JPWO2020090283A1 - Method and detection device for detecting abnormal detection - Google Patents

Abstract

In the method for detecting a detection abnormality in the present invention, a sample in the reagent storage region is transferred to the reaction region using a pipette tip for sucking or discharging the liquid mounted on the pipette nozzle, and the sample in the sample in the reaction region is covered. A step of detecting a substance to be detected, a step of measuring the pressure at the time of discharging the sample in the pipette tip after sucking the sample remaining in the reagent storage area with the pipette tip, information obtained by the measured pressure, and a reference. It includes a step of determining the detection result of the step of detecting by comparing with the value.

Description

The present invention relates to a method and a detection device for detecting a detection abnormality.

If a small amount of a substance to be detected such as protein or DNA can be detected with high sensitivity and quantitatively in a clinical test or the like, the patient's condition can be quickly grasped and treated. Therefore, there is a demand for a method capable of detecting a trace amount of a substance to be detected with high sensitivity and quantitatively. In order to detect a small amount of a substance to be detected with high sensitivity and quantitatively, it is necessary to accurately measure a sample containing the substance to be detected and a reagent used for detection.

In order to accurately measure a liquid such as a sample or a reagent, a method is known in which a gas is taken in and out from the tip of a pipette tip to detect the position of the tip of the pipette tip (see, for example, Patent Document 1).

Patent Document 1 describes a pipette tip when gas is sucked or discharged from the tip of the pipette tip in a state where the tip of the pipette tip and the surface of the liquid contained in the storage portion are separated from each other and in a state where they are brought close to each other. A detection method is described in which the pressure inside is measured and the position of the tip of the pipette tip with respect to the liquid surface is detected based on the measured pressure difference. In the technique described in Patent Document 1, the pipette tip is moved based on the detected tip position of the pipette tip to suck the liquid. Then, the substance to be detected in the aspirated sample is detected.

International Publication No. 2016/132793

However, in the technique described in Patent Document 1, when bubbles are contained in the liquid, the upper end of the bubbles may be erroneously detected as the surface of the liquid. As a result, even when the amount of the sucked liquid is extremely smaller than the specified amount, it is erroneously recognized that the sample is sucked according to the specified amount. If the device operates as it is with an extremely small amount of sample, it may not be possible to obtain a normal signal.

An object of the present invention is to provide a method and a detection device for detecting a detection abnormality capable of appropriately detecting a sample amount and detecting an abnormality in the detection result.

As one aspect for solving the above-mentioned problems, the method for detecting a detection abnormality of the present invention reacts a sample in a reagent storage area with a pipette tip for sucking or draining a liquid mounted on a pipette nozzle. The step of detecting the substance to be detected in the sample in the reaction region while moving to the region, and the pressure at the time of discharging the sample in the pipette tip after sucking the sample remaining in the reagent storage region with the pipette tip. It includes a step of measuring, a step of determining a detection result of the step to be detected by comparing the information obtained by the measured pressure with a reference value, and a step of determining the detection result of the step to be detected.

As one aspect for solving the above-mentioned problems, the detection device of the present invention has a pipette tip attached to a pipette nozzle, a liquid storage area for storing a sample, and a reaction area for reacting the sample. A detection device that detects a substance to be detected in a sample in the reaction region using a tip, and is a pipette including a tip holder for holding the reaction tip and a pipette nozzle to which the pipette tip can be attached and detached. And, after sucking the sample remaining in the reagent storage area with the pipette tip, the air pressure sensor for measuring the pressure at the time of discharging the sample in the pipette tip, and the control for controlling the pipette and the air pressure sensor. The control unit determines the detection result of the substance to be detected by comparing the information obtained by the measured pressure with the reference value.

According to the present invention, since the sample amount can be appropriately detected and an abnormality in the detection result can be detected, the reliability of the detection result can be improved.

