CN115575319A - Luminescence analysis device and method for adjusting sensitivity of light emission analysis device - Google Patents

Abstract

The present invention provides a light emission analyzer capable of accurately judging the sensitivity change of a photodetector without cost and realizing miniaturization of the device and a sensitivity adjustment method thereof. The luminescence analysis device obtains the amount of luminescence in the sample solution from the measurement solution (4) containing the sample solution and a luminescent reagent that emits light by reacting with the analyte component in the sample solution and a calibration curve. The component concentration is characterized in that it is equipped with: a photodetector (20) that detects light from the measurement liquid (4); When the calculation device (30) utilizes the luminescence detected by the photodetector (20) and the concentration of the analyte in the calibration curve when using a blank solution that does not contain the analyte as the sample solution in the measurement solution 4, it is zero. The luminescence amount is compared to confirm the sensitivity of the photodetector (20).

Description

Luminescence analysis device and method for adjusting sensitivity of light emission analysis device

technical field

The invention relates to a luminescence analysis device and a sensitivity adjustment method of the luminescence analysis device. More specifically, it relates to a luminescence analysis device suitable for luminescence analysis by bioluminescence and chemiluminescence, and a sensitivity adjustment method thereof.

Background technique

Since bioluminescence and chemiluminescence can quantify trace substances in a sample solution, they are used in various fields such as medicine, biochemistry, clinical examination, agriculture, and food.

For example, Patent Document 1 discloses a high-sensitivity bioluminescent method for detecting endotoxin, which releases a luminescent substrate from a synthetic substrate and makes the free luminescent substrate emit light by a reagent activated by endotoxin.

Most of the luminescence reagents used in luminescence analysis, especially in luminescence analysis based on bioluminescence, are biologically derived reagents, and the sensitivity of each batch is often different. Therefore, a calibration curve showing the relationship between the component to be measured and the amount of luminescence is obtained for each batch of the luminescence reagent.

prior art literature

patent documents

Patent Document 1: International Publication No. 2009/063840

Contents of the invention

The problem to be solved by the invention

However, the sensitivity of a photodetector used in a luminescence analysis device changes over time, and the amount of luminescence may not be accurately measured.

Therefore, it is conceivable to provide a reference light source in the device, detect the light emission amount from the light source with a photodetector, check the sensitivity change of the photodetector, and correct the measured light emission amount.

However, in this case, since it is necessary to install a reference light source in the device, not only the cost increases, but also a space for installing the light source needs to be prepared, making it difficult to reduce the size of the device. Furthermore, when the amount of light emitted by the light source itself as a reference changes with the passage of time, it is impossible to accurately determine the sensitivity of the photodetector.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a light emission analysis device and a sensitivity adjustment method thereof that can accurately determine a change in sensitivity of a photodetector without cost and can realize downsizing of the device.

Technical solution to the problem

In order to achieve the above objects, the present invention employs the following configurations.

A first aspect of the present invention is a luminescence analysis device, which is based on the amount of luminescence from a measurement solution containing a sample solution and a luminescence reagent that reacts with a component to be measured in the sample solution to emit light, and the luminescence reagent Calibration curve, obtain the component concentration to be measured in the described sample liquid, it is characterized in that, have:

a light detector that detects light from the assay liquid; and

an arithmetic means, which is input with the amount of luminescence detected by the photodetector,

The calculation device uses the luminescence amount detected by the light detector and the concentration of the analyte in the calibration curve when using a blank solution that does not contain the analyte as the sample solution in the measurement solution. The luminescence amount at zero time was compared to confirm the sensitivity of the photodetector.

A second aspect of the present invention is the luminescence analysis device according to the first aspect, wherein the calibration curve is a calibration curve determined for each batch of the luminescence reagent.

A third aspect of the present invention is a luminescence analysis device, which is based on the amount of luminescence from a measurement solution containing a sample solution and a luminescent reagent and a luminescent substance that reacts with a component to be measured in the sample solution to emit light, and the resulting The calibration curve of the luminescent reagent and the luminescent substance is used to obtain the concentration of the component to be measured in the sample solution, which is characterized in that it has:

a light detector that detects light from the assay liquid; and

an arithmetic means, which is input with the amount of luminescence detected by the photodetector,

The calculation device uses the luminescence amount detected by the light detector and the concentration of the analyte in the calibration curve when using a blank solution that does not contain the analyte as the sample solution in the measurement solution. The light emission amount at zero time was compared to confirm the sensitivity change of the photodetector.

