JPH11183372A - Spr sensor device, analysis system and detecting method using it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a surface plasmon sensor device having a small size and high detection accuracy and capable of detecting a plurality of materials. SOLUTION: A fluid feed pipe 1 is erected on the detection surface 2 of a prism 3. The feed pipe 1 is made linear and sufficiently long so that a fluid flows in it as a laminar flow. The outlet of the feed pipe 1 directly faces the detection surface 2 at a slight gap, and it is flared to guide the fluid laminar flow to the detection surface 2 and release it to the outside without disturbing it. A fluid discharge pipe 4 is a large-diameter circular pipe, it is arranged to locate the feed pipe 1 at the center axis position, and it is firmly crimped to the surface of the prism 3 by bots and nuts 6. A link section between the discharge pipe 4 and the surface of the prism 3 is smoothly curved not to disturb the fluid flow. Ring-like electrodes 9 are fitted to the feed pipe 1 and the discharge pipe 4 as electrochemical sensors.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sensor device utilizing surface plasmon resonance (SPR).
R sensor device ”), an SPR detection method and an analysis system.

[0002]

2. Description of the Related Art An SPR sensor device is capable of detecting a refractive index or a change in a refractive index of a liquid or a gas that comes into contact with a detection surface thereof, and is used for, for example, measurement of an antigen-antibody reaction by applying its principle. An example of the measurement method is as follows. 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, first, the buffer solution containing no antigen is first added to the sensor cell (buffer solution or A flow such as a test solution is brought into contact with the detection surface and flowed into the room, and the resonance angle of the surface plasmon at that time is measured. Next, a buffer solution containing the antigen of the subject is caused to flow through the sensor cell to cause an antigen-antibody reaction. Then, the sensor cell is washed with a buffer solution containing no antigen, and the resonance angle of the surface plasmon 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 that have reacted with the antibody, this is converted to the concentration of the antigen present as a solute.

[0003] The SPR sensor device is about 1 mm above the detection surface.
Since the change in the refractive index of the object within 00 nm is sensed with high sensitivity, an accurate resonance angle cannot be obtained if the flow is disturbed such as swirling of the fluid on the detection surface. Therefore, in the sensor cell of the conventional SPR sensor device, in order to make the flow of the fluid on the detection surface into a complete laminar flow, a long length having a certain length or more determined by parameters such as the flow path diameter of the sensor cell and the flow velocity of the fluid is used. A flow path is arranged parallel to the detection surface.

By the way, liquid chromatography (hereinafter, referred to as liquid chromatography)
LC) is a method of purifying a desired substance by utilizing the difference in the electrical properties or molecular size of low to high molecular substances, and is currently widely used mainly for the purification of physiologically active substances. . At this time, 260 nm
Alternatively, a 280 nm ultraviolet (hereinafter referred to as UV) absorbance meter is most widely used, and elution positions of various substances including a desired substance can be determined by a UV absorption chart.

[0005] However, in order to determine which peak on the UV absorption chart is the desired substance, it is necessary to measure the physiological activity of the eluate separated by a predetermined volume again after LC. However, in general, the measurement of these physiological activities (bioassay) involves complicated operations, requires a skillful operation, and requires a long time.

[0006]

Usually, the detection surface of the SPR sensor device is formed on a prism. In order to prevent turbulence on the detection surface, a linear flow path of an appropriate distance is required for the sensor cell as described above, so that the sensor cell and the prism are increased in size, and the SPR sensor device itself is also increased in size. At the same time, the prism is a relatively expensive component, which increases the cost. If the flow velocity of the fluid is reduced to avoid this, the time required for the measurement becomes longer and the practicality of the sensor is impaired.

Further, in the conventional SPR sensor device, a rubber gasket having a slot-like elongated hole and a prism serving as a sensor detecting section constitute a flow path of a sensor cell. For this reason, it is structurally poor in pressure resistance and is used only for low-pressure fluid measurement, and cannot be used for applications requiring high pressure, such as liquid chromatography.

In the flow path in the sensor cell, due to various restrictions,
The channel must be made at a right angle or at an angle close to it. In the bent portion of the flow path, fluid exchange is poor due to stagnation of the fluid, and furthermore, when bubbles are generated near the detection surface, the bubbles are difficult to escape and deteriorate the measurement accuracy.