FIG. 1 is a schematic view of a detection device. FIG. 2 is a schematic view of the detection chip. FIG. 3 is a flowchart showing the operation of the detection device. FIG. 4A is a flowchart showing the contents of the process of acquiring the position information, and FIG. 4B is a flowchart showing the contents of the determination of the detection result. FIG. 5 is a schematic graph showing the relationship between the amount of the sample and the pressure when the sample is discharged.

Hereinafter, an embodiment of the present invention will be described.

First, an example of the detection device 100 for implementing the method of detecting the detection abnormality will be described.

FIG. 1 is a diagram showing the configuration of the detection device 100. FIG. 2 is a schematic view of the detection chip 200. As shown in FIG. 1, the detection device 100 includes a liquid feeding unit 110, a transport unit 120, a position information acquisition unit 130, a detection unit 140, and a control unit 150. The detection device 100 is used with the detection chip 200 attached.

(Configuration of detection chip)
As shown in FIG. 2, the detection chip 200 has a reaction region 210 and a reagent storage region 220. The detection chip 200 may be a reproducible chip or a disposable chip. In this embodiment, the detection chip 200 is a disposable chip. Examples of the liquid stored in the reagent storage area 220 include a sample containing the substance to be detected (for example, blood, serum, plasma, urine, nasal fluid, saliva, semen, etc.) and a fluorescent substance. A labeling solution containing a trap and a cleaning solution are included.

The reaction region 210 is a region in which a reaction or the like is carried out in order to detect a substance to be detected in a sample. A flow path 212 may be arranged or a well may be arranged in the reaction region. In the present embodiment, the flow path 212 is formed in the reaction region 210.

The reagent storage area 220 is an area in which a sample, a labeling liquid, a washing liquid, and the like are stored. The reagent storage region 220 has a plurality of recesses 222. The number of recesses 222 is not particularly limited. In the present embodiment, the number of recesses 222 is two. A sample, a labeling liquid, a cleaning liquid, and the like are stored in the recess 222.

(Configuration of detection device)
Next, each component of the detection device 100 according to the present embodiment will be described. As described above, the detection device 100 includes a liquid feeding unit 110, a transport unit 120, a position information acquisition unit 130, a detection unit 140, and a control unit 150. The detection chip 200 can be held in the chip holder 121 of the transport unit 120.

The liquid feeding unit 110 includes a pipette 111, a pipette moving unit 112, and a liquid feeding pump driving mechanism 113. The liquid feeding unit 110 moves liquids such as a sample, a labeling liquid, and a washing liquid stored in the recess 222 of the reagent storage region 220 of the detection chip 200 held in the chip holder 121 into the flow path 212 of the reaction region 210. do. In addition, the liquid feeding unit 110 also discharges the liquid from the flow path 212 and agitates the liquid in the flow path 212. The liquid feeding unit 110 is used with the pipette tip 170 attached to the pipette nozzle 116 of the pipette 111. As the pipette tip 170, any commercially available pipette tip can be used. From the viewpoint of preventing impurities from being mixed in, the pipette tip 170 is preferably replaceable.

The pipette 111 sucks and discharges the liquid when injecting the liquid into the flow path 212 or removing the liquid from the flow path 212. The pipette 111 has a syringe 114, a plunger 115 capable of reciprocating in the syringe 114, and a pipette nozzle 116 connected to the syringe 114. Further, the pipette 111 can quantitatively suck and discharge the liquid by the reciprocating motion of the plunger 115. As a result, the pipette 111 can inject a liquid into the flow path 212 and remove the liquid from the flow path 212. Further, the pipette 111 can agitate the liquid in the flow path 212 by repeating suction and discharge of the liquid.

The pipette moving unit 112 moves the pipette nozzle 116 for sucking the liquid into the pipette tip 170 or for discharging the liquid from the pipette tip 170. The pipette moving portion 112, for example, freely moves the pipette nozzle 116 in the axial direction (for example, the vertical direction) of the pipette nozzle 116. The pipette moving unit 112 includes, for example, a solenoid actuator and a stepping motor.

