JP2007046979A - Dispensing amount automatic correction method and dispensing device having the function - Google Patents

Abstract

Dispensing accuracy is maintained even when it is difficult to restore dispensing accuracy only by exchanging a seal member between a piston and a cylinder.
A solution capable of measuring absorbance is prepared, and the absorbance is measured for one or more quantitative solutions in which the solution is dispensed into a predetermined container, and reference data (straight line O) is measured based on the measured absorbance. ), And the pipette that automatically dispenses at the target set dispensing volume is used to dispense the solution into the same type of container, and the actual dispensing volume dispensed to the set dispensing volume. Absorbance was measured for the solution of (2), and from the difference between the measured actual absorbance (0.712) and the reference absorbance (0.747) obtained from the reference data for the set dispensed amount, dispensing to the set dispensed amount was performed. A correction amount is obtained in advance, and when the set dispensing amount is dispensed with respect to the liquid sample by the pipette, the dispensing correction amount is corrected.
[Selection] Figure 6

Description

  INDUSTRIAL APPLICABILITY The present invention relates to an automatic dispensing amount correcting method and a dispensing apparatus having the function thereof, and particularly to an automatic dispensing amount correcting method and a function having the function which are suitable for maintaining dispensing accuracy over a long period of time. Note on the device.

  Conventional dispensing devices are equipped with pipettes (dispensing machines) for dispensing liquids such as specimens and reagents to microplates (reagent plates) on which reagents are fixed, especially the EIA method (enzyme immunoassay). Method) The analyzer is also equipped with a spectroscopic analyzer for measuring the inspection results.

  As said pipette, what is typically shown in FIG. 7 by patent document 1 is disclosed, for example. This mainly includes a piston 100, a cylinder 102, a seal member (not shown) such as an O-ring for maintaining airtightness between the piston 100 and the cylinder 102, a member for driving the piston 100, a stepping motor, etc. Piston driving means 104 and a nozzle portion (details omitted) 108 for connecting the tip 106 to the pipette.

  The pipette is electrically connected to a control unit (not shown), and the control unit controls injection / discharge operations of specimens, reagents, and the like. That is, when the piston 100 performs a suction operation, the inside of the cylinder 102 becomes negative pressure, and a sample, a reagent, and the like are sucked from the tip of the tip 106 connected to the nozzle portion of the pipette and stored in the tip 106.

When the piston performs a discharge operation, the inside of the cylinder 102 is pressurized, and the specimen, reagent, and the like in the chip 106 are discharged from the tip of the chip. At this time, the dispensing amount is equal to the volume change in the cylinder, that is, the product of the moving distance (= a ₀ −a ₁ ) of the piston 100 detected by the sensor 110 and the cross-sectional area of the cylinder. It is proportional to the rotation amount (movement amount) of the means 104.

  Therefore, in order to obtain the required target dispensing amount, it is realized by rotating (moving) the stepping motor 104 or the like according to a command from the control unit so that the volume change amount in the cylinder 102 becomes equal to it.

  However, the seal member between the piston and the cylinder inevitably wears due to its structure, but when this wear progresses, a leak occurs between the inside and the outside of the cylinder 102.

  Since this leak affects the generation of negative pressure and pressurization in the cylinder as the piston 100 moves, it also affects the dispensing amount. For this reason, the seal member is periodically replaced, and it is attempted to maintain the dispensing accuracy by replacing the parts.

JP-A-5-288757

  However, the method of maintaining the dispensing accuracy only by exchanging the sealing member, such as a pipette equipped in a conventional dispensing device (including an EIA analysis device), causes variations in the dimensions of the sealing member, piston Since the aging of the drive part and the aging of the piston part also occur, there is a problem that it is difficult to completely restore the original dispensing accuracy.

  The present invention has been made to solve the above-described conventional problems, and even when it is difficult to restore the dispensing accuracy only by replacing the seal member, the dispensing accuracy can be maintained over a long period of time. It is an object to provide an automatic quantity correction method and a dispensing apparatus.

