JPS62257048A - Measuring method using microplate - Google Patents

Abstract

PURPOSE:To improve a photometric system while the beneficial point of a conventional method in measuring sample forming work is put to practical use, by using a separable microplate at every one row and making it possible to perform photometry without interposing a liquid surface. CONSTITUTION:Each microplate 26 comprises a unit plate at every one row and is provided with several wells 22. The microplate 26 is subjected to the portionwise injection of a specimen or a reagent and incubation in a state incorporated in a holding frame 23 and subsequently applied to an apparatus to perform measurement. The microplate 26 to be measured is mounted on a specimen stand 27 and a unit plates 21 are successively fixed to a photometric holder 42 to be scanned as shown by a broken line and each well 22 is moved on the optical axis of a spectrometer 54 to successively measure the sample liquid allowed to fill each well 22. Measuring light is incident to the sample liquid from the lateral side of the well and, at the time of the measurement of absorbancy, lights emitted from the opposed side surfaces of the well are measured and, at the time of the measurement of fluorescence, the light emitted from the bottom surface thereof is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a measuring method using a microplate, and particularly to a measuring method and measuring device hl using a microplate for use in fluorescence photometry.

[Conventional technology]

In recent years, microplate-type measuring devices have been used in basic medical research and disease diagnosis in hospital laboratories. A microplate is a rectangular plate made of transparent synthetic resin such as styrene, with cylindrical or round-bottom reaction and side-light chambers (generally called wells, hereinafter referred to as wells) arranged at equal intervals. They are arranged in a vertical and horizontal matrix.

A microplate method like this? When using the 1I11 constant apparatus, pour the sample and reagent into each well, enter the constant temperature tank, and incubate at a constant temperature. Place the plate on the sample stage of the photometer and rotate the plate vertically and horizontally. The reaction solution is measured and analyzed by sequentially shining light on the cell. Fluorescence or absorption photometry is generally used as the photometric method.

This type of microplate type 1l11 constant device can measure many samples on one plate, so it is suitable for batch processing of many samples, and since many wells are arranged in a matrix, it is possible to measure many samples at once. be suitable for creating a dilution series for measuring multiple dilution series;
In addition, since measurements can be made with very small amounts of sample and reagent, measurements are frequently performed using dilution series, and expensive reagents such as antiserum and enzyme substrates are required. It is useful for immunological analysis based on antigen-antibody reactions and is widely used in this field.

FIG. 7 is an explanatory diagram of a typical example of a conventionally used microplate fluorometer. 1 indicates a microplate placed on a sample stage 3, 4 indicates a light source, and 9 indicates a detector. ing.

A microplate 1 has a large number of wells 2 arranged in a matrix. The size of the microplate 1 and the number of wells 2 vary depending on the usage, and there are various types.
For example, one with a total of 96 wells, 8 columns and 12 degrees horizontal column, is relatively often used. FIG. 1 shows a cross-section of the column.

In addition to the cylindrical shape shown in the figure, the shape of the well 2 may be round-bottomed, and the capacity of the well 2 is generally 0.
A material with a capacity of about 2 to 0.3 mQ is used. ? 11 Preparation of sample solution for determination], dispensing of specimens and reagents (antigens, enzyme-labeled antibodies, reaction reagents, etc.) to each well 2, and incubation are generally done manually or half-baked! l! I
When the reaction is completed, the microplate I is placed on the sample stage 3 of the device, and the sample liquid in each well 2 is sequentially measured by operating the microplate 1 vertically and horizontally. I do.

As the light source 4, a high brightness halogen tungsten lamp, xenon lamp, or mercury lamp may be used. light'g4
The light that exits the well 2 as monochromatic convergent light is passed through an input optical system 6 having an interference filter 5 that is replaceably provided.
is incident from above, and the fluorescence generated by the sample liquid passes through an interference filter 7 for selecting a detection wavelength and a condensing lens 8, and is measured by a detector 9. A photomultiplier or the like is used as the inspector 9. ]O so that the light that directly passes through well 2 will not interfere with the measurement.

It is a trap to catch them.

FIG. 8 is an explanatory diagram of another example of a conventionally used microplate fluorometer, in which the same parts as in FIG. 7 are given the same reference numerals. In this example, the light from the light source 4 is made to enter the well 2 through the input optical system 11 using an optical fiber, and the emitted light diagonally upward from the fluorescence generated from the sample liquid is passed through the receiving optical system 12 using the fiber. Detector 9
We are conducting measurements based on this information.

