JP4127814B2 - Analysis equipment - Google Patents

Description

  The present invention relates to an analyzer having a mechanism for mixing and reacting a sample and a reagent in a flat reaction vessel, and more particularly to an analyzer having a turntable on which a plurality of reaction vessels can be placed.

  In the qualitative / quantitative analysis of specific antigens, antibodies or genes in biological samples, a flat plate (called a chip) made of plastic, glass or the like may be used as a reaction surface. For example, a reagent is previously attached on a flat plate, and a sample is added to react. In the antigen / antibody reaction, the antigen / antibody, or in the gene detection method, the DNA fragment is bound to a labeling substance and used as a probe or a tracer.

  For example, Patent Document 1 discloses such a reaction chip.

  In the technique described in Patent Document 1, different DNA fragments are provided on a chip, a sample to be measured is introduced on the chip, and the fragment is used to measure hybridization.

JP 2003-121441 A

  The technique described in Patent Document 1 does not disclose how to stir the sample to be measured introduced on the chip to promote the reaction, but in the measurement using a normal chip, the sample is dropped, or It is considered that a method of providing a sample introduction port on the chip and pumping the sample is taken. In such a method, there is a concern that the reaction is not accelerated depending on the sample and the detection sensitivity is lowered.

  In the antigen, antibody reaction, or nucleic acid detection method using a flat reaction measurement container having a large bottom area compared to the height of the chip shape, the present invention promotes the reaction between the reagent and the sample and performs a measurement with good reproducibility. An object of the present invention is to provide an analysis apparatus that can be used.

  The configuration of the present invention for achieving the above object is as follows.

  A flat reaction vessel, a reaction vessel moving mechanism in which a plurality of the reaction vessels are mounted and the relative position of the reaction vessel can be moved, and a sample or a reagent is dispensed into the reaction vessel on the reaction vessel moving mechanism An analyzer comprising: a possible dispensing mechanism; and a control mechanism that controls the reaction container moving mechanism so that the reaction container on the reaction container moving mechanism is moved constantly at least during an analysis operation.

  Flat plate is a general term for containers having a large bottom area compared to their height. Preferably, the reaction vessel has a bottom area of 1 square centimeter or more, 100 square centimeters or less, and a height of 1 centimeter or less, and has a concave shape with an open top surface, but is not limited thereto. Further, the shape seen from above the container is not necessarily rectangular, and may be a perfect circle, an ellipse, or a polygon. It is a merit of the reaction vessel having a flat plate shape that the effect of stirring the sample and the reagent occurs only by moving the sample vessel. When there is a large amount of reaction liquid in a cylindrical container such as a test tube that has a large height relative to the bottom area, simply moving the reaction container does not produce a stirring effect. Even if configured to move, the same effect as the present invention cannot be obtained. In order to move constantly, it is effective not only to keep moving at a predetermined speed, but also to repeatedly stop moving and then move again. When the reaction vessel stops and starts moving again, a reverse acceleration is applied in the reaction vessel. This acceleration in both forward and reverse directions has the effect of promoting stirring. It is desirable to stop and move in a predetermined cycle, and to make a schedule so that the supply of the sample to the reaction vessel is completed at the time of stopping. The moving speed is selected from the viewpoint of obtaining an acceleration that maximizes the stirring effect when the reaction vessel is stopped and moved, and that the sample in the reaction vessel does not jump out of the reaction vessel. The optimum moving speed depends on the size of the reaction vessel and the like, but as a guide, an angular velocity of about 40 degrees / second is a guide. Needless to say, this does not limit the scope of the present invention.

