JP2011107069A - Surface plasmon resonance sensing system and surface plasmon resonance in-line measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a surface plasmon measuring technology to be incorporated in a process line and capable of performing the specific high-sensitivity in-line measurement of a particular substance in fluid having passed a preceding process. <P>SOLUTION: A measuring method includes steps of fixing a probe specifically combinable with the particular substance on a sensor surface of a surface plasmon measuring instrument 4; installing the surface plasmon measuring instrument 4 in the process line to continuously introduce the fluid having passed the preceding process to the sensor surface of the surface plasmon measuring instrument 4; and combining the particular substance in the fluid having passed the process with the probe to allow the specific in-line measurement of an amount of the particular substance. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a sensing system for a specific substance in a process line and an in-line measurement method using a measuring instrument (hereinafter also referred to as “SPR measuring instrument”) using surface plasmon resonance (SPR).

  There is a case where a high correlation is observed between diseases in the human body and mutations of proteins constituting the human body. For example, in cancer, influenza and other diseases, it is known that specific proteins increase in the body (such as in blood) as the disease progresses.

  Therefore, knowledge about the morbidity and progression of the disease can be obtained by examining the state of the specific protein (the presence or absence, amount, etc. of the specific protein). For example, biomolecules that increase with the progression of a tumor (cancer) are called tumor markers, and different tumor markers are specified depending on the tumor site.

  Surface plasmon resonance is used as a method for easily and accurately measuring the presence and amount of a specific protein including the tumor marker as described above. Surface plasmon resonance (SPR) is a resonance phenomenon caused by the interaction between free electrons on a metal surface and electromagnetic waves (light).

  FIG. 23 shows a biosensor for detecting a specific substance (for example, including a protein, a low-molecular compound such as a sugar, a lipid, an environmental hormone or a physiologically active substance, or a microorganism such as a pathogen) using surface plasmon resonance. The conceptual diagram of is shown.

  Here, consider a case where incident light enters the metal layer 100 from the lower left direction in FIG. 23A and is reflected in the lower right direction. Metal free electrons 101 exist in the metal layer 100 of FIG. Then, an electric field depending on the wavelength and incident angle of incident light acts on the metal free electrons 101, and the metal free electrons 101 vibrate. Then, the motion of the metal free electrons 101 and the electric field of the incident light resonate at a specific wavelength and incident angle, and the energy of the incident light transitions to the vibration motion of the metal free electrons 101. As a result, FIG. As shown by the solid line, the reflected light intensity at this wavelength decreases.

  In the resonance phenomenon between the movement of the metal free electrons 101 and light, an electric field 102 is generated outside the metal layer 100 in a range of about several hundred nm. A biosensor using surface plasmon resonance utilizes the fact that the resonance frequency of surface plasmon resonance changes due to the interaction between the electric field 102 and a specific substance (hereinafter also referred to as target) 103 which is a detection target. ing.

In order to specifically detect the detection target 103, first, the probe 104 that specifically binds to the target is immobilized on the measurement region. Thereafter, a sample containing the target is fed at a predetermined speed to flow the target 103. Then, the target 103 specifically binds to the probe 104 fixed on the surface of the metal layer 100, and the refractive index near the metal layer 100 changes. Then, due to the change in the refractive index in the vicinity of the metal layer 100 due to the coupling of the target 103, the resonance frequency or the resonance incident angle of the surface plasmon resonance changes as indicated by a broken line in FIG. That is, the target 10 is detected by detecting the movement of the wavelength (or the same incident angle of the incident light) at which the reflectance decreases as shown in FIG.
3 can be detected.

  Such sensors using surface plasmon resonance include a propagation type surface plasmon resonance sensor and a localized type surface plasmon resonance sensor. The principle of the propagation surface plasmon resonance sensor will be briefly described with reference to FIG. The propagation type surface plasmon resonance sensor 110 is formed by forming a metal film 112 of Au, Ag or the like having a thickness of about 50 nm on the surface of a glass prism 111 as shown in FIG. The propagation surface plasmon resonance sensor 110 irradiates light from the glass prism 111 side and totally reflects light at the interface between the glass prism 111 and the metal film 112. A target biomolecule is sensed by receiving the totally reflected light and measuring the reflectance of the light.

  That is, when the reflectance is measured while changing the incident angle θ of the light, the reflectance is greatly attenuated at a certain incident angle (resonance incident angle) as shown in FIG. When light incident on the interface between the glass prism 111 and the metal film 112 is totally reflected at the interface, as shown in FIGS. 24B and 24C, evanescent light (near-field light) generated at the interface is shown. And surface plasmon waves of the metal film 112 interact with each other. At a specific wavelength and a specific incident angle, the wave number of the incident light and the vibration of free electrons resonate so that the energy of the incident light is absorbed into the metal film 112 and the vibration of the free electrons in the metal film 112. Change to energy. As a result, the light reflectance is significantly reduced.

  Next, a localized surface plasmon resonance sensor using a nanogap structure will be described with reference to FIG. In the localized surface plasmon resonance sensor, free electrons in a plurality of groove-like metal nanogroove structures 120 as shown in FIG. 25A are vibrated by an alternating electric field of light (electromagnetic waves), and incident light is incident at a certain frequency. The free electrons resonate, the free electrons absorb light energy, and the absorbance reaches a peak at the resonance wavelength. Then, free electron vibrations generated by surface plasmon resonance are concentrated in a local region of a plurality of groove-shaped metal nanogroove structures 120 as shown in FIG. Therefore, the sensing region is very small compared to the propagation type surface plasmon resonance sensor, such as several tens of nm.

  Since the localized surface plasmon resonance sensor is also affected by the dielectric constant (refractive index) around the metal nanogroove structure 120, any dielectric that is the target 122 on the probe 121 previously dispersed in the metal nanogroove structure 120. When a substance (eg, antigen) is attached, the absorbance changes. For example, the presence / absence and amount of adhesion of the dielectric material can be detected by reading the wavelength at the peak value of the absorbance before and after the dielectric material as the target 122 adheres to the metal nanogroove structure 120.

  The SPR measuring instrument is used for measuring an antigen-antibody reaction, for example, by applying the above principle. As an example of the measurement method, when the analyte is an antigen present as a solute in the buffer solution and the antibody is immobilized on the detection surface of the sensor, the buffer solution not containing the antigen is first removed from the sensor cell (buffer solution or sample). A fluid such as a test solution is allowed to flow in contact with the detection surface, and the resonance angle of the surface plasmon resonance is measured. Next, after a buffer solution containing the antigen of the subject is flowed to the sensor cell to cause an antigen-antibody reaction, the sensor cell is washed with a buffer solution not containing the antigen, and the resonance angle of surface plasmon resonance is measured again. Since the difference in the surface plasmon resonance angle before and after the antigen-antibody reaction thus obtained corresponds to the number of antigens reacted with the antibody, this is converted to the concentration of the antigen present as a solute.

By the way, chromatography is a technique for purifying a desired substance by utilizing a difference in electrical properties or molecular sizes of low to high molecular substances, and is currently widely used mainly for purification of physiologically active substances. . At that time, 260 nm or 280 nm ultraviolet light (hereinafter referred to as UV) absorptiometers are most widely used for tracking biological substances, and the elution positions of various substances including specific substances are identified by a chart of UV absorption. can do.

  FIG. 26 shows a sensing system that combines chromatography and the above UV absorbance meter. FIG. 26A is an overall view of the sensing system, FIG. 26B is a diagram showing a measurement principle in the UV absorptiometer, and FIG. 26C is a diagram showing an example of a measurement result by the UV absorptiometer.

  As shown in FIG. 26A, in this system, a sample in which a measurement target (hereinafter also referred to as a target) and a noise component are mixed is introduced into a chromatography column 131. Then, the target and the contaminant are separated in the column based on the difference in molecular weight and the difference in affinity with the column. Then, the target and impurities are introduced into the UV absorbance meter 132 with a time difference. In the UV absorptiometer 132, the sample is irradiated with UV as shown in FIG. 26B, and light in a proportion corresponding to the amount of protein contained in the sample is absorbed. As shown in FIG. 26C, when the horizontal axis is the sample passage time and the vertical axis is the absorbance, a peak corresponding to the amount is observed when the target or impurities pass through. The content of the target is measured based on the presence or absence of this peak. In the example shown in FIG. 26, the sample discharged from the UV absorbance meter is introduced into the conductivity meter 133, and the conductivity is measured just in case.