  The present invention prepares a solution capable of measuring absorbance, measures the absorbance of one or more quantitative solutions in which the solution is dispensed into a predetermined container, and creates reference data based on the measured absorbance. At the same time, the pipette that automatically dispenses the target at an arbitrarily set dispensing volume is used to dispense the solution into the same type of container, and the absorbance of the actual dispensing volume of the solution dispensed with respect to the set dispensing volume. The dispensing correction amount for the set dispensing amount is obtained in advance from the difference between the measured actual absorbance measured and the reference absorbance obtained from the reference data for the set dispensing amount. When dispensing the dispensing amount, the problem is solved by correcting the dispensing correction amount.

  The present invention also provides a solution capable of measuring absorbance in a dispensing apparatus equipped with a pipette that sucks and discharges a liquid sample by controlling a driving means for moving the piston in the cylinder forward and backward by the control means. Then, the absorbance is measured for one or more fixed-quantity solutions in which the solution is dispensed into a predetermined container, and reference data is created based on the measured absorbance, and automatic dispensing is performed for any set dispensing volume as a target. Dispense the solution into the same type of container with a pipette to pour, measure the absorbance of the actual dispensed amount of the solution dispensed with respect to the set dispensed amount, and measure the measured absorbance and the set amount. A dispensing correction amount for the set dispensing amount is obtained in advance from the difference from the reference absorbance obtained from the reference data for the dispensing amount, and correction data relating to the determined dispensing correction amount is stored in the control means, When dispensing a set dispensing amount for a liquid sample by a pet, the problem is similarly solved by controlling the driving means based on the correction data and correcting the dispensing correction amount. It is.

  According to the present invention, even when the dispensing amount by the pipette changes over time, the error of the actual dispensing amount that is actually dispensed with the set dispensing amount as a target can be corrected using the absorbance of the solution. Therefore, even when the dispensing accuracy cannot be restored only by replacing the parts, the dispensing amount can be accurately corrected and maintained.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  In FIG. 1, the external appearance of the dispensing apparatus 10 of one Embodiment which concerns on this invention is shown. The dispensing device 10 includes a plurality of pipettes (dispensing machines) 12, and dispenses a liquid sample such as a specimen or a reagent into a plurality of wells W formed of concave portions formed in the microplate 14 by the pipette 12. It is like that.

  As shown in an enlarged view in FIG. 2, the pipette 12 includes a nozzle 16 for sucking and discharging a liquid sample, and a cylinder 20 in which a piston 18 is accommodated and communicated with the nozzle 16. ing.

  A feed screw 22 is coaxially attached to the upper end of the piston 18, and the feed screw 22 is screwed into a gear 24 having a female screw formed inside. A gear 26 is engaged with the gear 24, and an output shaft of a stepping motor (driving means) 28 is attached to the gear 26.

  Further, a piston seal (seal member) 18 </ b> A for sealing the piston 18 and the sliding portion of the cylinder 20 is attached to the inside of the upper end portion of the cylinder 20.

  The pipette 12 is controlled by the control unit 30, and the control unit 30 drives and controls the stepping motor 28 to rotate the gear 26 and raises the piston 18 through the rotation of the gear 24. By lowering the liquid, it is possible to suck and discharge (suction / discharge) the liquid, and it is possible to automatically correct a secular change or the like of the dispensed amount by a method described later.

  The dispensing apparatus 10 of the present embodiment is provided with an absorbance measuring apparatus as schematically shown in FIG. 3 as another facility.

  This absorbance measuring device has a lens holding member 40 having a substantially U-shaped cross section, and is mounted on a mounting base 42 made of a transparent substrate installed in parallel in the vertical middle position between the upper and lower frames 40A and 40B. A microplate 14 is placed. The mounting table 42 has holes formed in the lower portions of the wells W so that the wells W of the microplate 14 can be seen from above and below.

  In this embodiment, the mounting base 42 is movable by a motor and a control device (not shown) in the Y direction in the drawing, and the lens holding member 40 is movable by a motor and a control device (not shown) in a direction perpendicular to the paper surface of the drawing. By moving the mounting table 42 and the lens holding member 40, all the wells W of the microplate 14 are movable between the optical fibers 44A and 44B.