There are a plurality of objects to be detected in the mixed sample,
If one object is detectable by surface plasmon resonance and another object is detectable by an electrode-type sensor, the measurement must be performed using each sensor independently. This means that the configuration of the sensor device is increased due to the necessity of flowing the fluid through the sensor cell in a laminar flow as described above.

The operation procedure of the conventional SPR sensor device is as follows, taking as an example the measurement of an antigen which is a typical application. An antibody that specifically reacts with the antigen is immobilized on the detection surface. First, the resonance angle of the buffer solution containing no antigen is measured. Next, the detection surface is filled with a buffer solution containing an antigen for a certain period of time to cause an antigen-antibody reaction. After the reaction, the detection surface is washed with a buffer solution containing no antigen, and the resonance angle after the reaction is measured. Then, the difference between the resonance angles before and after the antigen-antibody reaction is determined, and the difference is converted into an antigen concentration. As described above, one measurement requires at least three steps: measurement before the reaction, measurement of the reaction, and measurement after the reaction, which increases the time required for the measurement and expects a real-time response. The attractiveness of the sensor is lost.

Regarding LC, as described above,
A great deal of labor and time are required for measuring the biological activity of each substance separated and purified by LC.

Accordingly, it is an object of the present invention to provide a small-sized SPR sensor device having high measurement accuracy.

Another object of the present invention is to provide a small-sized SPR sensor device capable of simultaneously detecting a plurality of substances.

Another object of the present invention is to provide an SPR detection method with high detection accuracy.

Still another object of the present invention is to separate and purify a physiologically active substance using LC and to measure a physiologically active substance,
It is an object of the present invention to provide an epoch-making analysis system capable of substantially simultaneously identifying a peak of a desired physiologically active substance by an R apparatus.

Still another object of the present invention is to provide a small-sized, time-consuming measurement result that simultaneously separates components in a mixed sample and determines components having specificity for a specific substance.
It is to provide a PR sensor device and an analysis system.

[0017]

An SPR sensor device according to a first aspect of the present invention has a supply pipe for supplying a fluid to a detection surface of an SPR sensor in a sensor cell.
The supply tube is long enough to allow the fluid to flow therethrough in a laminar flow, the fluid outlet of which is positioned in close proximity to the sensing surface. According to this configuration, the fluid such as the test liquid becomes a laminar flow without disturbance in the supply pipe, and exits from the supply pipe as it is in laminar flow, contacts the detection surface, and has a slight flow between the detection surface and the supply pipe outlet. It moves through the gap and is discharged. Although the flow is expected to be turbulent when the fluid moves for discharge, the effect of this turbulence is very small because the SPR sensor can detect only an object in the vicinity of only about 100 nm from the detection surface. Also, in order for the outlet of the supply tube to face the detection surface, the supply tube will typically be arranged in a posture standing perpendicular to the detection surface. With this arrangement, the length of the supply pipe can be made sufficiently long without increasing the size of the SPR sensor. Incidentally, the length of the supply pipe such that the flow of the fluid becomes laminar is determined from parameters such as the inner diameter of the supply pipe and the flow velocity of the fluid.

The inner diameter of the supply tube is desirably larger than the diameter of the spot of the surface plasmon excitation light applied to the detection surface. Semiconductor lasers and LEDs are used as excitation light sources for surface plasmons. It is easy to narrow down these lights to a spot diameter of usually 1 mm or less. And it is also easy to make the internal diameter of a supply pipe 1 mm or more. Since the inner diameter of the supply pipe is larger than the spot diameter of the surface plasmon excitation light, the laminar flow covers the entire detection surface where the surface plasmon is excited.

It is desirable that the inner diameter near the outlet of the supply pipe is smoothly enlarged like a trumpet toward the outlet. This facilitates smooth movement of the fluid in contact with the detection surface, and reduces turbulence in the flow. It is also desirable that the outlet face of the supply tube is parallel to the detection surface. This ensures a flow path parallel to the detection surface for movement of the fluid in contact with the detection surface, and reduces turbulence in the flow.