JP 2008-216055 A Japanese Patent Laid-Open No. 11-183372 Special table 2003-504638 gazette JP-T 9-503064

Takayuki Okamoto, “Investigative research on metal nanoparticle interactions and biosensors” (Fig. 2), Plasmonic Study Group, Grant-in-Aid for Scientific Research (Fundamental Research C), Research Report, http: // www .plasmon.jp / index.html N. Felidj et al. "Controlling the optical response of regular arrays of gold particles for surface-enhanced Raman scattering", Phys. Rev. B 65, 075419 (2002)

  In the above system, the target and the contaminant are separated by the column 131, but the detection sensitivity is low only by the information from the UV absorbance meter 132 and the conductivity meter 133, and only the target is specifically detected. It is difficult to obtain only results as shown in FIG. Therefore, it may be difficult to determine at which timing the data corresponds to the target. Therefore, it is necessary to fractionate the sample every time and to analyze the contents for each fraction.

  Moreover, although the UV absorbance changes depending on the amount of protein, there is no specificity for the type of protein. Furthermore, since the sensitivity is generally as low as about 10 μg / ml, it may be difficult to detect a target containing a very small amount.

  The present invention has been made in view of such technical problems, and the object of the present invention is to incorporate a specific substance in a fluid that has been incorporated into a process line and passed through a previous process, The object is to provide a surface plasmon measurement technique that can be measured in-line with high sensitivity.

  In the present invention, a probe that can specifically bind to a specific substance is fixed to the sensor surface of a surface plasmon measuring device, and the surface plasmon measuring device is provided in the process line, so that the fluid that has passed through the previous process Is continuously introduced on the sensor surface of the surface plasmon measuring instrument, and the specific substance in the fluid that has passed through the process is combined with the probe, so that the amount of the specific substance can be measured specifically and precisely in-line. The biggest feature is that it is possible.

More specifically, surface plasmon resonance can be generated on the surface of the sensor by irradiating the surface plasmon excitation light onto the sensor, and the surface plasmon resonance can be generated outside the surface of the sensor depending on the wavelength or incident angle of the excitation light. A surface plasmon measuring instrument provided in the process line, capable of measuring the amount of the substance present,
Fluid introduction means for continuously introducing fluid that has passed through a process prior to the surface plasmon measuring instrument in the process line onto the sensor surface of the surface plasmon measuring instrument;
With
A probe that is a substance that can specifically bind to a specific substance in the fluid is fixed to the surface of the sensor,
The amount of the specific substance can be measured in-line by combining the specific substance in the fluid that has passed through the process with the probe.

  According to this, it is possible to measure the concentration within the process line for only the substance that specifically binds to the probe. As a result, it is possible to measure the concentration of a specific substance with higher accuracy without measuring the concentration of impurities together. In addition, since measurement is performed within the process line, measurement can be performed more easily or more quickly.

Further, in the present invention, further comprising dissociation means for performing a dissociation operation for dissociating a bond between the probe on the surface of the sensor and the specific substance,
Measurement of the amount of the specific substance bound to the probe by the surface plasmon measuring instrument and dissociation of the specific substance bound to the probe by the dissociation means were alternately performed to bind to the probe. You may make it perform the measurement of several times continuously about the quantity of a specific substance.

  In general, in the surface plasmon measuring instrument, if all of the probes fixed on the sensor surface are combined with the target, the change in refractive index is saturated, and further measurement is impossible. On the other hand, in the present invention, by performing the dissociation operation, the probe and the target (specific substance) are dissociated in the sensor of the surface plasmon measuring device. This makes it possible to measure the concentration of a specific substance in a process line with high accuracy and continuously and specifically. In the present invention, when one measurement is completed, the dissociation operation may be performed at least twice. Then, the probe and the target can be dissociated more reliably, and the sensor surface can be regenerated. In that case, every dissociation operation of each of a plurality of measurements may be performed twice or more, or only when a part of the measurements are completed, dissociation operation is performed twice or more. May be performed.

  In the present invention, the dissociation operation may be an injection into the fluid of a dissociator that dissociates the bond between the probe and the specific substance on the surface of the sensor.

  This makes it easier to dissociate by simply injecting the dissociator into the fluid because the dissociator is introduced into the sensor surface and reaches the probe using the fluid flow that passed through the previous process. It is possible to complete the operation.

In the present invention, the fluid introduction means is a flow path for circulating the fluid that has passed through the process prior to the surface plasmon measuring instrument,
The surface of the sensor of the surface plasmon measuring device is arranged to face the fluid in the flow path,
The injection port for injecting the dissociating agent into the fluid is provided in the flow path substantially in the same plane as the surface of the sensor and on the upstream side of the surface of the sensor in the flow path. You may be allowed to.

  According to this, the dissociator injected from the inlet can be directly reached the sensor surface in a laminar flow state before diffusing into the fluid. Therefore, the probe and the target can be efficiently dissociated with a higher concentration. Moreover, according to this, since the usage-amount of a dissociator can be made smaller, the influence about the component of the fluid in a process line can be made smaller.

  In the present invention, a first measurement region in which a probe that binds to the specific substance is fixed and a second measurement region in which a probe that binds to the specific substance is not fixed are provided on the surface of the sensor. The amount of the specific substance may be measured by subtracting the measurement value measured in the second measurement region from the measurement value measured in the first measurement region.

  That is, in this invention, it has a some measurement area | region in the sensor surface of a surface plasmon measuring device. In the first measurement region, a probe that binds to a specific substance to be measured is fixed. In the other second measurement region, the above-described probe is not fixed. Here, consider a case where impurities other than the specific substance to be measured are mixed in the fluid. In this case, the data measured in the first measurement area measures the amount of specific substances and contaminants, and the data measured in the second measurement area measures the amount of contaminants. It is thought that there is. Therefore, by subtracting the measurement value measured in the second measurement region from the measurement value measured in the first measurement region, it is possible to obtain a measurement value only with a specific substance from which the influence of impurities is eliminated. It becomes possible.

  Therefore, according to the present invention, even if impurities other than the specific substance to be measured exist in the fluid that has passed through the previous process, the amount of the specific substance can be measured more accurately. Is possible. In this case, a substance having an optical property similar to that of the probe fixed to the first measurement region may be fixed to the second measurement region. By doing so, conditions other than the specific substance in the first measurement region and the second measurement region can be more approximated, so that the amount of the specific substance can be measured with higher accuracy.

In the present invention, the sensor of the surface plasmon measuring instrument is
A metal layer formed so as to cover at least a part of the surface of the sensor, and a groove structure including a plurality of grooves formed in a portion of the surface of the sensor where the metal layer is formed, A side wall surface of each groove in the groove structure forms a facing surface by the metal layer covering the side wall surface, and generates a localized surface plasmon resonance in a region sandwiched by the facing surface by the metal layer It may be a surface plasmon resonance sensor.

When a localized surface plasmon resonance sensor is selected as a sensor for a surface plasmon measuring instrument, the area covered by the electric field generated from the sensor surface is limited compared to when a propagation type surface plasmon resonance sensor is selected. There is a feature that. Therefore, when a localized surface plasmon resonance sensor is selected as the sensor of the surface plasmon measuring device as in the present invention, the region of the generated electric field can be limited to the region where the probe and the target exist, Can be reduced. As a result, it is possible to reduce the influence of contaminants with a simpler configuration and method without taking measures such as correcting the measured value obtained at the sensing unit with the measured value obtained at the reference unit. It becomes.

  When a propagation type surface plasmon resonance sensor is selected as the sensor of the surface plasmon measuring instrument, a prism is often bonded to the surface of the sensor opposite to the metal film. If it does so, it will become difficult to form the injection port of a dissociator in sensor itself. On the other hand, if a localized surface plasmon resonance sensor having a nano-groove structure is selected as the sensor of the surface plasmon measuring device as in the present invention, it is not necessary to attach a prism to the sensor itself. Then, it is easy to process the inlet of the dissociating agent in the sensor itself, and it is possible to realize a sensor that can perform the dissociation operation more easily and efficiently. In addition, the inlet can be formed at a location closer to the nano-groove structure where surface plasmon resonance actually occurs in the sensor, and the dissociation operation can be performed more efficiently.