A fourth aspect of the present invention is the luminescence analysis device according to the third aspect, wherein the calibration curve is a calibration curve determined for each batch of the luminescent reagent and the luminescent substance.

A fifth aspect of the present invention is the emission analysis device according to any one of the first to fourth aspects, wherein the photodetector is a photomultiplier tube.

A sixth aspect of the present invention is a sensitivity adjustment method of a luminescence analysis device, which is the sensitivity adjustment method of a luminescence analysis device according to the fifth aspect, wherein when the calculation device determines that the photodetector When the sensitivity of the photodetector fluctuates beyond a predetermined allowable range, the sensitivity of the photodetector is adjusted by changing the voltage applied to the photodetector.

A seventh aspect of the present invention is the sensitivity adjustment method of a luminescence analyzer according to the sixth aspect, characterized in that a reagent that emits light through a bioluminescence phenomenon is used as the luminescent reagent.

Invention effect

According to the emission analysis device and the sensitivity adjustment method thereof of the present invention, not only can the sensitivity change of the photodetector be accurately judged without cost, but also the size of the device can be realized.

Description of drawings

FIG. 1 is a schematic structural view of a light emission analysis device according to one embodiment of the present invention.

detailed description

[the first embodiment]

FIG. 1 is a light emission analysis device according to one embodiment of the present invention. The light emission analysis device of this embodiment includes: a measurement chamber 10 that can store the measurement container 1; a photodetector 20 that detects light from the measurement container 1 accommodated in the measurement chamber 10; The light emission amount detected by the photodetector 20 is input.

The measurement chamber 10 is composed of a bottomed cylindrical measurement chamber main body 11 having an opening 11a at its upper end, and a door 12 that covers the opening 11a in an openable and closable manner.

All parts of the measurement chamber main body 11 are light-shielded except for the part of the light-transmitting detection window 11b provided on the side surface. Furthermore, the door 12 is also light-shielded.

When the door 12 is in the open state (the state of the door 12 indicated by a dotted line in FIG. 1 ), the measurement container 1 can be taken out/into the measurement chamber main body 11 through the opening 11a. In addition, when the door 12 is in the closed state (the state of the door 12 indicated by the solid line in FIG. 1 ), the measurement chamber 10 can shield external light from entering the inside of the measurement chamber 10 .

As the photodetector 20, a photomultiplier tube, a photodiode, a phototransistor, an avalanche photodiode, or the like can be used as appropriate.

In this embodiment, the light detector 20 detects light only when the door 12 is in the closed state. For example, when the photodetector 20 is a photomultiplier tube, the voltage is applied to the photodetector 20 only when the door 12 is in the closed state.

The method of detecting light by the photodetector 20 is not particularly limited only when the door 12 is in the closed state. For example, the door 12 itself can be used as a switch, and the top end of the door 12 contacts the periphery of the opening 11a of the measurement chamber main body 11 to close. A circuit method for applying a voltage to the photodetector 20 . In addition, a touch sensor or the like that senses that the tip of the door 12 touches the periphery of the opening 11 a of the measurement chamber main body 11 may be used.

The measurement container 1 housed in the measurement chamber 10 is composed of a bottomed cylindrical container body 2 with an open upper end, and a cap 3 that liquid-tightly closes the upper end of the container body 2 .

The container main body 2 is made of a transparent material such as glass so as to sufficiently transmit light at least in the wavelength range of light to be detected by the photodetector 20 .

In the first embodiment, the measurement solution 4 accommodated in the measurement container 1 includes a sample solution and a luminescence reagent that reacts with analyte components in the sample solution to emit light.

In the present invention, the luminescence reagent means the reagent or the whole reagent group required to generate luminescence corresponding to the concentration of the component to be measured, and generally a reagent group composed of a plurality of reagents corresponds to the luminescence reagent.

For example, the components to be measured are microbial impurities such as endotoxin and β-glucan. When bioluminescence is used for analysis, a reagent that is activated by the microbial impurity and a reagent that activates the luminescent substrate is used. Photosynthetic substrates, luminescent enzymes that make the released luminescent substrates luminescent, and luminescent reagents for other compounds required for luminescent reactions.