A mechanism can be provided to adjust the distance between the detection surface and the outlet of the supply tube. When this distance exceeds a certain value, the fluid becomes turbulent and comes into contact with the detection surface. The distance required to reach the detection surface with laminar flow differs depending on the flow velocity. By adjusting the distance in accordance with the flow velocity, it is possible to guide the fluid to the detection surface in a laminar flow.

The discharge pipe for discharging the fluid in contact with the detection surface has a larger inside diameter than the supply pipe, and is arranged so as to surround the supply pipe, and the inlet face of the discharge pipe is arranged around the detection surface. To attach to the SPR sensor surface. By using such a discharge pipe, the discharge pipe itself becomes a casing of the sensor cell, and the structure of the sensor cell is simplified. Therefore, it is easy to reduce the size of the SPR sensor and the sensor cell. In addition, it is easy to form a sensor cell with a high withstand voltage by firmly adhering the inlet surface of the discharge pipe to the SPR sensor surface. From the viewpoint of pressure resistance, the discharge pipe and the supply pipe are desirably circular pipes in cross section.

It is desirable that the inner diameter near the inlet of the discharge pipe be curved and narrow so as to be continuously connected to the SPR sensor surface. As a result, the object after coming into contact with the detection surface is discharged through the discharge pipe without greatly disturbing the flow.

An electrode for an electrochemical sensor can be attached to one or both of the outer peripheral surface of the supply tube and the inner peripheral surface of the discharge tube. Some components in the mixed test liquid are measured by surface plasmons, and some components are simultaneously measured by the electrochemical sensor in the same sensor cell.

[0024] The SPR detection method according to the second aspect of the present invention provides a method of detecting an SPR while introducing a test solution into an SPR sensor.
The concentration of the target substance contained in the test liquid is obtained from the amount of change per unit time of the surface plasmon resonance angle detected by the SPR sensor. In an example in which the target substance is an antigen which is a typical application of the SPR sensor, a buffer solution containing no antigen is first flowed on the detection surface, and then a test solution containing the antigen is flown. While this test solution is flowing,
The resonance angle changes with time and settles to a substantially constant value after a certain time has elapsed. The amount of change per unit time when the resonance angle changes with time correlates with the antigen concentration. In the method of the present invention, the concentration of the target substance is determined from the amount of change in the resonance angle per unit time. Since the variance of the resonance angle change per unit time is small as described later, the measurement accuracy by this method is high.

An analysis system according to a third aspect of the present invention comprises a column for separating a plurality of substances contained in a test liquid, an absorbance meter into which an eluate from the column is introduced, and an eluate from the column. An SPR sensor device that outputs a signal responsive to the target substance. For example, in the case of a mixed test liquid containing a plurality of types of proteins, the mixed test liquid is first pushed into a column by a pump and separated into individual proteins in the column. An absorbance meter is connected after the column by a pipe. The separated proteins are confirmed by the absorbance meter to have come out of the column for each type. In addition, an SPR sensor device on which an antibody specifically reacting with a target protein is immobilized, is connected to the absorbance meter in parallel or in series with a pipe. The resonance angle signal output from the SPR sensor device changes each time the separated various proteins enter the SPR sensor device, but returns to the original base value when the protein passes. However, SPR
When a protein that specifically reacts with the antibody immobilized on the sensor device comes, the resonance angle does not return to the base value because a part of the protein is bound to the antibody even after the protein has passed through. This makes it possible to know when the target protein has entered the SPR sensor device from the mixed test liquid, and to know the characteristics of the target protein and the like from the signal change of the absorbance meter and the characteristics of the column.

[0026]

FIG. 1 shows the structure of a main part of an embodiment of an SPR sensor device according to the present invention.

On the upper surface of the prism 3, a detection surface 2 on which a gold thin film is deposited is formed. A light source 7 is arranged outside one inclined surface of the prism 3, and a lens 11 is arranged between the light source 7 and the prism 3. A light receiving element 10 such as a CCD line sensor is joined to the other inclined surface of the prism 3. The prism 3, light source 7, lens 11, and light receiving element 10 are housed and fixed in a prism case 12.