  In the present invention, a localized surface plasmon resonance sensor is selected as the sensor, and the inlet is located within 2 mm upstream of the groove structure on the sensor surface in the flow path. It may be provided. By doing so, it is possible to cause the dissociator in a high concentration laminar flow state to reach the sensor surface more reliably. As a result, the dissociation operation can be performed more efficiently. Further, the injection port has a circular cross section, and the diameter thereof is not less than the length in the width direction of the channel in the region where the probe is fixed on the surface of the sensor, and not more than the channel width. It may be. In this way, a highly concentrated laminar flow dissociator can reach the entire sensor surface more reliably.

  In the present invention, the dissociation operation by the dissociation means is based on a signal corresponding to the amount of the specific substance from the surface plasmon measuring device, and the magnitude of the signal exceeds a predetermined threshold value. It may be performed after a certain period of time.

  That is, when the signal from the surface plasmon measuring instrument is small, it can be said that there is almost no target in the fluid from the previous process. In such a state, it is not necessary to perform a dissociation operation. On the other hand, when the signal from the surface plasmon measuring device exceeds a predetermined threshold value, it indicates that the target starts to flow, and there is a possibility that the probe fixed on the sensor surface will be saturated with the target in the near future.

  On the other hand, in the present invention, when the signal from the surface plasmon measuring instrument exceeds a predetermined threshold value, the dissociation operation is performed after a predetermined time has passed. According to this, in the situation where the dissociation operation is not necessary, the dissociation operation is not performed, and the dissociation operation can be performed when it is really necessary. Therefore, waste of the dissociating agent can be suppressed, and unnecessary dissociation of the dissociating agent in the fluid in the process line can be suppressed. In the present invention, the predetermined threshold is a threshold at which the target starts to flow when the signal of the surface plasmon measuring instrument exceeds this, and it is considered that a dissociation operation is necessary. It is determined based on the result. Moreover, if the signal of the surface plasmon measuring instrument exceeds the threshold value and the dissociation operation is performed after this time has elapsed, the probe is saturated with the target and further measurement becomes difficult. This is the time during which it is difficult for the state to occur, and this is also determined in advance by experiments or the like.

Further, in the present invention, a valve is provided on the downstream side of the sensor in the flow path, and controls whether the fluid is recovered or discarded as waste liquid,
Based on a signal corresponding to the amount of the specific substance from the surface plasmon measuring instrument, the valve is operated after the signal exceeds a predetermined threshold until the dissociation operation by the dissociating means is performed. May be operated to recover a solution containing the product.

  Here, in the process line, the specific substance (target) to be measured should not be consumed as much as possible in the measurement. That is, after the measurement by the surface plasmon resonance sensor is finished, it should be collected and sent to a later process. However, the fluid after measurement by the surface plasmon resonance sensor may contain a dissociator. This dissociator should not be collected as much as possible, but is preferably discarded as a waste liquid.

  Therefore, the present invention has a valve that controls whether the fluid after measurement is recovered or discarded, and the valve is operated based on a signal from the surface plasmon resonance sensor. More specifically, after the signal from the surface plasmon resonance sensor exceeds a predetermined threshold and before the dissociation operation by the dissociating means is performed, the valve is operated to collect the fluid. It was decided.

  This is because when the signal from the surface plasmon resonance sensor is large to some extent, a certain amount of time has passed since the last dissociation operation, and there is a risk that the dissociation agent is mixed in the fluid. Therefore, in such a state, the fluid after the measurement by the surface plasmon resonance sensor is finished is collected. In other periods, since there is a high risk that the dissociation agent is mixed in the fluid after the measurement by the surface plasmon resonance sensor, this was discarded as waste liquid.

  According to this, it is possible to distinguish between a state in which the dissociating agent is not mixed in the fluid and a state in which the dissociating agent is mixed in the fluid with simpler control and with high accuracy. Waste can be reduced and mixing of the dissociator into the fluid can be more reliably suppressed. In the present invention, the predetermined threshold is that when a signal from the surface plasmon measuring instrument exceeds this, a specific substance as a target is bound to a probe on the sensor surface to some extent, The dissociator injected in the dissociation operation is a value of a signal that is determined to be no longer discarded, and is determined in advance by experiments or the like.

  In the present invention, the sensor further includes a flow sensor for detecting a flow rate of the fluid flowing through the flow path, and a signal corresponding to the amount of the specific substance from the surface plasmon measuring device and the flow sensor. The timing for collecting the fluid may be controlled based on the detected flow rate of the fluid. According to this, it is possible to determine whether or not the dissociator injected in the previous dissociation operation is no longer discarded in consideration of the flow rate of the fluid, and more accurately determine when to start recovery. be able to.

Further, in the present invention, the fluid introducing means is a flow path that circulates and circulates the fluid that has passed a predetermined process on the process line from the process line,
An opening / closing valve for flow rate control may be provided between the branch point of the flow path and the surface plasmon measuring instrument.

  According to this, the measurement of the amount of a specific substance can be performed independently of the control of the process line, and the degree of freedom of both the control of the process line and the measurement by the surface plasmon measuring instrument can be increased. .

  In this case, the surface plasmon measuring device may monitor the amount of the specific substance in the fluid at regular intervals by controlling the open / close valve at regular intervals. According to this, since it is sufficient to introduce a certain amount of fluid to the sensor surface at regular intervals, the amount of target used for measurement can be reduced and waste can be eliminated.

In the present invention, the process before the surface plasmon measuring instrument in the process line is purification chromatography, and the purified extraction state of the fluid that has passed through the purification chromatography is monitored in real time by the surface plasmon measuring instrument. It may be. Alternatively, the process before the surface plasmon measuring instrument in the process line is a cell culture tank, and the substance generated in the cell culture tank in the fluid is monitored in real time by the surface plasmon measuring instrument. Good.

Further, according to the present invention, surface plasmon resonance can be generated on the surface of the sensor by irradiating the surface plasmon excitation light to the sensor, and the surface of the sensor can be changed depending on the wavelength or incident angle of the excitation light. A surface plasmon resonance in-line measurement method using a surface plasmon measuring device capable of measuring the amount of a substance existing outside,
Introducing a fluid that has passed through a process prior to the surface plasmon meter in the process line onto a sensor surface of the surface plasmon meter;
By binding a specific substance in the fluid that has passed through the process to a probe that is a substance that can specifically bind to the specific substance immobilized on the surface of the sensor, the amount of the specific substance is in-line. Measuring process to measure in,
It may be a surface plasmon resonance in-line measurement method characterized by comprising:

  In that case, in this invention, you may make it further have a dissociation process which performs dissociation operation | movement which dissociates the coupling | bonding of the probe and the said specific substance in the surface of the said sensor.

  In the present invention, the dissociation operation may be an injection of a dissociating agent that dissociates a bond between the probe and the specific substance on the surface of the sensor into the fluid.

  In the present invention, the dissociation agent is injected into the fluid in substantially the same plane as the sensor surface in the flow path, and upstream of the sensor surface in the flow path. You may make it carry out from the inlet provided in.

  In the present invention, on the surface of the sensor, by subtracting the measured value when the probe that binds to the specific substance is not fixed, from the measured value when the probe that binds to the specific substance is fixed, You may make it measure the quantity of the said specific substance.

In the present invention, the sensor of the surface plasmon measuring instrument is
A metal layer formed so as to cover at least a part of the surface of the sensor, and a groove structure including a plurality of grooves formed in a portion of the surface of the sensor where the metal layer is formed, A side wall surface of each groove in the groove structure forms a facing surface by the metal layer covering the side wall surface, and generates a localized surface plasmon resonance in a region sandwiched by the facing surface by the metal layer It may be a surface plasmon resonance sensor.

  In the present invention, the dissociation operation in the dissociation step is constant after the amount of the specific substance exceeds a predetermined threshold based on the amount of the specific substance measured by the surface plasmon measuring instrument. It may be performed after time.

  Further, in the present invention, based on the amount of the specific substance measured by the surface plasmon measuring device, the dissociation operation by the dissociation step is performed after the amount of the specific substance exceeds a predetermined threshold. You may make it collect | recover the said fluid until it is performed.