Next, the luminescent reagent when the component to be measured is endotoxin will be described in detail.

The reagent activated by endotoxin is preferably a reagent containing factor C activated by binding to endotoxin, and more preferably contains factor B activated by activated factor C in addition to factor C and activated factor B to generate coagulation Reagent for proclotting enzymes. As a reagent containing factor C, factor B and procoagulase, limulus blood cell extract (lysing reagent) can be preferably used.

As the luminescent synthetic substrate, a luminescent synthetic substrate in which a luminescent substrate is bonded to a peptide can be used. As a luminescent synthetic substrate when the component to be measured is endotoxin, a substance having a structure that causes a luminescent substrate by the action (protease activity) of at least any one of active factor C, active B factor, and coagulation enzyme can be used. A structure in which the binding to the peptide is cut off.

As the luminescent substrate, aminofluorescein can be preferably used. The peptide to be bound to the luminescent substrate may be any one having an amino acid sequence that binds to the C-terminus of the peptide due to protease activity of at least one of active factor C, active factor B, and coagulase. The amide of aminofluorescein binds to the cleaved amino acid sequence.

A luminescent enzyme is an enzyme that acts as a catalyst for bioluminescence of a luminescent substrate released from a luminescent synthetic substrate to generate light. When the luminescent substrate is aminoluciferin, the luminescent enzyme is luciferase, and other compounds required for the luminescent reaction are ATP and divalent metal ions.

It should be noted that when the sample solution contains salt, the luminescent reagent may further contain NaCl in order to eliminate errors due to the concentration of the salt.

Preferably, the luminescent reagent is contained in the measurement container 1 before the sample solution is injected into the measurement container 1 . For example, the luminescent reagent may be attached in a lyophilized state to the bottom of the container body 2 in advance.

As long as the luminescence reagent is stored in the measurement container 1 in advance, it can be used in the analysis using the luminescence analysis device of this embodiment only by injecting the sample solution into the measurement container 1 .

When an unknown sample whose concentration of the analyte component is unknown is used as the sample solution in the measurement solution 4 , the computing device 30 obtains the concentration of the analyte component in the sample solution based on the amount of light emitted by the photodetector 20 .

Specifically, the amount of luminescence detected by the photodetector 20 when the measurement container 1 containing the measurement liquid 4 is stored in the measurement chamber 10 is compared with a calibration curve stored in advance to obtain the concentration of the sample solution. concentration of the component to be tested.

A calibration curve showing the relationship between the amount of luminescence and the concentration of the component to be measured is stored in advance in the computing device 30 . This calibration curve is determined for each batch of luminescent reagent, and when a new batch of luminescent reagent is used, it is updated by inputting the information of the calibration curve provided by the manufacturer or the like.

Preferably, the obtained concentration of the analyte can be displayed on the display device built in the emission analysis device of this embodiment or an independent display device, and can be output to a built-in or independent printer or an external computer.

Preferably, when the arithmetic unit 30 determines that the measurement container 1 is stored in the measurement chamber 10, it performs the operation of obtaining the concentration of the analyte component in the sample liquid.

Accordingly, only by storing the measurement container 1 in the measurement chamber 10, the calculation device 30 can automatically start the operation of obtaining the concentration of the component to be measured in the sample liquid.

For example, when the door 12 is opened and then closed, the computing device 30 can determine whether the assay container 1 has been stored in the assay chamber 10 according to whether the light detector 20 can detect the background luminescence of the luminescent reagent itself.

Specifically, whether or not the background light emission can be detected is determined based on whether or not the amount of light emission detected by the photodetector 20 is equal to or greater than a predetermined threshold.

The threshold value is the amount of luminescence at which it can be clearly determined that the background luminescence of the luminescent reagent itself has occurred.

It should be noted that the measurement container 1 itself may also generate extremely small natural luminescence, but the luminescence amount of such natural luminescence or a luminescence amount close to the luminescence amount should not be used as a threshold value. This is because, if an extremely small amount of luminescence such as the natural luminescence emitted from the measurement container 1 itself is used as a threshold value, a photodetector 20 with extremely high sensitivity has to be prepared, and it is difficult to accurately detect the presence or absence of background luminescence. judge.

