5 μL of the measurement sample was sucked from the well 605 and discarded into the waste liquid tank 513. 10 μL of buffer was aspirated from the reagent reservoir 503 and discharged into the well 605, and 10 μL was aspirated from the well 605 and discarded into the waste liquid tank 513. This operation was repeated 5 times to remove unfixed cells from the solution in the well 605 to stop the fixing reaction. The number of repetitions of this operation may be determined in advance, or may be continued until the stop of the fixation reaction is confirmed by monitoring the progress of the fixation reaction using the optical system control unit 122 and the analysis unit 121. Good. Also in this case, a condition for stopping the repetition may be provided by setting a timeout or an upper limit number.

Response observation is performed in process P6. The procedure of response observation is as shown in Example 1. Regarding the method of adding the reaction reagent to the measurement sample, first, the reaction sample is sucked in the same procedure as in Step P1, and added to the well 605 in which the measurement sample is fixed and filled with the buffer. The amount of the reaction reagent used in this step is 10 μL. After adding the reaction reagent, the mixing of the reaction reagent may be promoted by performing a pipetting operation using the nozzle 505. Alternatively, the reaction reagent may be added after discarding the buffer in the well 605 with the nozzle 505.

FIG. 7 shows a configuration around the irradiation unit and the detection unit according to the third embodiment. Other configurations are the same as those of the first embodiment. The feature of the present embodiment is that the measurement sample is observed using evanescent light. A series of observation processes according to this example will be described by taking, as an example, measurement of the response function of RBL-2H3 cells, which are a cell line derived from rat basophils, in response to stimulation with DNP-HSA antigen as in Example 1. RBL-2H3 cells are labeled in advance with AlexaFluor 546 (Life Technologies), and DNP-HSA antigens are labeled with AlexaFluor 647 (Life Technologies) in advance.

(Explanation of irradiation unit / detection unit structure)
Excitation light emitted from two types of light sources 701 to 702 having different wavelengths is arranged coaxially by a dichroic mirror 703, and the wavelength is selected by a filter unit 704. Incident light illumination is formed on the surface of the substrate 201 by being incident under a reflecting condition.
In general, evanescent light is formed in a range of several hundred nm from the surface. Therefore, it is possible to irradiate only the measurement sample existing in the very vicinity of the surface of the substrate 201. Light generated from the measurement sample by the evanescent light illumination is collected by the objective lens 706 and divided by the dichroic mirror 707 for each wavelength. From the divided light, only necessary wavelength components are extracted by the band-pass filters 708 to 709, respectively, and then imaged on the image sensors 711 to 713 by the lenses 710 to 711.

The material of the substrate 201 used in this example is synthetic quartz, and the shape is a plate shape of 20 × 20 mm in length and width and 1.0 mm in thickness. There is no gold film on the surface. The material of the prism 105 used in this embodiment is the same synthetic quartz as that of the substrate 201, and the shape thereof is a triangular prism shape having a height of 20 mm with an equilateral triangle having a side of 20 mm as the bottom. The substrate 201 and the side surface (square surface) of the prism 105 are in optical contact with matching oil (not shown). In this embodiment, glycerol (refractive index: 1.47) is used as the matching oil. A flow cell similar to that of Example 1 is formed on the surface of the substrate 201, but is omitted from the drawing.

In this example, a YAG laser with a wavelength of 532 nm was used as the light source 701, and a He—Ne laser with a wavelength of 633 nm was used as the light source 702, respectively. The dichroic mirror 703 used has a wavelength characteristic that transmits a component having a wavelength of 580 nm or longer and reflects a wavelength component shorter than that. The filter unit 704 holds two types of switchable states: a bandpass filter exG that transmits only a laser with a wavelength of 532 nm and a bandpass filter exR that transmits only a laser with a wavelength of 633 nm. A dichroic mirror 707 having a wavelength characteristic that transmits a component having a wavelength of 630 nm or longer and reflects a wavelength component shorter than that is used. As the band pass filter 708, a filter that selectively extracts the fluorescence wavelength component of AlexaFluor 546 was used. As the band pass filter 709, a filter that selectively extracts the fluorescence wavelength component of AlexaFluor 647 was used. The image sensors 712 to 713 are the same as those in the first embodiment.

(Description of operation)
The operation of this embodiment will be described. The same basic operation flowchart as that shown in FIG. 3 can be used. All operations are performed in accordance with instructions from the liquid feeding system control unit 120 and the optical system control unit 122 in the control PC 108 described in the first embodiment.