In the present invention, the timing for collecting the fluid may be determined based on the amount of the specific substance measured by the surface plasmon measuring instrument and the flow rate of the fluid.

Further, in the present invention, in the fluid introduction step, the fluid that has passed a predetermined process on the process line is branched from the process line and introduced onto the sensor surface of the surface plasmon measuring instrument,
The surface plasmon measuring device may monitor the amount of the specific substance in the fluid at regular intervals.

In the present invention, the process before the surface plasmon measuring instrument in the process line is purification chromatography,
The purified extraction state of the fluid that has passed through the purification chromatography may be monitored in real time by the surface plasmon meter.

  In the present invention, the process before the surface plasmon measuring instrument in the process line is a cell culture process, and the substance generated in the cell culturing process in the fluid is monitored in real time by the surface plasmon measuring instrument. You may make it do.

  Note that means for solving the above-described problems can be used in combination as much as possible.

  According to the present invention, in a process line, the amount of a specific substance in a fluid flowing between processes can be specifically and highly sensitively measured in-line.

It is a figure shown about schematic structure of the sensing system concerning Example 1 of the present invention. It is a figure which shows the structural example of the fraction sample which concerns on Example 1 of this invention. It is a graph which shows the measurement result of the light absorbency by the UV absorptiometer in Example 1 of this invention. It is a graph which shows the measurement result by the SPR measuring device which concerns on Example 1 of this invention. It is a conceptual diagram for demonstrating the countermeasure of the background noise by the foreign material which concerns on Example 1 of this invention. It is a graph which shows the signal obtained in each sensing part at the time of performing reference amendment concerning Example 1 of the present invention, and its difference. It is a figure which shows schematic structure of the sensor chip which concerns on Example 2 of this invention. It is a graph which shows the measurement result by the SPR measuring device using the localized surface plasmon resonance which concerns on Example 2 of this invention. It is the graph which put together the measurement result by the SPR measuring device using the localized surface plasmon resonance which concerns on Example 2 of this invention. It is a figure which shows schematic structure of the SPR measuring device which concerns on Example 3 of this invention. It is a figure which compares the local type sensor chip which concerns on Example 3 of this invention, and a propagation type sensor chip. It is explanatory drawing which compared the dissociator injection method which concerns on Example 3 of this invention, and the normal dissociator injection method. It is the graph which compared the relationship between the injection amount of ethanol and the signal variation | change_quantity in the dissociator injection method which concerns on Example 3 of this invention, and the normal dissociator injection method. It is a figure which shows schematic structure of the sensing system containing the SPR measuring device which concerns on Example 4 of this invention. It is a graph of the SPR signal obtained by the SPM measuring device in the sensing system according to Example 4 of the present invention. It is a flowchart about the SPR measurement routine performed in the control computer of the sensing system which concerns on Example 4 of this invention. It is a figure which shows the outline | summary of the control performed as a result of performing SPR measurement routine in the sensing system which concerns on Example 4 of this invention. It is a figure shown about the collection | recovery timing of the target in Example 4 of this invention. It is the schematic which shows the example of the process line which concerns on Example 5 of this invention. It is the schematic which shows another aspect of the example of the process line which concerns on Example 5 of this invention. It is the schematic which shows the 3rd aspect of the example of the process line which concerns on Example 5 of this invention. It is a figure which shows the distribution | circulation timing of the buffer solution in the 3rd aspect of the example of the process line which concerns on Example 5 of this invention, a sample, and a dissociator. It is a figure explaining the principle of the biosensor using generation | occurrence | production of surface plasmon resonance. It is a figure explaining the origin of propagation type surface plasmon resonance. It is a figure explaining the generation | occurrence | production origin of localized surface plasmon resonance. It is a figure for demonstrating the measuring apparatus by the combination of the conventional chromatography, UV absorption meter, and conductivity meter.

  DETAILED DESCRIPTION Exemplary embodiments for carrying out the present invention will be described in detail below with reference to the drawings.

<Example 1>
FIG. 1 shows a schematic configuration of a sensing system 1 used in this embodiment. The sample is introduced from column 2 of purification chromatography (hereinafter also simply referred to as chromatography). The column 2 is connected to the UV absorbance meter 3, and the sample after passing through the column 2 is introduced into the UV absorbance meter 3 and the absorbance is measured. The sample that has passed through the UV absorbance meter 3 is introduced into the SPR measuring device 4. In addition, the pipe | tube which connects the UV absorptiometer 3 and the SPR measuring device 4, and distribute | circulates a sample is equivalent to a fluid introduction means in a present Example. Moreover, the process in which the sample after passing through the column 2 is introduced into the UV absorbance meter 3 corresponds to a fluid introduction process in this embodiment. In the SPR measuring device 4, the sample is measured by an SPR sensor (not shown), and the measurement result is sent to the control computer 7 and recorded. This step corresponds to the measurement step in this example.

  For measurement, a dissociation agent that dissociates the target bound to the probe in the SPR sensor from the probe is introduced, and the dissociation operation that enables the capture of a new target is intermittently performed according to a command from the control computer 7. Done. In this embodiment, this step corresponds to a dissociation step. Further, the dissociating means is not shown in the figure, but includes a control computer 7. Further, in conjunction with the measurement result of the target by the SPR sensor, the control computer 7 controls the valve 8 to control the collection and disposal of the sample. Thereby, the target discharged from the SPR measuring device 4 selectively passes through the sample recovery tube 5 and is recovered, and the dissociator and the like pass through the waste tube 6 and are discarded.

In this example, a sample having a concentration gradient including impurities was prepared assuming a chromatography or pharmaceutical process. And the detection result by the UV absorbance meter 3 and the SPR measuring device 4 was compared. A fraction sample containing AFP (α-fetoprotein) as a target and BSA (Bovine Serum Albumin) as a contaminant was used. As shown in FIG. 2, this fraction sample was divided into fractions Nos. 10 and 12 for 100 μg / ml No. 11 contained 500 μg / ml. AFP is No. 3 and 7 are 0.1 μg / ml. 4 and 6, 0.5 μg / ml, 5 was included in the fraction sample so that the concentration was 3 μg / ml, which was a low concentration of 1/100 or less compared to BSA. A sample in which such AFP and BSA were mixed was actually measured with the UV absorbance meter 3 first.

In FIG. 3, the measurement result of the light absorbency by the UV absorptiometer 3 in a present Example is shown. A large signal change is obtained in fraction Nos. 10, 11, and 12 containing high-concentration BSA.
Fraction No. containing P In 3-7, it was buried in noise and no signal change was observed. This is because the detection limit of the UV absorbance meter 3 is as low as about 10 μg / ml, so that it is difficult to detect AFP with a concentration of 0.1 μg / ml. Furthermore, in the case of the UV absorbance meter 3, since there is no specificity depending on the type of protein such as AFP or BSA, even if the target has a high concentration, it is impossible to determine which peak corresponds to the target only with this data. is there.

  Next, using the SPR measuring device 4, a continuous measurement simulation experiment using the same fraction sample as described above was performed. A chip surface of an SPR sensor (not shown) is coated in advance with a polyethylene glycol film, and an anti-AFP antibody is immobilized on the surface layer as a probe (a method of adding a blocking agent for preventing nonspecific adsorption) There is also.)

  In order to realize continuous measurement, the surface was continuously regenerated by injecting a dissociator at regular time intervals onto the surface once bound with the target AFP. In order to completely remove non-specific adsorption, the dissociation agent was injected twice for one regeneration (may be performed three times or more). The obtained measurement results are shown in FIG. FIG. 4 (b) shows the result of summarizing the vertical axis as the signal value of the SPR measuring device 4. In the detection by the UV absorptiometer 3 described above, no. No signal change was detected in 3-7, but in the result using the SPR measuring instrument 4, In 3-7, a large signal change according to the concentration is observed, and the presence of the target AFP can be confirmed.

  By using the SPR measuring device 4 in this way, it is possible to measure a low concentration target that could not be measured by the conventional UV absorbance meter 3. Further, the conventional UV absorbance meter 3 had no selectivity at the time of protein detection, but a certain degree of selectivity was obtained by using the SPR measuring device 4.