The light source 7 emits, toward the detection surface 2, an excitation light beam for exciting surface plasmons on the gold thin film. The excitation light beam emitted from the light source 7 is conically collected on the detection surface 2 by the optical system. The diameter of the spot of the excitation light beam focused on the detection surface 2 is about 0.5 mm when the light source 7 is, for example, an LED, and is on the order of μm when the light source 7 is a laser oscillator. The excitation light beam is reflected from the detection surface 2, and the reflected light beam is received by the light receiving element 10. The light receiving element 10 is, for example, a CCD line sensor, and outputs a signal indicating a light receiving amount of each pixel. The pixel position where the output signal level of the light receiving element 10 is minimum corresponds to the plasmon resonance angle.

The sensor cell for flowing the fluid to the detection surface 2 includes a supply pipe 1 for supplying the fluid to the detection surface 2 and a discharge pipe 4 for discharging the fluid flowing on the detection surface 2 to the outside. Have.

The supply pipe 1 is a long straight pipe having a circular cross section, and is arranged in a posture standing at right angles to the detection surface 2. In the supply pipe 1, a fluid is caused to flow toward the detection surface 2 by the force of a pump (see FIG. 2). The supply pipe 1
It has a sufficient length determined from parameters such as the inner diameter of the supply pipe 1 and the flow velocity of the fluid so that the fluid flows in the interior as a laminar flow without turbulence. The inner diameter (diameter) of the supply pipe 1 is larger than the diameter of the light spot, for example, 1 mm.
It is.

The outlet of the supply tube 1 is directly opposite the part of the detection surface 2 where the light spot falls. The outlet of this supply tube 1 is close to the detection surface 2, and the distance of this outlet from the detection surface 2 can be adjusted by a tube lifting mechanism 5.
The inside diameter of the supply pipe 1 near the outlet is smoothly enlarged in a horn-shaped curve toward the outlet, and forms a parallel surface with the detection surface 2 at the outlet, and this parallel surface extends outward by a certain distance. It has spread.

Since the supply pipe 1 is sufficiently long, fluid is supplied from the supply pipe 1
A laminar flow is formed inside, and the laminar flow exits from the outlet of the supply pipe 1. The distance that the laminar flow reaches the detection surface 2 without being disturbed depends on the flow velocity of the fluid. Therefore, by adjusting the distance between the outlet of the supply pipe 1 and the detection surface 2 according to the flow velocity by the pipe lifting / lowering mechanism 5, the fluid can be guided to the detection surface 2 as laminar flow at any flow velocity. Since the inner diameter of the supply pipe 1 is larger than the diameter of the light spot, the laminar flow of the fluid covers the entire area of the light spot. The fluid that has hit the detection surface 2 flows outward, but the inside diameter near the outlet of the supply pipe 1 smoothly spreads in a horn-like manner, and at the outlet, it becomes parallel to the detection surface 2 and becomes parallel to the detection surface 2. Since the flow paths are formed in parallel, the laminar flow of the fluid flows outward from the detection surface 2 without being largely disturbed.

The discharge pipe 4 has an inside diameter much larger than that of the supply pipe 1, has a circular cross section, surrounds the supply pipe 1, and has a coaxial positional relationship with the supply pipe 1 so that the surface of the prism 3 can be formed. It is arranged in a posture perpendicular to. The flange 4A at the entrance end of the discharge tube 4 and the flange 12A at the end of the prism case 12 are fastened with bolts and nuts 6, whereby the entrance surface of the discharge tube 4 is firmly pressed against the surface of the prism 3. Have been. A packing 8 is interposed between the end face of the discharge pipe 4 and the surface of the prism 3 in a state where the packing 8 is crushed by the tightening pressure to seal a joint between the two. With this structure, a high withstand voltage sensor cell is realized. Discharge pipe 4 and supply pipe 1
Has a circular cross section, which also contributes to high breakdown voltage. Therefore, measurement of a high-pressure fluid is possible. The inner diameter of the inlet portion of the discharge pipe 4 is curvilinearly narrowed inward so as not to disturb the laminar flow of the fluid, and the inner peripheral surface of the inlet is continuously and smoothly connected to the surface of the prism 3.