The liquid feed pump drive mechanism 113 moves the plunger 115 to suck the external liquid into the pipette tip 170 or discharge the liquid in the pipette tip 170 to the outside. The liquid feed pump drive mechanism 113 includes a device for reciprocating the plunger 115 such as a stepping motor. Since the stepping motor can control the liquid feed amount and the liquid feed rate of the pipette 111, it is preferable from the viewpoint of controlling the residual liquid amount in the reagent storage region 220.

As described above, the liquid feeding unit 110 sucks various liquids from the recess 222 and injects them into the flow path 212 of the detection chip 200. At this time, while the tip of the pipette tip 170 is close to the bottom surface of the flow path 212 in the flow path 212, the reciprocating operation of the plunger 115 with respect to the syringe 114 is repeated, so that the liquid is liquid in the flow path 212 in the detection tip 200. Is agitated. As a result, it is possible to make the concentration of the liquid uniform and promote the reaction in the flow path 212.

The transport unit 120 transports the detection chip 200 to the detection position or the liquid feeding position, and holds the detection chip 200. Here, the "detection position" is a position where the detection unit 140 detects the substance to be detected in the sample. The "liquid feeding position" is a position where the liquid feeding unit 110 injects the liquid into the flow path 212 of the detection chip 200 or removes the liquid in the flow path 212 of the detection chip 200. The transport unit 120 includes a tip holder 121 and a transport stage 122.

The chip holder 121 is fixed to the transport stage 122 and holds the detection chip 200 in a detachable manner. The shape of the chip holder 121 is not particularly limited as long as it can hold the detection chip 200 and does not interfere with the detection of the substance to be detected. In the present embodiment, the shape of the chip holder 121 is configured so that the detection chip 200 can be held from the side surface direction.

The transport stage 122 moves the chip holder 121 in one direction and vice versa (left-right direction on the paper surface of FIG. 1). The transport stage 122 also has a shape that does not interfere with the detection of the substance to be detected. The transport stage 122 is driven by, for example, a stepping motor or the like.

The position information acquisition unit 130 acquires position information (hereinafter, also simply referred to as “position information”) regarding the position of the tip of the pipette tip 170 with respect to the liquid surface. The position information acquisition unit 130 includes an air pressure sensor 131. The air pressure sensor 131 is connected between the pipette nozzle 116 and the syringe 114. The type of the air pressure sensor 131 is not particularly limited as long as it can measure the air pressure (pressure) in the pipette tip 170. Examples of the types of the air pressure sensor 131 include a mechanical sensor using a Bourdon tube, an electronic sensor using a semiconductor, and the like.

The detection unit 140 detects the substance to be detected in the flow path 212 of the reaction region 210. The configuration of the detection unit 140 is not particularly limited as long as the substance to be detected can be detected. The configuration of the detection unit 140 is appropriately selected depending on the method for detecting the substance to be detected. For example, when the detection device 100 is a surface plasmon resonance excitation enhanced fluorescence spectroscopy (SPFS) device, the detection unit 140 may be configured to be able to detect the generated plasmon scattered light and fluorescence.

The control unit 150 controls the liquid feed pump drive mechanism 113, the transfer stage 122, the air pressure sensor 131, and the like. The control unit 150 is composed of, for example, a known computer or microcomputer including an arithmetic unit, a control device, a storage device, an input device, and an output device.

(Detection operation of detection device)
Next, the detection operation of the substance to be detected of the detection device 100 will be described. FIG. 3 is a flowchart showing an example of the operation procedure of the detection device 100. FIG. 4A is a flowchart showing the contents of the step of acquiring the position information (step S120 in FIG. 3), and FIG. 4B is a flowchart showing the contents of the step of determining the detection result (step S150 in FIG. 3).

First, preparation for measurement is performed (step S110). Specifically, the detection chip 200 is prepared, and the detection chip 200 is installed in the chip holder 121 at the set position of the detection chip 200. Further, the pipette tip 170 is attached to the tip of the pipette nozzle 116.