  However, even in this case, as can be seen from FIG. 4B, when high-concentration impurities (BSA) are mixed, No. A signal change occurs at 10-12, which is a noise component. This is because the BSA molecules floating in the vicinity of the SPR sensor chip enter the surface plasmon resonance sensitivity region (several hundreds of nanometers), thereby causing a background noise (bulk effect) due to a change in refractive index. ).

  Next, countermeasures against background noise due to impurities such as BSA will be described. If such impurities exist, prepare another sensor chip that does not bind the target AFP antibody, and correct the background amount at that sensor chip as a reference, so that It is possible to improve S / N.

FIG. 5 shows a conceptual diagram in this case. In order to reduce background noise, two or more measurement regions are prepared in the SPR sensor chip 10 in advance. A probe (in this case, an anti-AFP antibody) 12 for capturing the target AFP 11 is immobilized on one sensing unit 10a (corresponding to the first measurement region). On the other reference portion 10b (corresponding to the second measurement region), a probe (currently an anti-FLAG antibody) 13 that basically does not react with the molecules contained in the sample is immobilized. At this time, it is desirable to immobilize probes having the same physicochemical or optical properties as much as possible in the same amount for accurate correction. Since AFP11 binds only to the anti-AFP antibody 12 and does not bind to the anti-FLAG antibody 13, a signal change is obtained in the sensing unit 10a according to the concentration of AFP11. On the other hand, for a contaminant 14 such as BSA, a signal change commonly occurs as background noise in any of the measurement portions of the sensing unit 10a and the reference unit 10b. Therefore, an SPR signal from which background noise has been removed can be obtained by subtracting the signal obtained by the reference unit 10b from the signal obtained by the sensing unit 10a.

  FIG. 6 shows a signal obtained by the sensing unit 10a, a signal obtained by the reference unit 10b, and a signal obtained by subtracting the latter signal from the former signal when the reference correction is performed. As shown in FIG. 6A, a peak corresponding to the concentration of AFP11 and a peak due to BSA are seen in the signal obtained by the sensing unit 10a. As shown in FIG. 6B, only a peak due to BSA is seen in the signal obtained by the reference unit 10b. As shown in FIG. 6C, by taking the difference between the two, it is possible to eliminate the influence of BSA as a contaminant and obtain only a signal corresponding to the concentration of AFP 11 as a target.

<Example 2>
Next, a second embodiment of the present invention will be described. As in the previous example, it was confirmed that by connecting the SPR in-line to a process such as chromatography, detection can be performed with higher sensitivity and selectivity than when using a conventional UV absorptiometer. It was. However, when there is a large amount of impurities, there is a problem that background noise occurs and S / N decreases. On the other hand, when performing reference correction, an advanced immobilization technique for producing the reference portion is required, and there is a problem that the cost and size of the apparatus increase.

  Therefore, in this example, verification was performed in the case of using localized surface plasmon resonance in the SPR measuring device 4 in order to reduce background noise. If this method is used, the reference correction as described with reference to FIG. 5 becomes unnecessary, and the system can be simplified and the cost can be reduced.

  In this example, as shown in FIG. 7A, a metal nanogroove structure was formed in the sensor chip. Specifically, a transparent resin is dropped on the surface of the base 20 made of slide glass, and the nanogroove structure 21 is formed by nanoimprinting with a mold. Here, a UV curable acrylic resin was used as the resin. The nano-groove structure 21 on the resin has a parallel groove shape, the pitch is 300 nm, and the depth is 50 to 60 nm. The groove width is 100 to 140 nm. In this embodiment, as shown in FIG. 7B, a metal film 22 having a thickness of 100 nm is formed on the grooves of the nano-groove structure 21 by gold sputtering. The nanogroove structure may be produced by thermal nanoimprinting or injection molding. In this embodiment, a localized surface plasmon resonance sensor such as a general gold colloid may be used.

  About the same fraction sample as Example 1, the same measurement was performed using the SPR measuring device using localized surface plasmon resonance. The obtained measurement results are shown in FIG. In addition, the horizontal axis represents the fraction No. FIG. 9 shows the result of summarizing the measurement results with the vertical axis as the signal value of the SPR measuring device. A large signal change was similarly obtained for AFP, but the background noise due to BSA was smaller than that of the SPR measuring device in Example 1, and it was confirmed that the level was hardly affected by the measurement. This is because the influence of the background can be reduced by the localization of the sensing area by using the localized surface plasmon resonance.

  As described above, it was confirmed that in-line measurement was possible with almost no influence of the background by using the SPR measuring device using localized surface plasmon resonance. Furthermore, the background noise can be further reduced by optimizing the pattern. Conversely, only specific signal changes can be increased. By using this method, it is possible to realize a compact, simple, low-cost, specific, continuous and highly sensitive in-line sensing system.

<Example 3>
Next, a third embodiment of the present invention will be described. In Example 1 and Example 2, in order to realize continuous sensing, the target on the sensor chip surface was dissociated intermittently from the probe using a dissociator. In this way, the dissociator is an effective means for regenerating the sensor chip surface, but it may cause denaturation of the protein itself. It is desirable to have as few as possible.

  Conventionally, the dissociation agent is injected so as to fill the entire flow path of the sample, but in this embodiment, an injection inlet is provided on the same surface as the surface of the sensor chip in the flow path of the sample. Thus, a thin dissociator layer (laminar flow) was formed along the surface of the sensor chip, and effective dissociation was performed with a small amount of dissociator. In particular, a sensor chip using localized surface plasmon resonance does not require a prism on the back side of the sensor chip, unlike a sensor chip using conventional propagation surface plasmon resonance. It is possible to provide an inlet for the dissociator. Therefore, efficient surface regeneration using a very small amount of dissociator is possible.

  In FIG. 10, schematic structure of the SPR measuring device 30 in a present Example is shown. FIG. 10A is a cross-sectional view of the SPR measuring device 30. In FIG. 10A, the sample including the target flows in the direction of the white arrow in the sample channel 35. Further, a dissociator injection port 32 for injecting a dissociator into the sample is provided immediately upstream of the sensor chip 31 in the sample flow path 35. The dissociating agent is injected into the sample from the dissociating agent inlet 32 by the injector 33.

  FIG. 10B shows a cross-sectional view of the injector 33. The injector 33 in this embodiment includes a bracket 43 made of an epoxy resin composite material and an injection portion 36. Further, a water repellent film 40 is provided on the surface of the injecting portion 36 so that the dissociating agent does not easily adhere. The bracket 43 and the injecting portion 36 are provided with a dissociating agent flow path 37, and the dissociating agent flow path 37 is filled with the dissociating agent by supplying the dissociating agent in the direction of the arrow. . The dissociator channel 37 is narrowed at the tip to form a nozzle 41. Further, the upstream side of the nozzle 41 of the dissociator channel 37 is a pressure chamber 42 having a relatively wide channel cross-sectional area. The thickness of the upper side of the pressure chamber 42 in the injection part 36 is thin, and the vibration film 38 is formed. By periodically pressing the vibration film 38 with the PZT element 39, the vibration film 38 is vibrated. As a result, the volume of the pressure chamber 42 changes periodically, and the dissociating agent is continuously discharged from the nozzle 41.

The dissociator injected into the sample channel 35 from the dissociator injection port 32 does not diffuse into the sample immediately after injection, but the liquid flow behaves as a layer and gradually spreads (laminar flow, Sheath) -flow). On the other hand, since the mixing rate of the dissociating agent into the sample is required to be as small as possible, it is desirable that the dissociating agent inlet 32 be as close as possible to the sensor chip 31. In addition, by providing the dissociator injection port 32 immediately upstream of the same surface as the sensor chip 31, the dissociator in the laminar flow state before diffusion reaches the sensing part of the sensor chip 31 at a high concentration, and even a small amount of dissociator Efficient dissociation operation is possible (the reaction efficiency per unit solution can be increased, and this mechanism can be applied to increase the reaction efficiency of antigen-antibody reaction).