On the outer peripheral surface of the supply pipe 1 and the inner peripheral surface of the discharge pipe 4,
A plurality of ring-shaped electrodes 9 are provided. These electrodes 9 can function as an electrochemical sensor. For example, in this embodiment, there are three ring-shaped electrodes 9,
One of them has a surface of silver chloride and operates as a reference electrode, and the other two has a surface of platinum and operates as a working electrode and a counter electrode, respectively. By immobilizing, for example, glucose oxidase, which is an enzyme, on the surface of the working electrode, the electrochemical sensor using these three electrodes 9 can function as a glucose sensor. On the other hand, by immobilizing, for example, an antibody on the detection surface 2 of the SPR sensor, the SPR sensor can function as a protein sensor. In this way, it is possible to simultaneously detect two types of components such as glucose and protein.

FIG. 2 shows a sectional structure of an example of a pump applicable to the SPR sensor device shown in FIG.

This pump is a syringe pump. By reciprocating a piston 23 in the syringe 21 by a motor 25, an external fluid is sucked into the liquid reservoir 22 in the syringe 21, Can be sent out to the outside. The syringe 21 includes a liquid storage chamber 22.
And a plurality of, for example, six, external flow paths (see the plan view in FIG. 3) 29A to 29F that open to the outside of the syringe and face in different directions. .
By rotating the rotary joint 31 in the syringe 21 by the motor 33 and switching its direction, the central flow path 2
7 can be selectively communicated with one of the six external flow paths 29A to 29F.

FIG. 4 shows an overall configuration of an example of a detection system in which the SPR sensor device shown in FIG. 1 and the pump shown in FIG. 2 are combined.

The buffer liquid tank 43, the test liquid tank 45, the dissociating liquid tank 47, the waste liquid pipe 49, and the buffer liquid tank 43 are provided in five (29B to 29F) of the six external flow paths of the pump 41 shown in FIG. The supply pipe 1 of the SPR sensor device 51 shown in FIG. 1 is connected. Motors 25 and 33 of pump 41
(See FIG. 2) is driven by a motor driver 55 under the control of the microcomputer 53. The buffer solution, the test solution, and the dissociation solution can be selectively supplied to the SPR sensor device 51 through the pump 41. An output signal 57 from the light receiving element 10 of the SPR sensor device 51 is taken into the microcomputer 53 (or taken into another computer (not shown)) and is automatically processed.

FIG. 5 is a flowchart showing one embodiment of the SPR detection method of the present invention. FIG. 6 shows a change in the plasmon resonance angle in the SPR sensor device during the execution of this detection method. This detection method is shown in FIG.
2 can be used, or a conventional general detection system can be used.
When the system shown in FIG. 5 is used, the procedure shown in FIG.

First, a buffer solution is sent to the SPR sensor device, for example, for 2 minutes (step S1). During this time, the plasmon resonance angle is settled at the base value. Next, the feeding of the test liquid (for example, albumin solution) to the SPR sensor device is started (S2, time t0 in FIG. 6). Then, the plasmon resonance angle starts to change. The plasmon resonance angle approaches a specific value corresponding to the albumin concentration in the test solution as time elapses while the test solution is continuously supplied. The plasmon resonance angle is measured when a first fixed time, for example, 20 seconds, has elapsed (time t1 in FIG. 6) from the start of the supply of the test liquid (S3). Subsequently, at the time when a second fixed time, for example, 40 seconds, has elapsed from the start of the supply of the test liquid (time t2 in FIG. 6).
The plasmon resonance angle is measured again (S3). And 2
The albumin concentration is determined from the difference between the two plasmon resonance angles measured after 0 seconds and 40 seconds (that is, the amount of change in the resonance angle during the 20 seconds) (S5). Thereafter, the supply of the test solution is stopped, and then the dissociation solution is allowed to flow, for example, for 3 minutes (S6), and then the buffer solution is allowed to flow for 4 minutes (S7). Thereby, the plasmon resonance angle returns to the base value.