The incident angle is adjusted in process P1. As in the first embodiment, the flow cell on the substrate 201 is filled with a buffer, and then irradiated with excitation light, and the incident angle is continuously changed. When the angle exceeds a certain angle, the excitation light is totally reflected at the interface between the substrate 201 and the buffer. At that time, the angle (critical angle) is set as the incident angle in the subsequent observation.

In step P2, the cell fixation amount at the time of observation is set. The setting method and operation are the same as those in the first embodiment.

In step P3, cells are fixed on the surface of the substrate 201. The fixing method and operation are the same as those in the first embodiment.

In step P4, the number of cells attached to the surface of the substrate 201 is monitored. The filter unit 704 is operated to switch to the band pass filter exG to perform fluorescence observation. Since RBL-2H3 cells are labeled with AlexaFluor 546, they are excited by evanescent light having a wavelength of 532 nm and emit fluorescence. Detection is performed by the image sensor 712. As described above, since evanescent light is limited to a range of several hundred nanometers from the surface of the substrate 201, only cells attached to the surface of the substrate 201 can be detected substantially.
In step P5, a fixed amount is determined. The determination method and operation are the same as those in the first embodiment.
In step P6, the response is observed. The addition method and operation of the reaction reagent containing the DNP-HSA antigen are the same as in Example 1. The filter unit 704 is operated to switch to the bandpass filter exR to perform fluorescence observation. Since RBL-2H3 cells are within the irradiation range of evanescent light, when labeled DNP-HSA antigen binds to RBL-2H3 cells, AlexaFluor 647, which is a labeled molecule, is excited by evanescent light having a wavelength of 633 nm and emits fluorescence. . Detection is performed by the image sensor 713.

In this example, AlexaFluor 546 and AlexaFluor 647 were used as fluorescent dyes for labeling, but these may be different fluorescent dyes. Examples of the fluorescent dye include Cy3 and Cy5. Also, fluorescent dyes having different wavelength characteristics can be used. In this case, the light sources 701 to 702 and various optical components are selected according to the wavelength characteristics of the fluorescent dye to be used. Moreover, you may use a quantum dot instead of a fluorescent pigment | dye. In addition, a labeled compound containing a radioisotope may be used.

In this embodiment, two types of light sources 701 to 702 having different wavelengths are used. However, a desired wavelength component may be extracted by a filter unit 704 from one light source that oscillates a plurality of wavelengths. The light source is, for example, a xenon lamp. In this case, since only one light source unit is required, there is an effect of cost reduction.

In this embodiment, the fluorescence separated for each wavelength by the dichroic mirror 707 is imaged on each of the two image sensors 712 to 713. For example, the transmitted fluorescence is connected to the left half of the light receiving element of the image sensor 713. The reflected and reflected fluorescence can be folded back by a total reflection mirror to form an image on the right half of the light receiving element of the image sensor 713. In this case, the image sensor 712 is unnecessary, and there is an effect of cost reduction. As another method of detecting two types of fluorescence with one image sensor 713, for example, a filter unit capable of switching the bandpass filters 708 to 709 without using the dichroic mirror 712 can be used. Further, it is also possible to perform wavelength dispersion using a diffraction grating or the like without using the dichroic mirror 712 and form an image on another region on the image sensor 713.

(Another form of fluorescence detection method)
Fluorescence resonance energy transfer (FRET) can be used. AlexaFluor 546, which labels RBL-2H3 cells, is used as a donor, and AlexaFluor 647, which labels DNP-HSA antigen, is used as an acceptor. In this method, since the donor and the acceptor can be detected by exciting the donor, the light source 702 and various optical components for exciting the acceptor are not necessary, and the cost can be reduced. FRET may be a combination of different fluorescent dyes, or quantum dots may be used.

(Another form of evanescent lighting)
FIG. 8 is a schematic diagram of another optical system other than the above. While FIG. 7 shows a prism type evanescent irradiation method, FIG. 8 adopts an objective evanescent irradiation method. Objective evanescent illumination is realized by converging the excitation light through the lens 705 and the multi-edge dichroic mirror 801 at the rear focal position of the objective lens 706. Other configurations are the same as those of the prism type evanescent irradiation method described above. In addition, although this system includes an optical axis adjustment mechanism of excitation light for adjusting the incident angle, it is omitted in the figure. This system is relatively easy to adjust the optical axis, etc., compared to the prism-type evanescent irradiation system. In addition, since the irradiation unit and the detection unit are partially overlapped, the optical system becomes compact, and there is an effect of reducing the size of the entire apparatus.