  Here, in the conventional propagation type surface plasmon resonance sensor, as shown in FIG. 11A, it is necessary to attach the prism 46 to the opposite side of the sensor chip 45 from the metal film 45a via the matching oil 46a. Therefore, it is difficult to inject the dissociating agent in the vicinity of the sensing part by providing a through hole in the sensor chip 45 itself. On the other hand, since a prism is not required by forming a nano-groove structure and using the sensor chip 47 using the localized SPR generated in the nano-groove structure, the sensor portion 47 in the vicinity of the sensing portion 48 that is the nano-groove structure portion is used. The through hole 49 can be provided. Therefore, it is possible to inject the dissociating agent from the through hole 49 and efficiently dissociate the target bonded to the probe of the sensing unit 48. When the sensor chip 47 in this embodiment is manufactured by injection molding, a through hole 49 may be formed in the vicinity of the sensing unit 48 at the time of injection molding.

  Next, the effect of laminar flow when a dissociator was injected from the vicinity of the sensing portion of the sensor chip was evaluated. In FIG. 12, the conceptual diagram of the dissociator injection method in a normal case and the dissociator injection method in a present Example is contrasted and shown. As shown in FIG. 12A, in the normal injection method, the dissociating agent is injected from the dissociating agent channel 52 at a part of the sample channel 51 that is away from the upstream side of the sensor chip 50. Accordingly, the dissociating agent diffuses to some extent in the sample channel 51 before reaching the sensor chip 50, and it is difficult to efficiently dissociate the target on the sensor chip 50. On the other hand, in the present embodiment, the dissociating agent channel 53 is disposed immediately upstream of the sensing unit 50 a of the sensor chip 50, and the dissociating agent is directly injected from the dissociating agent channel 53.

The specific contents of the apparatus used for the evaluation are as follows. First, a through hole 54 was provided in the sensor chip 50 on the upstream side of the sensing part 50a having a nanogroove structure manufactured by nanoimprinting, and a dissociator was injected into the through hole 54 from the back side. In addition, water (n = 1.33) is always flowed through the sample channel, and ethanol (n = n = 3) is used as a simulated dissociator.
1.36) was injected. And when the amount of ethanol injection as a simulated dissociator was reduced, the signal change in the normal injection method and the injection method from the through hole 54 in this embodiment was observed.

FIG. 13 shows a graph of the results. The horizontal axis represents the amount of ethanol injected, and the vertical axis represents the magnitude of signal change before and after ethanol injection. The magnitude of signal change (actually, the shift amount of the SPR generation wavelength due to target detection) is normalized with the magnitude of the signal when 50 μl of ethanol is passed as 1. In the normal injection method, the size of the dissociator flow path is 2.0 mm wide and 25 μm high, and the flow rate of the dissociator is 40 μl / min.
It was. In general, a commercially available injector (type 9735, manufactured by Leodyne) was connected to the front of the sensor chip 50 for use. From FIG. 13, in the normal injection method, when the amount of ethanol becomes a very small amount (20 μl or less), liquid mixing and diffusion occur and the concentration decreases, and the original signal change amount cannot be obtained. I understand.

  On the other hand, in the injection method from the through hole 54 in the present embodiment, the diameter of the through hole 54 is about 1.5 mm, and a syringe pump is used from the back side of the sensor chip 50 (the flow rate is adjusted at 40 μl / min). And ethanol was injected. In this case, even if the injection amount is as small as 5 μl, a signal change almost equivalent to that in the case of 50 μl is obtained. In other words, since the ethanol solution layer exists on the surface of the sensing unit 50a in the sensor chip 50 in a laminar flow and the distance between the sensing unit 50a and the through hole 54 is short, liquid diffusion and mixing do not occur. I understand that. That is, if the dissociating agent is injected using this configuration, the mixing ratio of the dissociating agent can be reduced.

  In the present embodiment, the dissociating means includes an injector, a dissociating agent inlet, and a control computer (not shown) that controls the injector. Further, the fluid introducing means in the present embodiment is configured to include a fluid flow path. From the results of this example, it is possible to perform a suitable dissociation operation if the dissociating agent injection port (through hole 54) is disposed at least 2 mm upstream from the sensing unit 50a having the nano-groove structure. It turns out that it becomes. The diameter of the inlet (through hole 54) is preferably equal to or larger than the size of the region where the probe is fixed on the surface of the sensor. Considering that the dissociating agent injected from the injection port moves downstream while slightly diffusing, it is more desirable to have a size equivalent to the size of the region where the probe is fixed. For example, when the probe is actually fixed using a spotting device, the diameter is about 20 to 500 μm, and therefore the diameter of the dissociator injection port is preferably about 20 to 500 μm. Of course, even if it is 1.5 mm as in this embodiment, there is no problem as long as the area where the probe is fixed on the surface of the sensor can be covered.

<Example 4>
Next, a fourth embodiment of the present invention will be described. In the present embodiment, as described above, it is necessary to reduce the amount of the dissociating agent for regenerating the sensing unit surface as much as possible. Moreover, when measuring together with chromatography etc., it is desirable to take out and collect | recover the sample in which the target was contained using the signal obtained with the SPR measuring device. Therefore, a system that feeds back to the dissociation device and the recovery device based on the measurement result is required.

  In FIG. 14, schematic structure of the sensing system 60 containing the SPR measuring device in a present Example is shown. A sample is supplied to the SPR measuring device 61 from the preceding process. Data relating to the concentration of the target measured by the SPR measuring device 61 is sent to the control computer 63 for analysis and recording. The control computer 63 determines the injection timing of the dissociating agent based on the received data, and drives the dissociating agent injection pump 62 to perform the dissociation operation. Further, the control computer 63 determines the valve drive timing for collecting the target based on the received data, and includes many targets among the samples discharged from the SPR measuring device 61 by driving the valve 64. The part is recovered, and the part containing a large amount of dissociator is discarded as waste liquid. In this embodiment, the recovery means includes a control computer 63 and a dissociator injection pump 62.

  In FIG. 15, the graph of the value of the SPR signal obtained by the SPM measuring device 61 in the sensing system 60 of the present embodiment is shown. The horizontal axis is time, and the vertical axis is the value of the SPR signal. This SPR signal is actually a signal corresponding to a shift in the generation wavelength of surface plasmon resonance due to the coupling between the target and the probe in the sensing unit, and is a signal corresponding to the amount (concentration) of the target in the sample. In FIG. 15, it is not necessary to inject the dissociator when the target is not flowing. Therefore, after the target flows out, the dissociator is injected for the first time after the elapse of a predetermined ΔT, starting from the time when the magnitude of the SPR signal becomes equal to or greater than the predetermined threshold A. For example, the threshold A may be a baseline + 9σ (σ is a signal standard deviation between ΔT). The value of ΔT is determined by the concentration of the target, the liquid feeding speed, etc., but may be fixed at 1 minute, for example. Here, the signal change amount ΔS is defined as a value obtained by subtracting the SPR signal value before ΔT ′ from the SPR signal value immediately before injecting the dissociator. Here, the value of ΔT ′ is a predetermined time under the condition that 0 <ΔT ′ <ΔT.

As shown in FIG. 15, after the injection of the dissociating agent is started, the dissociating agent is continuously injected at ΔT intervals until ΔS becomes a certain value or less. At that time, the dissociator may be injected twice at intervals of ΔT. When ΔS becomes equal to or smaller than a certain value, it is determined that there are no targets, and the injection of the dissociator is stopped. The threshold value of ΔS in this case is defined by 9σ, for example. The stop condition may be that ΔS is equal to or less than the threshold value (9σ) twice or more. This cycle may be repeated any number of times.

  FIG. 16 shows a flowchart of an SPR measurement routine executed at the time of SPR measurement in the control computer 63 of the sensing system 60. When this routine is executed, it is first determined in S101 whether or not the SPR signal is greater than A. Here, when it is determined that the SPR signal is A or less, the process proceeds to the process of S101. That is, the process of S101 is repeatedly executed until it is determined in S101 that the SPR signal is greater than A. On the other hand, if it is determined in S101 that the SPR signal is greater than A, the process proceeds to S102.