The luminescence amount of the background luminescence of the luminescent reagent itself can be confirmed by measuring the luminescence amount of the measurement solution 4 containing the blank solution and the luminescent reagent using a sample solution not containing the test component as a blank solution.

The threshold is preferably 10%-100% of the background luminescence of the luminescent reagent itself, more preferably 30%-70%, for example, about 50%.

When the sensitivity of the photodetector 20 is confirmed, a blank solution containing no component to be measured is used as the sample solution in the measurement solution 4 . Then, the operator stores the measurement container 1 containing the measurement solution 4 including the blank solution in the measurement chamber 10 and performs an input operation requesting confirmation of sensitivity.

Then, the computing device 30 compares the luminescence detected by the photodetector 20 with the luminescence of the pre-stored calibration curve when the concentration of the analyte is zero, and confirms the sensitivity change of the photodetector 20 .

Here, the luminescence of the calibration curve stored in advance when the concentration of the analyte is zero (hereinafter sometimes referred to as "zero reference luminescence") corresponds to the background luminescence of the luminescent reagent itself. For example, the background luminescence of the luminescent reagent itself is produced by the luminescence of the free luminescent substrate contained in the luminescent synthetic substrate due to the luminescent enzyme.

The arithmetic unit 30 compares the light emission amount detected by the photodetector 20 with the zero reference light emission amount. For example, the comparison is performed by obtaining the ratio of the light emission amount detected by the photodetector 20 to the zero reference light emission amount or the difference between the two.

When obtaining the ratio of the luminous amount detected by the photodetector 20 to the zero reference luminous amount, if the obtained ratio is less than or exceeds the ratio of the preset specified range, the computing device 30 determines that the light detection The sensitivity of the sensor 20 has changed beyond the specified allowable range.

In addition, when the difference between the two is obtained, if the obtained difference exceeds the difference within the preset specified range, the arithmetic device 30 determines that the sensitivity of the photodetector 20 exceeds the specified allowable range, and a fault has occurred. change.

The predetermined range can be appropriately set according to the required measurement accuracy and the like, and can be stored in the computing device 30 .

When the calculation device 30 confirms that the sensitivity of the photodetector 20 exceeds the specified allowable range and changes, the operator can take measures such as adjusting the sensitivity of the photodetector 20 or replacing the photodetector 20 by outputting an alarm. .

If the photodetector 20 is a photomultiplier tube, the sensitivity of the photodetector 20 can be adjusted by changing the voltage applied to the photodetector 20 .

[the second embodiment]

The device configuration itself of the light emission analysis device of the second embodiment is the same as that of the first embodiment, and as shown in FIG. The light in the measuring container 1 in the measuring container 1 is detected;

In the emission analyzer of the second embodiment, the measurement solution 4 contained in the measurement container 1 is different. In addition, the operation of the computing device 30 is also different according to the difference in the measurement liquid 4 . Other matters are the same as those of the emission analysis device of the first embodiment, and therefore description thereof will be omitted.

In the second embodiment, the measurement solution 4 contained in the measurement container 1 includes a sample solution, a luminescent reagent and a luminescent substance that emit light by reacting with the analyte component in the sample solution.

As described in the first embodiment, the luminescent reagent means the reagent or the entire reagent group required to generate luminescence corresponding to the concentration of the component to be measured, and generally a reagent group composed of a plurality of reagents corresponds to a luminescent reagent. .

In the present invention, the luminescent substance is a substance that emits light due to a luminescent reagent in the absence of a component to be measured, or a substance that emits light by itself, and may be a reagent group composed of a plurality of reagents.

The wavelength range of the light emitted by the luminescent substance is preferably equal to, more preferably the same as, the wavelength range of the light emitted by the component to be measured acting on the luminescent reagent. Accordingly, when the blank solution is used as the sample solution, the amount of light emitted by the photodetector 20 can be increased.

In the absence of the component to be measured, the substance that emits light due to the luminescent reagent may be a luminescent substrate that emits light due to a luminescent enzyme contained in the luminescent reagent, for example, luciferin when the luminescent enzyme is firefly luciferase, etc. .