Incidentally, the detection method according to the present invention comprises:
The amount of change in the resonance angle during a certain period of time (for example, after 20 seconds to 40 seconds) while the resonance angle of the test solution is being changed, in other words, the average amount of change in the test solution during the transfer. Resonance angle change per unit time (time derivative of resonance angle, ie change speed)
Therefore, the method is used to determine the concentration of the detection target in the test solution, and this method is hereinafter referred to as “differential method”. In contrast,
In the conventional detection method, the resonance angle (base value) before the start of liquid supply of the test liquid and the resonance angle (end point) at the time when the resonance angle after the test liquid has been sent for a sufficient time have completely changed have been changed. This is a method for determining the concentration of a target substance in a test liquid based on the difference from the above point), and this conventional method is hereinafter referred to as an “endpoint method”.

FIGS. 7 and 8 show the actual calibration curves of albumin using the same SPR sensor device and the same albumin standard solution, respectively, by the differential method and the end point method. Note that one pixel in the number of pixels on the vertical axis in the figure corresponds to 1/100 degree in the resonance angle.

In order to prepare these calibration curves, albumin standard solutions having five concentrations (1, 3, 10, 30, 100 μg / ml) were prepared. Buffer solution 2 per measurement
Test solution (albumin standard solution) for 1 minute, buffer solution 2
For 3 minutes, dissociation solution for 3 minutes and buffer solution for 4 minutes,
During that time, the resonance angle was measured every two seconds. Five measurements were performed for each concentration.

With respect to the differential method, 2
From the amount of change in the resonance angle for 20 seconds from 0 seconds to 40 seconds, this 2
The amount of change in the resonance angle per unit time in 0 seconds was calculated. Then, the amount of change in the resonance angle per unit time is calculated five times for each concentration, the CV (coefficient of variation) value based on the standard deviation is calculated, and the abscissa represents the albumin concentration (logarithm),
The calibration curve shown in FIG. 7 was created by taking the amount of change on the vertical axis. For the end point method, the difference between the resonance angles before and after the test solution was sent five times for each concentration was determined, and the C
The V value was determined, and the calibration curve shown in FIG. 8 was created by taking the albumin concentration (log) on the horizontal axis and the above difference on the vertical axis. FIG.
Is the differential method and the endpoint method
The V value is shown in comparison.

As can be seen from FIGS. 7 and 8, a sufficiently practical calibration curve could be obtained by either the differentiation method or the end point method. However, as can be seen from FIG. 9, the CV value is smaller in the differential method than in the end point method. In particular, when the CV value of a low-concentration albumin solution is compared, for example, 1 μg
The endpoint method was 8.8% for the / ml albumin solution, but was significantly improved to 4.28% for the differential method of the present invention. Therefore, the differential method can perform detection with higher accuracy than the endpoint method.

FIG. 10 to FIG.
Shows the configuration of some embodiments of the analysis system.

10 to 13, reference numeral 60 denotes a column (for example, TSKg) packed with a carrier having a molecular sieve action.
el G3000PWxl) HPLC device (high pressure LC)
The test liquid is sent to the column 65 at a high pressure from the test liquid tank 61 by the pump 63. In the embodiment of FIG. 10, an absorbance meter 67 is connected downstream of the column 65, and an SPR sensor device 71 for detection in which a substance that specifically reacts with the target substance is immobilized on the detection surface of the SPR sensor downstream thereof, Another reference SPR sensor device 69 provided with a reaction prevention film on the sensor detection surface that does not react with any substance is connected in parallel. FIG.
In one embodiment, an absorbance meter 67, a detection SPR sensor device 71, and a reference SPR sensor device 69 are connected in parallel downstream of the column 65. In the embodiment of FIG. 12, an absorbance meter 67, a reference SPR sensor device 69, and a detection SPR sensor device 71 are connected in series downstream of the column 65. In the embodiment of FIG. 13, a reference SPR sensor device 69, an absorbance meter 67, and a detection SPR sensor device 71 are provided downstream of the column 65.
And are sequentially connected in series. Since the concentration of the target substance in the test liquid decreases after passing through the detection SPR sensor device 71, the detection SPR sensor device 71 and another measurement device are connected in series as shown in FIGS. In such a case, the detection SPR sensor device 71 should be arranged at the most downstream. As the SPR sensor devices 69 and 71 used here, for example, those shown in FIG. 1 that can withstand high-pressure liquid sending are preferable.