  In S102, sample collection is started. Specifically, the valve 64 is driven to the collection side according to a command from the control computer 63. When the process of S102 ends, the process proceeds to S103. In S103, a waiting process of ΔT is performed, and an elapsed time of ΔT is secured. When the process of S103 ends, the process proceeds to S104. In S104, the dissociator is injected. When the process of S104 ends, the process proceeds to S105. In S105, it is determined whether or not the value of ΔS is less than 9σ. Here, when the value of ΔS is 9σ or more, the process returns to the process before S103, and the processes of S103 and S104 are executed. In other words, the dissociator is repeatedly injected at intervals of ΔT until the value of ΔS becomes less than 9σ in S105. If the value of ΔS is less than 9σ in S105, the process proceeds to S106. In S106, sample recovery is terminated. Specifically, the valve 64 is driven to the waste liquid side. When the processing of S106 ends, the process returns to the previous step of S101 and waits for the next target to flow.

  In the above SPR measurement routine, the fluid introduction process and the measurement process are executed as the routine starts. And a dissociation process is performed in S104.

  FIG. 17 shows the control performed as a result of the execution of the SPR measurement routine in the sensing system 60. As shown in FIG. 17, the SPR signal increases with the inflow of the target, and when it exceeds A, the recovery of the sample (target) and the injection of the dissociator at the ΔT interval are executed. Thereafter, the SPR signal increases as the target flow rate increases, and the SPR signal decreases as the target flow rate decreases. When ΔS becomes less than 9σ, recovery of the target is completed, the sample is discarded as waste liquid, and injection of the dissociating agent is completed.

  For sample recovery, the target is efficiently recovered by switching the valve 64 to the recovery side from when the magnitude of the SPR signal exceeds A until the dissociator injection is stopped. Can be implemented. Further, in order to reduce the mixing of the dissociating agent into the sample including the collected target, the time zone in which the sample containing the dissociating agent passes through the valve 64 is identified from the SPR signal of the SPR measuring device 61, and the dissociating agent is dissociated. The valve 64 may be switched to the collection side only in a time zone when no agent is mixed. As the determination for specifying the mixing of the dissociating agent, for example, it may be determined that the dissociating agent is mixed in a time zone in which the SPR signal is A or less. FIG. 18 shows the target recovery timing in this case.

<Example 5>
Next, a fifth embodiment of the present invention will be described. In the present embodiment, an example of construction of a process line when the sensing system according to the present invention is applied to pharmaceutical process, concentration control in a liquid state such as culture, foreign matter inspection, and the like will be described. In a pharmaceutical process or the like, mixing of a dissociator into a sample may cause a process problem. In such a case, the valve on the downstream side is switched in accordance with the dissociation operation, and only the solution portion containing the dissociator is separated as waste liquid. FIG. 19 shows an example of a process line in such a case.

  In this line, the sample process pipe 75 connects the former process P1 and the latter process P2. The pre-stage process P1 may be, for example, a cell culture tank (cell culture process). The same applies to the following embodiments. The sample pipe 75 is provided with an SPR measuring device 70. The SPR measuring device 70 is provided with a sample flow path 70a, and connects the sample pipe 75 on the upstream side and the downstream side. The sample channel 70a is provided with a sensor chip 71, and a through hole 72 for dissociating agent injection is provided immediately upstream thereof. Further, the SPR sensor 70 is provided with an injector 73 and discharges a dissociating agent to the through hole 72.

  A valve 74 is provided on the downstream side of the SPR measuring instrument 70 in the sample pipe 75. By driving this valve 74, it is possible to select whether the sample flowing out from the SPR measuring instrument 70 is introduced into the subsequent process P2 or discarded as waste liquid. A flow sensor 76 as a flow sensor for measuring the flow rate of the sample is provided on the upstream side of the SPR measuring instrument 70 in the sample pipe 75. In this embodiment, in order to separate the dissociator from the sample with high accuracy, the valve switching timing is calculated based on the information of the flow sensor 76. Specifically, when the flow rate of the sample is large, the time taken for the injected dissociator to flow downstream is shortened. Therefore, the threshold value of the SPR signal for starting recovery, which is A in FIG. You may make it do. That is, as the flow rate of the sample increases, the threshold value of the SPR signal for starting collection is decreased. The relationship between the increase amount of the flow rate and the decrease amount of the threshold value of the SPR signal may be linear, or may be a curvilinear relationship such as convex upward or convex downward. In the above, the sample pipe 75 and the sample flow path 70a constitute a fluid introducing means. Further, the injector 73 and the through hole 72 constitute dissociation means.

  FIG. 20 shows another aspect of the process line. In this embodiment, the pre-stage process P1 and the post-stage process P2 are the same, and the sample is circulated. A pump 77 for circulating the sample is also provided. The pump 77 may be any system such as a verista pump, a plunger pump, and an electroosmotic pump. This embodiment can be used for management of a culture tank or the like.

  FIG. 21 is a diagram showing a third mode in the present embodiment. In this embodiment, in order to completely eliminate the mixing of the dissociating agent into the sample, the branch pipe 78 is branched from the main process line P3, and the monitoring pipe 79 is provided with the SPR measuring device 80, the pump 81, and the valve 82. ing. In the case of the SPR measuring device 80, it is possible to detect even a small amount of sample of 100 μl or less. Further, in this embodiment, when the sample is always branched to the monitoring pipe 79, the loss of the sample becomes large. Therefore, the valve 82 is switched at regular intervals (for example, every hour), as shown in FIG. As described above, it is possible to increase the collection efficiency of the sample by flowing the sample or the dissociating agent to the SPR measuring device 80 and feeding the buffer solution for the other time.

  In this embodiment, the influence of background noise can be completely eliminated by determining the concentration of the target after switching the flow of the monitoring pipe 79 from the sample to the buffer solution. The fluid introduction means in this case includes a branch pipe 78, a monitoring pipe 79, a pump 81, and a valve 82. In FIG. 22, the period during which the sample is supplied corresponds to the fluid introduction process. The period from the supply of the sample to the dissociation operation corresponds to the measurement process. The period during which the dissociation operation is performed corresponds to the dissociation process.

The process line of the embodiment shown in FIG. 21 can be applied to concentration control of antibodies produced by culture, antibody leak check, etc. in a production line for antibody drugs and the like. In such a case, in order to detect antibodies, it is desirable to form a probe layer containing protein A or a probe layer containing anti-Fc antibodies such as protein G, L and Anti-IgG on the surface of the sensor chip. . A similar system can also be constructed in a process line where the target object is an expressed protein. In this case, on the surface of the sensor chip, anti-GST antibody, anti-His-tag antibody, anti-FLAG antibody, anti-biotin antibody, streptavidin,
It is desirable to form a probe layer capable of binding the expressed protein such as neutravidin, avidin, Ni-NTA (nickel-nitrilotriacetic acid).

  In the above-described embodiment, an example in which the sample is a liquid including a target that has passed through the previous process has been described. However, the present invention applies to a sample that is any fluid (including gas) other than the above in the process line. It is applicable.

DESCRIPTION OF SYMBOLS 1 ... Sensing system 2 ... Column 3 ... UV absorbance meter 4 ... SPR measuring instrument 5 ... Sample collection pipe 6 ... Waste pipe 10 ... SPR sensor chip 11 ... Target (AFP)
12 ... Probe (anti-AFP antibody)
13 ... Probe (anti-FLAG antibody)
14 ... Foreign matter (BSA)
DESCRIPTION OF SYMBOLS 20 ... Base 21 ... Nano-groove structure 22 ... Metal film 30 ... SPR measuring device 31 ... Sensor chip 32 ... Dissociator injection port 33 ... Injector 60 ... Sensing system

Claims (27)