As a substance that itself emits light, there may be a combination of a luminescent enzyme other than the luminescent enzyme contained in the luminescent reagent and a luminescent substrate that emits light due to the other luminescent enzyme, for example, a combination of tap beetle luciferase and luciferin, The combination of Renilla luciferase and coelenterazine, etc.

When the luminescent substance is a luminescent substrate that emits light due to the luminescent enzyme contained in the luminescent reagent, it is preferably a luminescent substrate that emits light in a wavelength range equal to that of the luminescent substrate freed from the luminescent synthetic substrate contained in the luminescent reagent, and is particularly preferably the same as that obtained from the luminescent synthetic substrate in the luminescent reagent. The luminescent synthetic substrate contained in the luminescent reagent is the same luminescent substrate as the free luminescent substrate.

For example, aminofluorescein is preferably used as the luminescent substance when the luminescence reagent contains a luminescence synthesis substrate that releases aminofluorescein and the luminescence enzyme contains luciferase.

When the luminescent substance is a combination of another luminescent enzyme different from the luminescent enzyme contained in the luminescent reagent and a luminescent substrate that emits light due to the other luminescent enzyme, it is preferable that the wavelength range emitted by the combination is free from the luminescent synthetic substrate contained in the luminescent reagent. The luminescent matrix equals the light.

For example, when the luminescence reagent contains a luminescence synthesis substrate that releases aminoluciferin and luciferase is used as the luminescence enzyme, it is preferable to use a combination of beetle luciferase and luciferin.

Preferably, the luminescent substance and the luminescent reagent are contained in the measurement container 1 before the sample solution is injected into the measurement container 1 . For example, a luminescence reagent and a luminescence substance may be attached in a lyophilized state to the bottom of the container body 2 in advance.

As long as the luminescent reagent and the luminescent substance are stored in the measurement container 1 in advance, the sample solution can be used in the analysis using the luminescence analysis device of this embodiment only by injecting the sample solution into the measurement container 1 .

Similarly, in the present embodiment, when an unknown sample whose concentration of the component to be measured is unknown is used as the sample liquid in the measurement liquid 4, the calculation device 30 obtains the sample liquid concentration based on the amount of luminescence detected by the photodetector 20. The concentration of the analyte in .

Specifically, the amount of luminescence detected by the photodetector 20 when the measurement container 1 containing the measurement liquid 4 is stored in the measurement chamber 10 is compared with a calibration curve stored in advance to obtain the concentration of the sample solution. concentration of the component to be tested.

A calibration curve showing the relationship between the amount of luminescence and the concentration of the component to be measured is stored in advance in the computing device 30 . The calibration curve is determined for each batch of luminescent reagent and luminescent substance combination, and when a new batch of luminescent reagent and luminescent substance is used, it is updated by inputting the information of the calibration curve provided by the manufacturer or the like.

Preferably, the obtained concentration of the analyte can be displayed on the display device built in the emission analysis device of this embodiment or an independent display device, and can be output to a built-in or independent printer or an external computer.

Likewise, in the present embodiment, it is preferable that the computing device 30 performs the operation of obtaining the concentration of the analyte component in the sample liquid when it determines that the measurement container 1 is stored in the measurement chamber 10 .

Accordingly, only by storing the measurement container 1 in the measurement chamber 10, the calculation device 30 can automatically start the operation of obtaining the concentration of the component to be measured in the sample liquid.

For example, when the door 12 is in an open state and then becomes a closed state, the computing device 30 can determine whether the light detector 20 can detect the luminescence that combines the background luminescence of the luminescent reagent itself and the luminescence of the luminescent substance to determine whether the measurement container 12 is in the open state. Whether it has been stored in the measurement chamber 10 or not.

The method for determining the open/close state of the door 12 by the arithmetic unit 30 is the same as that of the first embodiment.

Compared with the case of only the background luminescence of the luminescent reagent itself, the luminescence amount of the luminescence combining the background luminescence of the luminescent reagent itself and the luminescence of the luminescent substance is larger. Therefore, it is possible to more easily determine whether or not the measurement container 1 is stored in the measurement chamber 10 .

Specifically, whether or not the luminescence combining the background luminescence and the luminescence of the luminescent substance can be detected is determined based on whether or not the amount of luminescence detected by the photodetector 20 is equal to or greater than a predetermined threshold.