(1) According to the present invention, in an antigen, antibody reaction or gene detection method using a reaction measurement container having a large bottom surface compared to the height of the chip shape, by moving a plurality of containers in a stationary manner. As a result, uniform shaking is given to the chips, and measurement with good reproducibility is performed without greatly different liquid stirring effects between the chips.
(2) According to the present invention, a simple apparatus is provided in an antigen, antibody reaction or gene detection method using a reaction measuring container having a large bottom surface compared to the height of the chip shape. Mixing of sample / reagent / diluent does not require a mixing / stirring system, and mixing / stirring is performed by moving the disk or belt required to move the sample / reagent / diluent / washing liquid to the tip for suction dispensing. Is made.
(3) The object of the present invention is to define the bottom surface and height of the chip, ensuring sufficient mixing and stirring efficiency, and the chip wall surface does not interfere with the measurement during measurement. Provided is a biomarker measurement chip that can easily and safely measure a reaction without spilling a reagent, a diluent, or a cleaning solution.

  In the conventional automatic analyzer, although the reaction vessel is moved by moving the gantry on which the reaction vessel is placed, it is not expected that the sample, reagent, or reaction solution is stirred and mixed by this operation. This is presumably because the height of the reaction vessel and the reaction solution is larger than the bottom area. Therefore, conventionally, the liquid in the reaction vessel has been forcibly stirred, and a dedicated stirrer has been provided. Examples of the stirring method include vortexing, ultrasonic wave, repeated pipetting, or stirring with a stirring rod. Such a system for agitation increases the cost of equipment.

  As a result of intensive studies on an analyzer that does not require a stirring mechanism, the present inventors have reached the present invention. That is, it was found that a sufficient stirring effect can be obtained by using a flat reaction vessel and alternately and regularly stopping and moving the reaction vessel.

  The present inventors have also found that measurement can be performed quickly and with high reproducibility by alternately stopping and moving the reaction vessel.

  Embodiments of the present invention will be described below with reference to FIGS. In the examples, biological samples such as nucleic acids, antigens, and antibodies are used as samples. However, the present invention is applicable to any analyzer that can react a sample with a reagent and measure the reaction. Needless to say.

  A concave plastic chip shown in FIG. 1 is produced. What is indicated by the left circle is a spot (spot) where an antigen, antibody reaction or nucleic acid hybridization is performed, and does not necessarily indicate that the chip has a boundary with the chip bottom surface.

  If the reaction vessel shape of the present invention is a flat plate shape having a smaller container height than its bottom area, the effect of the present invention is achieved although there is a difference in degree. As a more preferable container shape, it is desirable that X, Y, and Z in FIG. 2 have the following relationship.

Maximum length of bottom surface (square root (X ² + Y ² ))> = Z × 2
Meet.

  FIG. 3 shows a system outline of the chip cleaning apparatus and the chip measuring apparatus. The chip is subjected to antigen, antibody reaction or nucleic acid hybridization in a chip washing apparatus. Thereafter, spot analysis is performed using a chip measuring apparatus. The analytical measurement data is displayed on the PC or data analysis is performed.

  FIG. 4 shows an outline of the system reaction unit. The chip is mounted on a rotating disk of the cleaning device and moved to the position of a nozzle for dispensing and sucking antibody solution, cleaning solution or waste solution according to the reaction protocol.

  FIG. 5 shows a schematic diagram of a chip mounting disk. Multiple chips can be mounted.

  FIG. 6 shows an example of a turntable rotation time chart.

  The cleaning liquid 1 and the cleaning liquid 2 perform discharge suction of the cleaning liquids 1 and 2.

  Dispense the sample and antibody solution every 30 seconds. The turntable stops rotating every 30 seconds.

  The rotation stop of the turntable after 30 seconds is repeated until the processing of the cleaning liquid 2 with the total number of mounted chips is completed. However, it is not necessary that the operation and stop be all at regular intervals, and it is sufficient to set a preferable time for each operation together with the liquid dispensing suction.

  The rotation start / stop and rotation speed should be rapid or fast as long as there is no liquid spillage.

  The direction of rotation is due to device limitations, and may be either forward or reverse. As a result, an intermittent and steady turntable operation is performed. The optimum rotation speed depends on the radius of the turntable, but for example, an angular speed of about 40 ° / second is a standard.