By irradiating the surface plasmon excitation light to the sensor, surface plasmon resonance can be generated on the surface of the sensor, and a substance existing outside the surface of the sensor according to the wavelength or incident angle of the excitation light. A surface plasmon measuring device provided in the process line,
Fluid introduction means for continuously introducing fluid that has passed through a process prior to the surface plasmon measuring instrument in the process line onto the sensor surface of the surface plasmon measuring instrument;
With
A probe that is a substance that can specifically bind to a specific substance in the fluid is fixed to the surface of the sensor,
A surface plasmon resonance sensing system characterized in that the amount of the specific substance can be measured in-line by combining the specific substance in the fluid that has passed through the process with the probe. Further comprising dissociation means for performing a dissociation operation for dissociating a bond between the probe and the specific substance on the surface of the sensor;
Measurement of the amount of the specific substance bound to the probe by the surface plasmon measuring instrument and dissociation of the specific substance bound to the probe by the dissociation means were alternately performed to bind to the probe. The surface plasmon resonance sensing system according to claim 1, wherein a plurality of measurements are continuously performed for the amount of the specific substance.   3. The surface plasmon resonance sensing system according to claim 2, wherein the dissociation operation is an injection of a dissociating agent that dissociates a bond between the probe and the specific substance on the surface of the sensor into the fluid. . The fluid introducing means is a flow path for circulating the fluid that has passed through the process before the surface plasmon measuring instrument,
The surface of the sensor of the surface plasmon measuring device is arranged to face the fluid in the flow path,
The injection port for injecting the dissociating agent into the fluid is provided in the flow path substantially in the same plane as the surface of the sensor and on the upstream side of the surface of the sensor in the flow path. The surface plasmon resonance sensing system according to claim 3.   A first measurement region in which a probe that binds to the specific substance is fixed and a second measurement region in which a probe that binds to the specific substance is not fixed are provided on the surface of the sensor, and the first measurement is performed. The amount of the specific substance is measured by subtracting the measurement value measured in the second measurement region from the measurement value measured in the region. Surface plasmon resonance sensing system described in 1. The sensor of the surface plasmon measuring instrument is:
A metal layer formed so as to cover at least a part of the surface of the sensor, and a groove structure including a plurality of grooves formed in a portion of the surface of the sensor where the metal layer is formed, A side wall surface of each groove in the groove structure forms a facing surface by the metal layer covering the side wall surface, and generates a localized surface plasmon resonance in a region sandwiched by the facing surface by the metal layer The surface plasmon resonance sensing system according to any one of claims 1 to 5, wherein the surface plasmon resonance sensing system is a surface plasmon resonance sensor. The sensor of the surface plasmon measuring instrument is:
A metal layer formed so as to cover at least a part of the surface of the sensor, and a groove structure including a plurality of grooves formed in a portion of the surface of the sensor where the metal layer is formed, A side wall surface of each groove in the groove structure forms a facing surface by the metal layer covering the side wall surface, and generates a localized surface plasmon resonance in a region sandwiched by the facing surface by the metal layer A surface plasmon resonance sensor,
5. The surface plasmon resonance sensing system according to claim 4, wherein the inlet is provided in the flow path at a location within 2 mm upstream of the groove structure on the surface of the sensor.   The injection port has a circular cross section, and a diameter of the injection port is equal to or greater than a length in a width direction of the flow channel in a region where the probe is fixed on the surface of the sensor, and is equal to or smaller than the flow channel width. The surface plasmon resonance sensing system according to claim 4 or 7.   The dissociation operation by the dissociation means is performed after a certain time after the magnitude of the signal exceeds a predetermined threshold based on a signal corresponding to the amount of the specific substance from the surface plasmon measuring device. The surface plasmon resonance sensing system according to any one of claims 2 to 8. A valve provided on the downstream side of the sensor in the flow path for controlling whether the fluid is recovered or discarded as a waste liquid;
Based on a signal corresponding to the amount of the specific substance from the surface plasmon measuring instrument, the valve is operated after the signal exceeds a predetermined threshold until the dissociation operation by the dissociating means is performed. The surface plasmon resonance sensing system according to any one of claims 2 to 9, wherein the fluid is recovered by actuating the sensor.   A flow rate sensor for detecting the flow rate of the fluid flowing through the flow path; a signal corresponding to the amount of the specific substance from the surface plasmon measuring device; and a flow rate of the fluid detected by the flow rate sensor. The surface plasmon resonance sensing system according to claim 10, wherein the timing for collecting the fluid is controlled based on: The fluid introduction means is a flow path for branching and circulating a fluid that has passed a predetermined process on the process line from the process line,
The surface plasmon resonance sensing system according to any one of claims 1 to 11, further comprising an on-off valve for flow rate control between a branch point of the flow path and the surface plasmon measuring instrument.   The surface plasmon according to claim 12, wherein the surface plasmon measuring device monitors the amount of the specific substance in the fluid at a constant interval by controlling the open / close valve at a constant interval. Resonance sensing system. The process before the surface plasmon measuring instrument in the process line is purification chromatography,
The surface plasmon resonance sensing system according to any one of claims 1 to 13, wherein a purified extraction state of the fluid that has passed through the purification chromatography is monitored in real time by the surface plasmon measuring device.   The process before the surface plasmon measuring instrument in the process line is a cell culture tank, and a substance generated in the cell culture tank in the fluid is monitored in real time by the surface plasmon measuring instrument. Item 14. The surface plasmon resonance sensing system according to any one of Items 1 to 13. By irradiating the surface plasmon excitation light to the sensor, surface plasmon resonance can be generated on the surface of the sensor, and a substance existing outside the surface of the sensor according to the wavelength or incident angle of the excitation light. A surface plasmon resonance in-line measurement method using a surface plasmon measuring instrument capable of measuring a quantity,
Introducing a fluid that has passed through a process prior to the surface plasmon meter in the process line onto a sensor surface of the surface plasmon meter;
By binding a specific substance in the fluid that has passed through the process to a probe that is a substance that can specifically bind to the specific substance immobilized on the surface of the sensor, the amount of the specific substance is in-line. Measuring process to measure in,
A surface plasmon resonance in-line measurement method characterized by comprising:   The surface plasmon resonance in-line measurement method according to claim 16, further comprising a dissociation step of performing a dissociation operation for dissociating a bond between the probe on the surface of the sensor and the specific substance.   The surface plasmon resonance in-line measurement according to claim 17, wherein the dissociation operation is an injection into the fluid of a dissociator that dissociates a bond between the probe and the specific substance on the surface of the sensor. Method.   Injecting the dissociator into the fluid is in substantially the same plane as the surface of the sensor in the flow path, and from an inlet provided on the upstream side of the surface of the sensor in the flow path. 19. The surface plasmon resonance in-line measurement method according to claim 18, wherein the method is performed.   On the surface of the sensor, the amount of the specific substance is obtained by subtracting the measurement value when the probe binding to the specific substance is not fixed from the measurement value when the probe binding to the specific substance is fixed. The surface plasmon resonance in-line measurement method according to any one of claims 16 to 19, wherein measurement is performed. The sensor of the surface plasmon measuring instrument is:
A metal layer formed so as to cover at least a part of the surface of the sensor, and a groove structure including a plurality of grooves formed in a portion of the surface of the sensor where the metal layer is formed, A side wall surface of each groove in the groove structure forms a facing surface by the metal layer covering the side wall surface, and generates a localized surface plasmon resonance in a region sandwiched by the facing surface by the metal layer The surface plasmon resonance in-line measuring method according to any one of claims 16 to 20, wherein the surface plasmon resonance sensor is a surface plasmon resonance sensor.   The dissociation operation in the dissociation step is performed after a certain time after the amount of the specific substance exceeds a predetermined threshold based on the amount of the specific substance measured by the surface plasmon measuring instrument. The surface plasmon resonance in-line measurement method according to any one of claims 17 to 21.   Based on the amount of the specific substance measured by the surface plasmon meter until the dissociation operation by the dissociation step is performed after the amount of the specific substance exceeds a predetermined threshold, The surface plasmon resonance in-line measurement method according to any one of claims 17 to 22, wherein the fluid is recovered.   24. The surface plasmon resonance in-line according to claim 23, wherein a timing for collecting the fluid is determined based on an amount of the specific substance measured by the surface plasmon measuring device and a flow rate of the fluid. Measuring method. In the fluid introduction step, the fluid that has passed a predetermined process on the process line is branched from the process line and introduced onto the sensor surface of the surface plasmon measuring instrument,
The surface plasmon resonance in-line measurement method according to any one of claims 16 to 24, wherein the surface plasmon measurement device monitors the amount of the specific substance in the fluid at regular intervals. The process before the surface plasmon measuring instrument in the process line is purification chromatography,
The surface plasmon resonance in-line measurement method according to any one of claims 16 to 25, wherein the purified extraction state of the fluid that has passed through the purification chromatography is monitored in real time by the surface plasmon measurement device.   The process before the surface plasmon measuring instrument in the process line is a cell culture process, and the substance generated in the cell culturing process in the fluid is monitored in real time by the surface plasmon measuring instrument. Item 26. The surface plasmon resonance in-line measurement method according to any one of Items 16 to 25.

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