In the analysis system shown in FIGS. 10 to 13, the identification of which peak of the UV absorption chart output from the absorbance meter 67 corresponds to a desired substance is performed by the SPR sensor devices 69 and 71. This can be done in real time using the change in the angle difference.

An example in which the separation and detection of the Escherichia coli σ32 factor were actually performed using the embodiment of FIG. 10 will be described below. Escherichia coli K-1 in Davis minimal medium supplemented with 0.1% yeast extract
Two strains were cultured at 37 ° C. for 8 hours. Then, the cells were reduced to 0.1
The resultant was washed by centrifugation using an M phosphate buffer (pH 7.0), and finally suspended in 0.1 M phosphate buffer (pH 7.0) of 1/5 volume of the culture solution ( Cell concentration; about 10
10th power / ml). Dispense 5 ml of this suspension into test tubes, one in a constant temperature water bath at 37 ° C and the other in 42 ° C.
After keeping the temperature for 10 minutes, keeping the temperature and then cooling on ice, the cells were immediately crushed using a French press, and the supernatant (cell-free extract) after centrifugation was collected.

Subsequently, as column 65, TSKgel G3000PW
xl equipped with HPLC device 60, flow rate 1.0 ml
Each cell-free extract was separated using a 0.2 M / min 0.2 M phosphate buffer as a mobile phase.

In the subsequent stage of the column 65, an absorbance meter 67 for monitoring the absorbance at 280 nm was used.
The absorbance of the eluate from 4 was monitored. In the subsequent stage, a detection SPR sensor device 71 in which an anti-σ32 factor mouse monoclonal antibody is immobilized on the sensor detection surface.
SP of the reference SPR sensor device 69 provided with a non-specific adsorption prevention film on the sensor detection surface without immobilizing the antibody
R sensor output difference (hereinafter referred to as SPR response)
Changes were monitored.

The result of the monitoring is shown in FIG.
This is shown in FIG. 15 (for cells kept at 42 ° C.) and FIG. 15 (for cells kept at 42 ° C.). In FIGS. 14 and 15, the dotted lines show the change with time of the output of the absorbance meter, and the solid lines show the SP.
The change with time of the R response is shown. FIG.
As can be seen from FIG. 4, almost no change in response due to binding to the anti-σ32 factor mouse monoclonal antibody was observed in the cell-free extract of the cells incubated at 37 ° C. On the other hand, as indicated by the arrow in FIG.
In cell-free extracts of the cells incubated at ℃, changes in response due to binding to anti-σ32 factor mouse monoclonal antibody were clearly observed (ie, it was confirmed that σ32 factor is a heat shock protein) ). in this way,
By overlapping the pattern of the UV absorption from the absorbance meter 67 with the pattern of the SPR response, it was possible to simultaneously separate and identify the component corresponding to the σ32 factor from the cell-free extract of E. coli.

Although several embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, and various other modifications and improvements may be made without departing from the scope of the invention. But it can be implemented.
For example, it goes without saying that the same effects as described above can be obtained even if the detection target is another protein, glycoprotein, nucleic acid, or the like. In FIG. 15, after the response of the SPR sensor changes, the analyte adsorbed on the detection portion of the sensor may be separated and regenerated using hydrochloric acid or the like, and the subsequent measurement may be continued.

[0054]

As described above, the SPR sensor device according to one aspect of the present invention has a high measurement accuracy because a laminar flow can flow on the sensor detection surface even though it is small.

[0055] The SPR sensor device according to another aspect of the present invention has a small built-in electric sensor, and therefore can simultaneously detect a plurality of substances even if it is small.

The differential method, which is the SPR detection method of the present invention, has high detection accuracy.

The analysis system of the present invention comprises a separation and purification of a physiologically active substance using an LC, a measurement of a physiologically active substance,
Since the identification of the peak of the desired physiologically active substance by the R apparatus can be performed substantially simultaneously, the efficiency of analysis using LC is improved.

[Brief description of the drawings]

FIG. 1 is a sectional view of a main part of an embodiment of an SPR sensor device of the present invention.

FIG. 2 is a sectional view of an example of a pump applicable to the SPR sensor device shown in FIG.