[Problem that the invention seeks to solve]

The light measurement method of the conventional microplate fluorometer mentioned above is such that, because the wells are arranged in a matrix, the incident light is applied from above the wells, and the light is directed diagonally downwards or upwards of the wells. However, such a photometry method has the following drawbacks.

That is, the surface of the sample liquid filled in the well becomes a concave surface due to the adsorption phenomenon of the liquid to the wall surface of the well. Since the wells of a microplate have a small diameter, this degree is particularly strong, and there are also variations from one well to the next. For this reason, as the incident light is bent or reflected at the liquid surface, light that interferes with measurement enters the detector, and the background of the detection signal fluctuates, reducing the accuracy of the 8Iq light. This has been a cause of limiting the detection limits of conventional microplate fluorometers.

An object of the present invention is to eliminate the problems of the prior art and to provide a measuring method and a measuring device using a microplate that can efficiently achieve highly accurate 81 measurements.

[Means for solving problems]

The configuration adopted in the present invention in order to eliminate the problems of the prior art described above is that in the measurement method using a microplate of the first invention, the microplate in which the reaction and photometry chambers are provided is fixed. In a measurement method using a microplate, in which a photometric holder is moved and the small chambers are sequentially moved to a photometric position of a photometer for measurement,
Microplates that can be separated into rows and have multiple reaction and photometry chambers integrally molded in each row are assembled into a multi-row holding frame to form a vertical and horizontal matrix. Then, with the unit microplate assembled in the holding frame, samples and reagents are dispensed into each of the small chambers and incubated.

The unit microplates are placed on a sample stage of a measuring device, and during measurement, the unit microplates are separated one by one row by row.

The light from the light source is incident on the side surface of each of the small chambers, and the light emitted from the bottom surface of the small chamber or the side surface in a direction different from the position of the light source is detected. In the measuring device using a microplate according to the second aspect of the invention, the photometry holder on which the microplate is fixed is moved, and the small chambers are opened. In a measuring device that uses a microplate that is sequentially sent to the photometry position of a photometer for measurement, a unit microplate that can be separated into rows and has multiple reaction and photometry chambers integrally molded in each row. a microplate holding frame that holds microplates forming a vertical and horizontal matrix by incorporating a plurality of rows of a moving device that positions the photometer at a specific position; and a moving device that separates the row of unit microplates positioned at the specific position from other rows and pushes it upward, and moves the photometer from the side of the small chamber. means for transporting the row of small chambers to a position of a photometry holder where light from the light source enters, and means for fixing the row of small chambers to the photometry holder and sequentially transporting each of the small chambers to a photometry position of a photometer for measurement. It is characterized by this.

[Effect]

In the measuring method and measuring device using a microplate of the present invention, a plurality of rows of independently molded separation-integrated unit microplates (hereinafter referred to as unit plates) are held so that each row of microplates can be separated. It is assembled into a frame to form a vertical and horizontal array of wells, and preparation of measurement sample liquid, such as dispensing specimens and reagents and incubation, can be carried out with the assembly in place. That is, since a holding frame incorporating wells arranged vertically and horizontally is used, the work of preparing a measurement sample can be performed in the same way as in the conventional case.

During measurement, the unit plates of each row of microplates placed on the sample stage of the device are sequentially separated from other rows and transported to the photometry section, where the unit plates are moved in the longitudinal direction of the rows. This allows light from the light source to enter from the side of each well.

Therefore, it is possible to perform measurements by receiving light emitted from the bottom of the well or from the side in a direction different from the position of the light source, eliminating the cause of limiting the detection limit of fluorometers, which has been a problem in the past. 6 [Example] Examples will be described below.

1 and 2 are a side view and a plan view showing the structure of a microplate fluorometer that is an example of a measuring device using a microplate of the present invention, and FIG. 73 is (a), (b),
(c) is a plan view and a side view in two different directions of the microplate that is also used; Figure 4 is an explanation of the plate holding mechanism of the photometric holder; Figure 5 is an explanatory diagram of the optical system. .

The microplate 26 shown in FIG. 3 consists of unit plates (independently molded microplates with built-in separation and m-position) for each row, and has two wells.
A plurality of unit plates 21 having a plurality of microplates 2 are assembled into a holding frame 23 to form one microplate 2.
6' is constructed. A unit plate 21 is attached to the holding frame 2;
a positioning pin 24 for pushing the lower plate 2j into the holding frame 23, and a cover 25 for preventing it from falling off when transporting the lower plate 2j assembled in the holding frame 23, or when performing manual operations such as dispensing reagents. is the provision. Since the cover 25 is 411 fixed, it must be removed when installing the holding frame 23 in the device. Using such a microplate 26, after dispensing samples and reagents (antigens, enzyme-labeled antibodies, reaction reagents, etc.) and incubating them while assembled in the holding frame 23,
Apply the measurement to the device. Note that the number of wells 22 in the microplate 26 is 8 vertically and 1 horizontally in FIG.
2, a total of 96 wells have been shown, but in order to simplify the drawing, in the operation explanatory diagram of FIG. 2, the wells are simplified to 6 in the vertical direction and 8 in the horizontal direction.