Next, the position information is acquired (step S120). First, the first pressure in the pipette tip 170 is measured (step S121). Specifically, the control unit 150 drives the pipette moving unit 112 to move the tip of the pipette tip 170 directly above the liquid level of the liquid stored in the recess 222. Then, the control unit 150 drives the liquid feed pump drive mechanism 113 to advance the plunger 115 with respect to the syringe 114, and while continuously ejecting air from the tip of the pipette tip 170, the pipette is operated by the air pressure sensor 131. The first pressure in the chip 170 is measured.

Next, the second pressure in the pipette tip 170 is measured (step S122). Specifically, the control unit 150 drives the pipette moving unit 112 and makes the tip of the pipette tip 170 closer to the liquid level side of the liquid stored in the recess 222 than in the step of measuring the first pressure (step S121). Move to. Then, the control unit 150 drives the liquid feed pump drive mechanism 113 to advance the plunger 115 with respect to the syringe 114, and while continuously ejecting air from the tip of the pipette tip 170, the pipette is operated by the air pressure sensor 131. The second pressure in the chip 170 is measured.

Next, the difference between the first pressure and the second pressure is obtained (step S123). Specifically, the control unit 150 obtains the difference between the first pressure and the second pressure by subtracting the second pressure (first pressure) from the first pressure (second pressure). Then, the control unit 150 detects the position of the tip of the pipette tip 170 with respect to the liquid level or the height of the liquid level due to the difference between the first pressure and the second pressure. That is, the control unit 150 acquires the position information of the tip of the pipette tip 170 with respect to the liquid level or the height of the liquid level by detecting the pressure by the air pressure sensor 131. The steps of moving the tip of the pipette tip 170 and measuring the second pressure are repeated until the difference between the first pressure and the second pressure becomes equal to or higher than a predetermined threshold value.

In the step of acquiring the position information (step S120), the pipette tip is continuously or intermittently ejected from the tip of the pipette tip 170, and the tip of the pipette tip 170 is brought close to the liquid level by the pipette tip 131. The pressure in 170 may be measured. In this case, the pressure before moving the pipette tip 170 becomes the first pressure. Further, the pressure inside the pipette tip 170 measured by the air pressure sensor 131 while bringing the tip of the pipette tip 170 close to the liquid level becomes the second pressure.

Next, the substance to be detected in the sample is detected (step S130). In this embodiment, the sample and the reagent are mixed to detect the substance to be detected contained in the sample. The control unit 150 operates the transport stage 122 to move the recess 222 in which the sample is stored to directly below the pipette tip 170. Then, based on the acquired position information, the tip of the pipette tip 170 is moved toward the recess 222 in which the sample is stored, and the sample is sucked into the pipette tip 170. Then, the control unit 150 drives the pipette moving unit 112 to move the tip of the pipette tip 170 into the flow path 212, and injects the sample into the flow path 212.

At this time, as shown in FIG. 2, when the bubbles B are present on the surface of the sample, the control unit 150 recognizes the upper end of the bubbles B as the liquid level. In this case, since the tip of the pipette tip 170 is not sufficiently moved into the recess 222, it may not be possible to inhale the sample amount required for detection. Then, if the substance to be detected is detected while the amount of the sample is small, an inaccurate detection result will be obtained. Therefore, in the present embodiment, it is determined whether or not the detection step is properly performed by measuring the sample amount after the detection step in the reagent storage region 220.

Further, the control unit 150 operates the transfer stage 122 to move the recess 222 in which the reagent is stored to directly below the pipette tip 170. Then, based on the acquired position information, the tip of the pipette tip 170 is moved toward the recess 222 in which the reagent is stored, and the reagent is sucked into the pipette tip 170. Then, the control unit 150 drives the pipette moving unit 112 to move the tip of the pipette tip 170 into the flow path 212, and injects the reagent into the flow path 212.