The threshold value is the amount of luminescence at which it can be clearly determined that the background luminescence of the luminescent reagent itself and the luminescence of the luminescent substance are combined.

It should be noted that, in the present embodiment as well, the luminescence amount of natural luminescence emitted from the measurement container 1 or a luminescence amount close to the luminescence amount should not be used as the threshold value.

By using a sample solution that does not contain the component to be measured as a blank solution, and measuring the luminescence amount of the measurement solution 4 containing the blank solution, the luminescent reagent, and the luminescent substance, it is possible to confirm the background luminescence and the luminescent substance combined with the luminescent reagent itself. The amount of luminescence of the luminescence.

The threshold value is preferably 10%-100% of the luminescence amount that combines the background luminescence of the luminescent reagent itself and the luminescence of the luminescent substance, more preferably 30%-70%, for example, about 50%.

When the sensitivity of the photodetector 20 is confirmed, a blank solution containing no component to be measured is used as the sample solution in the measurement solution 4 . Then, the operator stores the measurement container 1 containing the measurement solution 4 including the blank solution in the measurement chamber 10 and performs an input operation requesting confirmation of sensitivity.

Then, the computing device 30 compares the luminescence detected by the photodetector 20 with the luminescence of the pre-stored calibration curve when the concentration of the analyte is zero, and confirms the sensitivity change of the photodetector 20 .

Here, the luminescence amount of the calibration curve stored in advance when the concentration of the analyte component is zero corresponds to the luminescence combining the background luminescence of the luminescent reagent itself and the luminescence of the luminescent substance.

The comparison method and the utilization method of the comparison result are the same as those of the first embodiment.

According to the emission analysis device of each of the above-described embodiments, it is possible to accurately determine the sensitivity change of the photodetector 20 without providing a light source in the device. Therefore, miniaturization and cost reduction of the device can be achieved.

Explanation of reference signs

1 Assay container

2 container body

3 covers

4 measuring solution

10 measuring room

11 Main body of the measuring chamber

11a Opening

11b Detection window

12 doors

20 light detector

30 computing device.

Claims (7)

1. A luminescence analysis device, which obtains the luminescence amount from a measurement solution containing a sample solution and a luminescence reagent that emits light by reacting with a component to be measured in the sample solution, and a calibration curve of the luminescence reagent. The concentration of the component to be measured in the sample solution is characterized in that it has: a light detector that detects light from the assay liquid; and an arithmetic means, which is input with the amount of luminescence detected by the photodetector, The calculation device uses the luminescence amount detected by the light detector and the concentration of the analyte in the calibration curve when using a blank solution that does not contain the analyte as the sample solution in the measurement solution. The luminescence amount at zero time was compared to confirm the sensitivity of the photodetector. 2. The emission analysis device according to claim 1, wherein: The calibration curve is a calibration curve determined for each batch of the luminescence reagent. 3. A luminescence analysis device based on the amount of luminescence from a measurement solution containing a sample solution, a luminescent reagent and a luminescent substance that emits light by reacting with a component to be measured in the sample solution, and the luminescent reagent and the The calibration curve of the luminescent substance is used to obtain the concentration of the component to be measured in the sample solution, which is characterized in that it has: a light detector that detects light from the assay liquid; and an arithmetic means, which is input with the amount of luminescence detected by the photodetector, The calculation device uses the luminescence amount detected by the light detector and the concentration of the analyte in the calibration curve when using a blank solution that does not contain the analyte as the sample solution in the measurement solution. The light emission amount at zero time was compared to confirm the sensitivity change of the photodetector. 4. The emission analysis device according to claim 3, wherein: The calibration curve is a calibration curve determined for each batch of the luminescent reagent and the luminescent substance. 5. The luminescence analysis device according to any one of claims 1 to 4, characterized in that, The light detectors are photomultiplier tubes. 6. A sensitivity adjustment method for a luminescence analysis device, which is the sensitivity adjustment method for a luminescence analysis device according to claim 5, wherein: When the computing device determines that the sensitivity of the photodetector has fluctuated beyond a predetermined allowable range, the sensitivity of the photodetector is adjusted by changing a voltage applied to the photodetector. 7. The method for adjusting the sensitivity of a luminescence analyzer according to claim 6, wherein: A reagent that emits light by a bioluminescence phenomenon is used as the light-emitting reagent.

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