As shown in FIGS. 1 and 2, this microplate measuring device includes a pulse motor 28, a pulley 29. so
, pinion 3], movement a of the sample stage 27 having the rack 32
Mechanism, motor 38, pin 39. push-up means 37;
A push-up mechanism having a guide shaft 40 and a support arm 41, a photometric holder 42, a motor 44, a drive system 45, a holding mechanism having a holding means 46, a pulse motor 51, a pinion 49, a rack 50. A unit plate scanning mechanism having a guide shaft 52, 53, a light source 56, a photometer 54, an interference filter 57, an input optical system 58, an interference filter 59. It consists of an optical system having a condenser lens 60 and a detector 61.

In this measurement method using a microplate, a microplate 26 for determining M1g is mounted on a sample stage 27.

The sample stage 27 is moved to a pulley 29 by a pulse motor 28.
．． :30. Operates via pinion 31 and rack 32,
It slides in engagement with the guide shafts 33 and 34, and sequentially moves the unit plates 21 of each row of the microplate 26 to the push-up position; 35. 236 is the movement of the sample stage 27! [! I
This is a photosensor for detecting the position of the J range, that is, the starting point and the ending point. Push up position! 1'? 35 is provided with a push-up means 37, and the push-up means 37 is moved up and down along a guide shaft 40 by a push-up pin 39 operated by a motor 38, and the fourth position plate 21 placed at the push-up position [35] is supported by the support arm 41 and pushed up to the position of the first Mitsukawa holder 42. Reference numeral 40 denotes a holding plate for preventing unit putots 2 in the first and next rows from being lifted together when pushing up the unit plate 21. The pushed-up unit plates 1-2 are fixed to the photometric holder 42 by a holding means 46 operated by a motor 44 via a drive system 45. As shown in the fourth example, the pushed up unit plate 21 touches the frame 4 of the photometry holder 42.
A presser means 46 that is fitted into the photometer 7 and operated by a drive system 45 is pressed against the photometric holder 42 from below by a presser metal 48 and fixed. , the photometry holder 42 is connected via a pinion 49 and a rack 5o.

Operated by pulse motor 51, guide shaft 52°5:
1, the unit plate 21 is scanned, each well 22 is moved onto the optical axis of the photometer 54, and the sample liquid filled in the well 22 is sequentially measured. Reference numeral 55 denotes a photosensor for detecting the operating range of the photometric holder 42, that is, the J origin and end point of the operation. Accurate positioning of each well 22 of the unit plate 21 on the optical axis is controlled by the number of pulses of the pulse motor 51.

In the case of a fluorescent photometer, the light source 56 of the photometer 54 is usually a high-brightness halogen tungsten lamp, xenon lamp, or mercury lamp. The emitted light from the light source 56 enters from the side of the well 22 of the unit plate 21 via an incident optical system 58 having an interference filter 57 for selecting the wavelength of the incident light, which is provided replaceably, and is generated by the sample liquid. Measurement is performed by detecting the fluorescent light emitted downward from the well.

As shown in the light receiving optical system of FIG. 5, the light emitted downward from the well 22 is measured by a detector 61 via an interference filter 59 for selecting the detection light wavelength and a condensing lens 60. A photomultiplier or the like is normally used as the detector 61. As a detection method in the case of fluorescence photometry, in addition to the method shown in FIG. 1, a method of receiving light emitted diagonally to the side of the well is also possible.

When the 3111 fixing is completed, the pulse motor 51 reverses to return the m-position plate 21 to its origin (the position where it is pushed up and fixed), and then the motor 44 operates to move the presser and second presser 4.
e is reversed, the presser foot 48 is opened, and the unit plate 21 is dropped downward by the presser foot 62, which is received by the support arm 41 which has been raised and is waiting, and lowered to its original position on the microplate 26. Return to Next, the sample stage 27 is moved one step by the pulse motor 28.

The next unit play 21 is sent to the push-up position 35.

By repeating the above-described procedure, all the samples on the microplate 26 are measured.