FIG. 9 shows a configuration around the irradiation unit and the detection unit according to the fourth embodiment. Other configurations are the same as those of the first embodiment. The feature of the present embodiment is that the measurement sample is observed using coaxial epi-illumination. In this embodiment, by using the objective lens 706 having a shallow depth of field, there is an effect that the observation can be limited to the measurement sample existing in the vicinity of the surface of the substrate 201. The depth of field is preferably 100 μm or less, more preferably 10 μm or less. The measurement method is the same as in Example 1. Although the upright arrangement is shown in FIG. 9, an inverted arrangement may be used. In addition, the light after passing through the multi-edge dichroic mirror 801 is once condensed by a lens, a pinhole is set at the focal position, and converted back to parallel light by the lens, so that a so-called confocal microscope structure is obtained. An effect can be obtained. In addition, as described in the third embodiment, various methods can be used for the type of excitation light source, the detection method, and the combination of components associated therewith.

FIG. 10 shows a configuration around the irradiation unit and the detection unit according to the fifth embodiment. Other configurations are the same as those of the first embodiment. The feature of this embodiment is that the measurement sample is observed using transmitted light illumination. In the present embodiment, similarly to the fourth embodiment, by using the objective lens 706 having a shallow depth of field, there is an effect that the observation can be limited to the measurement sample existing in the vicinity of the surface of the substrate 201. The depth of field is preferably 100 μm or less, more preferably 10 μm or less. The measurement method is the same as in Example 1. In FIG. 10, an inverted arrangement is used, but an upright arrangement may be used. Similarly to the fourth embodiment, the light before passing through the dichroic mirror 707 is once condensed by the lens, a pinhole is set at the focal position, and then returned to the parallel light by the lens again, so-called confocal microscope. It becomes a structure and the same effect can be acquired.

As described in the third embodiment, various methods can be used for other types of excitation light sources, detection methods, and combinations of components associated therewith.

In Examples 1 to 5, the measurement sample was observed using optical means, but the observation method is not limited to optical means. For example, a surface acoustic wave sensor or observation with an electric signal can be used. A micro electrode array is used as an observation substrate using electrical signals. Examples of observation using such a substrate are disclosed in non-patent literature (Anal. Chem. 2011, 83, 571-577). More specifically, voltammetry, amperometry, measurement of impedance and capacitance, and the like may be used. Furthermore, by measuring the change of each particle of the measurement sample one by one, and further, the localization of the change inside one particle by measuring the pitch between the electrodes sufficiently small with respect to the particulate measurement sample, It becomes possible to observe. For example, for a cell having a diameter of about 10 μm, the state of each cell can be measured by using an electrode array in which the pitch between the electrodes is 30 μm or less, more preferably 10 μm or less, and even more preferably 5 μm or less. Similarly, by using an electrode array having an interelectrode pitch of 3 μm or less, more preferably 1 μm or less, and even more preferably 0.5 μm or less, the localization of the intracellular state can be observed. According to the method of this embodiment, since the presence or absence of the measurement sample on the substrate surface can be observed, the fixed amount can be evaluated. Further, according to the method of this embodiment, it is possible to observe the change in the state of the measurement sample on the substrate surface, so that the response to the stimulus can be evaluated. According to the system of the present embodiment, it is possible to observe the response of the measurement sample without using optical means. Moreover, you may combine the observation by this system and the above-mentioned optical observation.

As mentioned above, although the example of this invention was demonstrated, this invention is not limited to this, It is understood by those skilled in the art that various changes are possible in the range of the invention described in the claim. The It is also within the scope of the present invention to appropriately combine the embodiments.

DESCRIPTION OF SYMBOLS 101 Analysis apparatus 102 Irradiation part 103 Detection part 104 Flow cell 105 Prism 106 Liquid sending part 107 Temperature control apparatus 108 Control PC
109 Monitor 110 CO 2 Gas Cylinder 111 Light Source 112 Optical Fiber 113 Condensing Lens 114 Polarizing Element 115 Imaging Lens 116 Image Sensor 117 Excitation Light 118 Measurement Area 119 Reflected Light 120 Liquid Supply System Control Unit 121 Analysis Unit 122 Optical System Control Units 123 and 124 , 125 Reagent reservoir 126, 127 Liquid feed tube 128, 129 Switching valve 130 Liquid feed pump 131 Waste liquid tank 201 Substrate 202 Flow path component 203 Holder 204 Groove 205, 206 Vertical flow path 207, 208, 209, 210, 212 Opening 211 CO2 gas piping P1 Device preparation P2 Fixed amount target value setting P3 Measurement sample fixed reaction P4 Fixed amount measurement P5 Fixed amount determination P6 Response observation 501, 502, 503 Reagent observation 504 Pipetter mechanism 505 Nozzle 506 Robot arm 507 Pipe 508 Syringe pump 509 Liquid feed pump 510 Switching valve 511 Water tank 512 Cleaning tank 513 Waste liquid tank 601 Well substrate 602 Well part 603 Holder 604 Liquid holding site 605 Well 606, 608 Opening 607 Septa 701, 702 Light source 703 Dichroic mirror 704 Filter unit 705 Lens 706 Objective lens 707 Dichroic mirrors 708 and 709 Band pass filters 710 and 711 Lenses 712 and 713 Image sensor 801 Multi-edge dichroic mirror