  A projection lens 46 connected to a projector (not shown) via an optical fiber 44A is disposed on the upper frame 40A above the microplate 14. A light receiving lens 48 connected to a spectroscopic analyzer (not shown) via an optical fiber 44B is disposed on the opposed lower frame 40B. The absorbance of the liquid dispensed in the well W of the microplate 14 located between both lenses 46 and 48 can be measured.

  In the present embodiment, correction at the time of dispensing of the pipette 12 is realized by the above-described absorbance measuring apparatus according to the principle and method described in detail below.

  First, as a solution capable of measuring absorbance, water colored with a colorant, that is, colored water having a predetermined concentration is prepared. As the colorant, for example, Azo Rubin. Extra or Brilliant Crimson Red, which is generally used as food red, can be used.

  The color density of the colored water is determined in proportion to the amount of the colorant. Further, when water having a certain color concentration is put into a container such as a 96-well microplate in which 8 × 12 wells W as shown in FIGS. 1 and 3 are formed, for example, the wells of the microplate When the (predetermined container) W is viewed from above and below, since the horizontal cross-sectional area of the well W is substantially constant, the color density changes in proportion to the amount of colored water, that is, the height. Looks. That is, even if the color density is the same, if the amount of colored water is doubled, the density appears to be double. In other words, the amount of colored water can be quantified by the color density.

  Therefore, as shown in the image in FIG. 4, 100 μl of water that is not colored in advance is dispensed in a predetermined amount into several locations (seven adjacent locations in the drawing) in the empty well W of the microplate 14. Further, 200 μl of colored water is dispensed into another well W in the empty state (adjacent right end in the figure). This quantitative dispensing is performed accurately using, for example, a calibrated handheld pipette. In particular, colored water is used as a standard, so dispense accurately and accurately.

  First, 10 μl of colored water is dispensed into the well in which 100 μl of water is dispensed at a time using one pipette 12 provided in the dispensing apparatus 10 of the present embodiment. Similarly, dispense 20 μl of colored water into another well. In this way, 10, 20, 30, 40, 50, and 100 μl of each colored water is automatically and sequentially applied to the well of FIG. 4 in which water is quantitatively dispensed as shown in FIG. Dispense in a batch. In this way, only the fixed amount of 100 μl of water is placed in the left end well, and 10 μl of colored water + 100 μl of water, 20 μl of colored water + 100 μl of colored water, 30 μl of colored water + 100 μl of colored water, 40 μl of colored water + 100 μl of colored water, 50 μl of colored water + 100 μl of water 100 μl of colored water + 100 μl of water, and 200 μl of colored water at the right end enter each well of the microplate.

  Thereafter, the microplate is shaken, and the colored water and water are mixed. As shown in FIG. 3, the wells containing the colored water evenly mixed are arranged between the light projecting lens 46 and the light receiving lens 48, and the absorbance in the specific wavelength region is measured using a spectroscopic analyzer. Measure.

  Thus, the light absorbency which measures the applicable well of the microplate 14 from the up-down direction by spectroscopic analysis is proportional to coloring density. Therefore, by comparing the absorbance, the amount of the colored water contained in the well can be relatively compared and quantified.

  Absorbance of wells in which 100 μl of uncolored water has been dispensed in a fixed amount and wells in which only 100 μl of colored water has been dispensed are dispensed, and water is dispensed quantitatively, and colored water is automatically dispensed with pipette 12 of the dispensing device. Compare the absorbance of each injected well.

  That is, the absorbance of each well in which colored water and water are mixed with the measurement result (reference data) of each well in which only 100 μl of water is dispensed in a well and well in which only 200 μl of colored water is dispensed in a fixed amount. By proportionally (comparing) calculating the measurement result (actual absorbance), the amount automatically dispensed by the pipette 12 of the dispensing device can be accurately obtained.