Measurement using this '14Ff is started by setting the microplate 26 on the sample stand 27 and pressing the start switch, and all samples placed on the microplate are automatically measured. Measurement condition.

Arithmetic expressions and the like can be set in advance using a prepared program. Regarding the control and calculation of such a computer-based automatic device, a detailed explanation will be omitted since it is possible to use the same general methods as those used in other similar analytical instruments.

1ll using a microplate using the fluorophotometry method.
Although the embodiment of the measurement device has been described, the present invention can also be applied to a measurement device based on absorptiometry.

FIG. 6 shows the optical system in that case, in which a tungsten lamp is normally used as the light source 6:3, and an incident optical system 58 having an interference filter 57 for wavelength selection, which is replaceably installed, focuses the focused light. is incident from the side of the well 22, and the light transmitted through the sample liquid is detected by the detector 64 to measure the absorbance. For example, a silicon photodiode or the like is used as the detector 64. In the case of absorbance measurement and fluorescence measurement, the overall system of the device is almost the same except for the light reception and detection system when using the optical system, so it is easy to create a device for both absorbance and fluorescence that has both functions. .

The measuring device using the microplate of this embodiment is a conventional microplate type 2II! It is possible to improve the measurement performance, which had been considered a problem in the past, without changing the features of "Handling, such as the convenience of preparing a single reaction solution in the assay method." The purpose of the present invention is to provide a measuring method and a measuring device that are useful for analysis that requires.

〔Effect of the invention〕

The present invention eliminates the problems of the prior art and makes it possible to provide a measuring method and a measuring device using a microplate that can efficiently achieve highly accurate measurements, and has great industrial effects. It is.

[Brief explanation of drawings]

FIG. 1 is a side view showing the structure of a microplate fluorometer that is an example of a measuring device using a microplate of the present invention, FIG. 2 is a plan view of the same, and FIG. A plan view of the plate, FIGS. 3(b) and 3(c) are side views in two different directions, FIGS. 4 and 5 are explanatory views of different main parts, and FIG.
The figures are also explanatory diagrams of main parts of an example different from FIG. 5, FIG. 7 is an explanatory diagram of a conventional microplate fluorometer, and FIG. 8 is an explanatory diagram of another example. 21... Separate built-in unit microplate (, It
1st place Pre-plate 22...Small chamber (well) for reaction and a+++ light, 23...Holding frame, 26...Microplate, 27...Sample stand, 28...Pulse motor
137... Push-up means, 338... Motor, 4
2...ilp+light holder, 44...motor,
46... Holding means, 51... Pulse motor 15
4... Photometer, 61... Detector (for fluorescence), [i4
...Detector (for absorbance). Agent Patent attorney Fuao Nagasaki: (2 others) Figure 3 (Ni 4) Dream 4fjJ 7th story

Claims (1)

[Claims] 1. A microplate is used in which the photometry holder in which the microplate is fixed is moved and the small chambers are sequentially moved to the photometry position of a photometer for measurement. In the measurement method, a separate built-in unit microplate, which can be separated into rows and has a plurality of integrally molded reaction and photometry chambers in each row, is assembled into a multi-row holding frame to form a vertical and horizontal matrix. Using a microplate, with the unit microplate assembled in the holding frame, a sample and a reagent are dispensed into each of the small chambers, incubation is performed, and then placed on a sample stage of a measuring device, In the measurement, the unit microplates are separated one by one in sequence and transported to the photometry position, and the light from the light source is made to enter from the side of each of the chambers, and the light from the light source is incident on the bottom of the chamber or in a direction different from the position of the light source. A measurement method using a microplate, characterized in that light emitted from the side of the plate is detected and measurement is performed by fluorescence photometry or absorption photometry. 2. In a measuring device using a microplate, the photometry holder in which the microplate in which the reaction and photometry chambers are installed is moved, and the chambers are sequentially moved to the photometry position of the photometer for measurement. A microplate holding frame that holds microplates forming a vertical and horizontal matrix by incorporating multiple rows of separate built-in unit microplates that can be separated into two cells and each row has a plurality of integrally molded reaction and photometry chambers. a moving device that moves the microplate placed on the microplate holding frame and sequentially positions the unit microplates in each row at a specific position on the photometer; means for separating the row of unit microplates from other rows and pushing them upwards to a position of a photometry holder where light from the light source of the photometer enters from the side of the small chamber; A measuring device using a microplate, characterized in that it has means for fixing a row of microplates to a photometric holder and sequentially transporting each of the small chambers to a photometric position of a photometer for measurement.