Claims (23)

1. A substrate and a liquid feeding means for providing a measurement sample and a reaction reagent to the substrate;
   Observation means for observing the measurement sample on the substrate surface;
   Based on the observation results, analysis means for analyzing information on the amount of the measurement sample fixed on the substrate;
   Based on the information, control means for adjusting the amount by which the measurement sample is fixed on the substrate;
   An interaction analysis apparatus characterized by comprising:
2. The interaction analysis apparatus according to claim 1,
   The apparatus is characterized in that the information is information relating to the number of particles or the particle density of the measurement sample fixed to the substrate.
3. The interaction analysis apparatus according to claim 1,
   The control means adjusts the amount by which the measurement sample is fixed on the substrate by stopping the step of providing the measurement sample.
4. The interaction analysis apparatus according to claim 1,
   The apparatus characterized in that the control means adjusts the amount by which the measurement sample is fixed on the substrate by controlling the number of repetitions of the step of providing the measurement sample.
5. The interaction analysis apparatus according to claim 1,
   The control means adjusts the amount by which the measurement sample is fixed on the substrate by controlling the change of the concentration of the measurement sample in the fluid containing the measurement sample.
6. The interaction analysis apparatus according to claim 1,
   The observation device observes the binding reaction of the measurement sample with respect to the substrate.
7. The interaction analysis apparatus according to claim 1,
   The observation means observes a response reaction of the measurement sample bound to the substrate to the reaction reagent.
8. The interaction analysis apparatus according to claim 1,
   The observation means observes a binding reaction of the measurement sample to the substrate and observes a response reaction of the measurement sample bound to the substrate to the reaction reagent.
9. The interaction analysis apparatus according to claim 1,
   Based on the information, the analysis means
   An apparatus comprising an algorithm for instructing the control means to perform the next operation.
10. The interaction analysis apparatus according to claim 1,
    The control means controls the operation of one or both of the liquid feeding means and the observation means.
11. The interaction analysis apparatus according to claim 1,
    The measurement sample is a molecule, a biological sample, or a cell.
12. The interaction analysis apparatus according to claim 1,
    The apparatus, wherein the observation means includes a light source for irradiating the substrate with the first light and a detector for measuring the second light generated as a result of the irradiation.
13. The interaction analysis apparatus according to claim 12,
    The apparatus wherein the second light is reflected light of the first light.
14. The interaction analysis apparatus according to claim 12,
    An apparatus for measuring the intensity of reflected light in a surface plasmon resonance phenomenon.
15. The interaction analysis apparatus according to claim 12,
    An apparatus for measuring a change in resonance angle in a surface plasmon resonance phenomenon.
16. The interaction analysis apparatus according to claim 12,
    The apparatus according to claim 1, wherein the first light irradiation method is evanescent light illumination, epi-illumination, or transmitted light illumination.
17. A substrate and liquid feeding means for providing a measurement sample to the substrate;
    Observation means for observing the measurement sample on the substrate surface;
    Control means for controlling the liquid feeding means to stop providing the measurement sample when the amount of the measurement sample fixed to the substrate is not less than a predetermined value set in advance;
    An interaction analysis apparatus characterized by comprising:
18. In an interaction analysis method in which a reaction reagent is brought into contact with a measurement sample and the response of the measurement sample is observed,
    Providing the measurement sample on the substrate surface;
    Measuring information about the amount of the measurement sample fixed to the substrate surface;
    Based on the measurement results, the amount of the measurement sample fixed to the substrate is adjusted.
    A method characterized by that.
19. The method of claim 18, wherein
    The method is characterized in that the information is the number of particles or the particle density of the measurement sample fixed to the substrate.
20. The method of claim 18, wherein
    In the adjustment, the method of adjusting the amount of the measurement sample fixed to the substrate by stopping the step of providing the measurement sample.
21. The method of claim 18, wherein
    In the adjustment, the amount of the measurement sample fixed to the substrate is adjusted by adjusting the number of repetitions of the step of providing the measurement sample.
22. The method of claim 18, wherein
    In the adjustment, the amount of the measurement sample fixed to the substrate is adjusted by changing the concentration of the measurement sample of the fluid including the measurement sample.
23. The method of claim 18, wherein
    The method, wherein the measurement sample is a molecule, a biological sample, or a cell.

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