Next, the detection unit 140 detects the substance to be detected. Specifically, the control unit 150 operates the transport stage 122 to move the detection chip 200 to the detection position. Then, the detection unit 140 detects the substance to be detected in the sample.

The sample may be stored in another container. In this case, the detection chip 200 is formed with a storage hole for housing the container.

The types of the sample and the substance to be detected are not particularly limited. Examples of specimens include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, semen and dilutions thereof. Examples of substances to be detected include nucleic acids (such as DNA and RNA), proteins (such as polypeptides and oligopeptides), amino acids, sugars, lipids and modified molecules thereof. For example, blood and the like are generally highly viscous, so bubbles are likely to be generated.

Next, the pipette tip 170 sucks the sample remaining in the recess 222, and then the pressure at which the sample in the pipette tip 170 is discharged is measured (step S140). Specifically, the control unit 150 operates the transport stage 122 to move the flow path 212 in which the sample is stored directly below the pipette tip 170. Then, the tip of the pipette tip 170 is moved to the bottom of the flow path 212 in which the sample is stored, and all the samples remaining in the flow path 212 are sucked into the pipette tip 170. The height of the bottom of the flow path 212 has been determined in advance.

The control unit 150 operates the transfer stage 122 to move the flow path 212 directly below the pipette tip 170. Then, the sample in the pipette tip 170 is discharged to the flow path 212. At this time, the control unit 150 measures the air pressure in the pipette tip 170 by the air pressure sensor 131.

Then, the detection result of the detection step (step S130) is determined by comparing the information obtained from the measured pressure with the reference value (step S150). In the present embodiment, when the measured pressure is equal to or higher than the reference value (step S151; Yes), the detection result of the detection step (step S130) is determined to be "no abnormality in detection" (step S152). When the measured pressure is less than the predetermined value (step S152; No), the detection result of the detection step (step S130) is determined to be "abnormal in detection" (step S153).

When the amount of residual sample estimated based on the measured pressure is equal to or greater than the reference value, the detection result of the detection step (step S130) is determined to be "no abnormality in detection", and the estimated residual sample is used. When the amount is less than the reference value, the detection result of the detection step (step S130) may be determined to be "abnormal in detection".

Here, the concept of the default value will be described. The specified value is determined corresponding to the minimum amount of sample that should remain in the recess 222 after the detection step (step S130). As the amount of sample in the pipette tip 170 increases, the pressure at which the sample is ejected from the pipette tip 170 increases. Therefore, when determining by the pressure at the time of discharging the sample from the pipette tip 170, the pressure at the time of discharging the minimum amount of the sample that should remain in the recess 222 may be set as the specified value. Further, when determining the amount of the sample estimated from the pressure when the sample is discharged from the pipette tip 170, the minimum amount of the sample that should remain in the recess 222 may be set as the specified value.

FIG. 5 is a schematic graph showing the relationship between the amount of the sample and the pressure when the sample is discharged.

In order to obtain the reference value, first, the relationship between the amount of the sample in the pipette tip 170 and the pressure when the sample is discharged is obtained. Specifically, for example, 0 μL, 40 μL, 90 μL, 140 μL, and 150 μL are sucked. Then, after suction, all the samples are discharged into the flow path, and the maximum pressure at the time of discharge is obtained. By repeating this process 6 times, the average value of the pressure at the time of discharge is obtained. Next, the relationship between the amount of the sample discharged to the flow path 212 and the average value of the pressure when the sample is discharged is obtained. As shown in FIG. 5, the amount of the sample discharged into the recess 222 and the pressure at the time of discharging the sample are in a proportional relationship.

For example, when there are no bubbles in the sample, the lower limit of the amount of the sample for determining that the detection result in the detection step (step S130) is “abnormal in detection” may be about 146 μL. Then, in the protocol that uses the sample most, 110 μL of the sample is used, and the minimum liquid amount of the remaining sample amount is 36 μL from the lower limit amount and the used amount. The calculated reference value of the residual sample amount is 36 μL, but 40 μL is set as the reference value including the variation in the liquid amount. The average value (actual measurement value) of the pressure when 40 μL of the liquid is discharged is about 1.7 kPa. However, considering the variation in discharge pressure (3SD), the lower limit value (reference value) of the pressure is 0.67 kPa.