FIG. 3 is a plan view showing an arrangement of external flow paths of the pump of FIG. 2;

4 is an overall configuration diagram of an example of a detection system in which the SPR sensor device shown in FIG. 1 and the pump shown in FIG. 2 are combined.

FIG. 5 is a flowchart showing one embodiment of the SPR detection method of the present invention. The top view which shows arrangement of the external flow path of 2 pumps.

FIG. 6 is a diagram showing a change in a plasmon resonance angle during the execution of the detection method of FIG. 5;

FIG. 7 is a diagram showing a calibration curve of albumin according to the measurement method of the present invention.

FIG. 8 is a diagram showing a calibration curve of albumin according to a conventional method.

FIG. 9 shows a comparison between CV values obtained by the differential method and the end point method.

FIG. 10 is a configuration diagram of a first embodiment of the analysis system of the present invention.

FIG. 11 is a configuration diagram of a second embodiment of the analysis system of the present invention.

FIG. 12 is a configuration diagram of a third embodiment of the analysis system of the present invention.

FIG. 13 is a configuration diagram of a fourth embodiment of the analysis system of the present invention.

FIG. 14: UV absorption pattern of the eluate of the cell-free extract of Escherichia coli subjected to heat treatment at 37 ° C. for 10 minutes and SPR for detecting σ32 factor
The figure which showed the pattern of the response.

FIG. 15: UV absorption pattern of eluate of E. coli cell-free extract heat-treated at 42 ° C. for 10 minutes and SPR for detecting σ32 factor
The figure which showed the pattern of the response.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Supply pipe 2 SPR sensor detection surface 3 Prism 4 Discharge pipe 5 Pipe lifting mechanism 9 Electrode 41 Pump 51, 69, 71 SPR sensor apparatus 60 HPLC apparatus 65 Column 67 Absorbance meter

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Keisuke Kanzaki, 2-1-1 Nakajima, Kokurakita-ku, Kitakyushu-shi, Fukuoka Prefecture Totoki Equipment Co., Ltd. (72) Shuji Sonezaki 2, Nakajima, Kokurakita-ku, Kitakyushu-shi, Fukuoka (1-1) Emi Ogawa 2-1-1 Nakajima, Kokurakita-ku, Kitakyushu-city, Fukuoka Prefecture

Claims (10)

[Claims] An SPR sensor surface having a detection surface to which surface plasmon excitation light is irradiated, and a tube for supplying a fluid to the detection surface, wherein the fluid flows through the inside as a laminar flow. A supply pipe having an outlet that is sufficiently long and has an outlet disposed in close proximity to the detection surface. 2. The SPR sensor device according to claim 1, wherein an inner diameter of the supply pipe is larger than a diameter of a spot of the surface plasmon excitation light applied to the detection surface. 3. The SPR sensor device according to claim 1, wherein an inner diameter of the supply pipe near the outlet smoothly expands in a trumpet shape toward the outlet. 4. The SPR sensor device according to claim 1, wherein an outlet surface of the supply pipe is parallel to the detection surface. 5. The SPR sensor device according to claim 1, wherein a distance between the detection surface and an outlet of the supply pipe is variable. 6. The SPR sensor device according to claim 1, wherein the pipe is for discharging the fluid, has an inner diameter larger than the supply pipe, and is arranged so as to surround the supply pipe. An SPR sensor device further comprising an outlet tube having an inlet surface coupled to the SPR sensor surface around a detection surface. 7. The SPR sensor device according to claim 6, wherein an inner diameter near an inlet of the discharge pipe is the SPR.
An SPR sensor device that narrows in a curved manner so as to be continuously connected to the sensor surface. 8. The SPR sensor device according to claim 6, wherein an electrode for an electrochemical sensor is attached to at least one of an outer peripheral surface of the supply pipe and an inner peripheral surface of the discharge pipe. . 9. A method for obtaining a concentration of a target substance contained in the test liquid from a change amount per unit time of a surface plasmon resonance angle detected by the SPR sensor while introducing the test liquid into the SPR sensor. SPR detection method. 10. A column for separating a plurality of substances contained in a test liquid, an absorbance meter into which an eluate from the column is introduced, and a signal responsive to a target substance, into which an eluate from the column is introduced. An analysis system comprising: an SPR sensor device that outputs a signal;