  An example of alpha fetoprotein (AFP) measurement using antigen and antibody reactions is shown.

Create a spot with a diameter of about 2 mm on the chip, and dispense 2 μL of anti-human AFP antibody (manufactured by Japan Biotest Laboratories) diluted to 5 micro (μ) g / mL with 0.1 M carbonate buffer (pH 9.6). And static coating at 4 ° C overnight. Then, after washing with 0.05% Tween 20-added physiological saline, 1% BSA (bovine serum albumin) and 2% sucrose-added carbonate buffer (pH 9.1) 200 μL are added and allowed to stand at 37 ° C. After 1 hour, 0.05% Tween 20 (manufactured by Sigma) was washed with physiological saline, and with 1% BSA physiological saline, human AFP standard (DACO
(Immunoglobulins) is diluted 10-fold, and 200 μL is added to the chip. Mounted on the apparatus, shake for 1 hour continuously or dispense and suck 10 chips of reaction solution, leave it for the rest of the time, and wash with 0.05% Tween 20 physiological saline. Thereafter, biotin-labeled anti-AFP antibody (manufactured by DACO Immunoglobulins) is diluted to 1 μg / mL with 1% BSA-added physiological saline, and 200 μL thereof is dispensed to each chip and shaken with the apparatus for 1 hour or left still. Let Then, it is washed with 0.05% Tween 20-added physiological saline, and 200 μL of streptavidin-labeled fluorescent dye is dispensed onto the chip.

  After standing at room temperature for 30 minutes, it was washed with 0.05% Tween 20-added physiological saline. The plate is measured for light emission with a xenon lamp light source fluorescence measurement device (Hitachi High-Technologies).

FIG. 7 shows a calibration curve when human AFP standard and biotin-labeled anti-AFP antibody are dispensed on 20 chips, respectively, and each 10 is shaken or left standing. The vertical axis represents the light intensity (signal intensity), and the horizontal axis represents the human AFP standard product concentration. The mean value (mean) at each measured concentration (n = 10) is plotted, and the standard deviation (SD) is shown on the bar axis.

  When the chip is continuously shaken, it is found that the calibration curve range is widened and the SD at each measured concentration is small.

Left figure in Fig. 7 AFP calibration curve when the chip is shaken continuously A right figure in Fig. 7 AFP calibration curve when only 10 chips of the reaction solution are dispensed and aspirated and washed, and left for rest line

Outline of chip shape container. Outline of chip shape container. The schematic diagram of the apparatus by a chip-shaped container. Cleaning device flow path block diagram. Disc turntable overview. Schematic diagram of turntable operation time chart. AFP calibration curve.

Claims (3)

A reaction vessel having a bottom area of 1 square centimeter or more, 100 square centimeters or less, a height of 1 centimeter or less, and a concave shape with an open top surface ;
A plurality of the reaction vessels, and a reaction vessel moving mechanism capable of moving the relative position of the reaction vessels;
A dispensing mechanism capable of dispensing a sample or a reagent into the reaction container on the reaction container moving mechanism;
When a sample or a reagent is dispensed from the dispensing mechanism to at least one of the reaction containers on the reaction container moving mechanism, whether or not the sample is dispensed to another reaction container on the reaction container moving mechanism. Regardless, a control mechanism for controlling the reaction vessel moving mechanism so as to move the reaction vessel on the reaction vessel moving mechanism constantly ,
An analyzer characterized by comprising: The reaction container moving mechanism according to claim 1, wherein a plurality of the reaction containers are arranged in a line on the circumference of a disk, and a mechanism for rotating the disk is provided. apparatus. 3. The analyzer according to claim 1 , further comprising a cleaning liquid supply mechanism for supplying a cleaning liquid to the reaction container on the reaction container moving mechanism, and a cleaning liquid suction mechanism for sucking the cleaning liquid in the reaction container. Analysis equipment.

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