The method of determining the amount of the remaining sample from the discharged pressure may be obtained from the relationship between the amount of the sample discharged into the recess 222 described above and the pressure at the time of discharging the sample. In this case as well, it is necessary to consider the variation in discharge pressure (3SD).

(effect)
As described above, according to the present invention, since the presence or absence of the detection result is determined by comparing the information obtained by the pressure and the reference value, the abnormality of the detection result can be appropriately detected.

This application claims priority under Japanese Patent Application No. 2018-205571 filed on October 31, 2018. All the contents described in the application specification and drawings are incorporated in the specification of the present application.

The method for detecting a detection abnormality according to the present invention can improve the reliability of the detection result of the substance to be detected, for example. Therefore, it is expected to contribute to the development, dissemination and development of the detection system for trace substances to be detected.

100 Detection device 110 Liquid transfer unit 111 Pipette 112 Pipette movement unit 113 Liquid transfer pump drive mechanism 114 Syringe 115 Plunger 116 Pipette nozzle 120 Transfer unit 121 Chip holder 122 Transfer stage 130 Position information acquisition unit 131 Pneumatic sensor 140 Detection unit 150 Control unit 170 Pipette tip 200 Detection tip 210 Reaction area 220 Reagent storage area 212 Channel 222 Recess

Claims (6)

A step of transferring the sample in the reagent storage area to the reaction area using a pipette tip for sucking or discharging the liquid mounted on the pipette nozzle, and detecting a substance to be detected in the sample in the reaction area.
A step of measuring the pressure at the time of discharging the sample in the pipette tip after sucking the sample remaining in the reagent storage area with the pipette tip, and
A step of determining the detection result of the step to be detected by comparing the information obtained by the measured pressure with a reference value, and a step of determining the detection result of the step to be detected.
How to detect detection anomalies, including. In the determination step, when the measured pressure is equal to or higher than the reference value, the detection result of the detection step is determined to be "no abnormality in detection", and when the measured pressure is less than the reference value. Determines that the detection result of the detection step is "abnormal in detection".
The method for detecting an abnormality in the detection according to claim 1. In the determination step, when the amount of residual sample estimated based on the measured pressure is equal to or greater than the reference value, the detection result of the detection step is determined to be "no abnormality in detection" and estimated. When the amount of the remaining sample is less than the reference value, the detection result of the detection step is determined to be "abnormal in detection".
The method for detecting an abnormality in the detection according to claim 1. Using a pipette tip attached to a pipette nozzle and a reaction tip having a reagent storage area for storing a sample and a reaction area for reacting a sample, a substance to be detected in the sample is detected in the reaction area. It is a detection device that
A chip holder for holding the reaction chip and
A pipette including a pipette nozzle to which the pipette tip can be attached and detached, and
An air pressure sensor that measures the pressure at the time of discharging the sample in the pipette tip after sucking the sample remaining in the reagent storage area with the pipette tip.
It has a control unit that controls the pipette and the air pressure sensor.
The control unit determines the detection result of the substance to be detected by comparing the information obtained by the measured pressure with the reference value.
Detection device. When the measured pressure is equal to or higher than the reference value, the control unit determines that the detection result of the substance to be detected is "no abnormality in detection", and when the measured pressure is less than the reference value, the control unit determines. Judge that the detection result of the substance to be detected is "abnormal in detection",
The detection device according to claim 4. When the amount of residual sample estimated based on the measured pressure is equal to or greater than the reference value, the control unit determines that the detection result of the substance to be detected is "no abnormality in detection" and estimates the residual amount. If the amount of the sample is less than the standard value, the detection result of the substance to be detected is judged to be "abnormal in detection".
The detection device according to claim 4.

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