  The difference between this calculation result and the set dispensing amount (10, 20, 30, 40, 50, 100 μl) set as the target is the error amount of automatic dispensing. By calculating the dispensing error amount for each set dispensing amount, the dispensing error in each set dispensing amount is set as a dispensing correction amount and stored in the control unit 30.

  The correction amount for each set dispensing amount obtained and stored in this way is converted into the rotation amount (movement amount) of the stepping motor 28 when automatically dispensing the set dispensing amount, and is output from the control unit 30. The correction amount is added to the target command value, and the stepping motor 28 is driven and controlled so that the movement amount of the piston 18 becomes an added value of the specified movement amount and the correction movement amount with respect to the set dispensing amount.

  The drive control for the stepping motor described in detail above can be similarly executed for the remaining pipettes by obtaining the dispensing assistant amount.

  Next, a method of calculating the correction value (dispensing correction amount) based on the absorbance described above will be described in detail.

  The absorbance at each dispensing amount is determined from the absorbance of 100 μl of water and 200 μl of colored water. As shown in FIG. 6, the ideal value (reference data) of the absorbance in each dispensed amount is obtained by connecting the absorbance of 100 μl of water and 200 μl of colored water with a straight line O. Although there is no substantial difference, 100 μl of water may be added in the case of 200 μl of colored water in order to bring it closer to the ideal value.

  In order to improve accuracy, the absorbance of 100 μl of water and 200 μl of colored water, and the absorbance in each dispensed amount of 100 μl of water + colored water should be determined by averaging a plurality of operations.

In the calculation
Absorbance per 1 μl = ((absorbance of 200 μl of colored water) − (absorbance of 100 μl of water)) / 200
Can be obtained as

  The difference between the ideal (reference) absorbance thus obtained and the actually measured absorbance of 100 μl of water + colored water injection is the amount of error in dispensing. The percentage of the ideal absorbance corresponding to this error amount is calculated, and this is used as a correction amount. This correction amount is replaced with the rotation amount (movement amount) of piston drive means such as a stepping motor during automatic dispensing by a pipette, and the movement amount of the piston is determined by adding the correction amount to the command of the control unit 30. Dispensing is performed as the specified movement amount with respect to the amount + the correction movement amount.

In the example of FIG.
Absorbance per 1 μl = ((absorbance of 200 μl of colored water) − (absorbance of 100 μl of water)) / 200
= (3.002-0.014) / 200
= 0.01494
It becomes.

The ideal absorbance on the straight line O when dispensing 50 μl is
0.01494 × 50 = 0.747
The difference from the actual measurement data (actual absorbance) on the straight line R, that is, the dispensing error amount is
0.747-0.712 = 0.035
It is. This is 0.035 / 0.712 = 4.915% of the ideal absorbance.
This is a lack of the ratio.

If the stepping motor rotation amount at the time of 50 μl dispensing is, for example, 1000 pulses, the corrected amount of movement will be 1000 × 4.915% = 49.15 pulses, and 1000 + 49 pulses is the ideal stepping motor rotation amount at the time of 50 μl dispensing. Dispensing according to value is possible.

Alternatively, the ratio of the actually measured absorbance in the actual dispensing amount (actual dispensing amount) to the ideal absorbance at the time of 50 μl dispensing is 0.712 / 0.747 = 95.315%
Therefore, if the stepping motor rotation amount at the time of 50 μl dispensing is, for example, 1000 pulses, in order to obtain the ideal dispensing amount,
Stepping motor rotation after correction = 1000 + 1000 × 100% / 95.315%
= 1000 + 49.15 = 1049 pulses.

  As described above, for each set dispensing amount, the ratio of the error amount of the absorbance measurement value in the actual dispensing amount to the ideal absorbance is obtained, and this data or this data is used as the rotation amount (movement of the piston driving means of the stepping motor 28). The data replaced with (quantity) is stored in the control unit 30. Alternatively, the ratio of the actually measured absorbance value in each actual dispensing amount with respect to the ideal absorbance is obtained, and this data or data obtained by replacing this data with the rotation amount (movement amount) of the piston driving means such as a stepping motor is stored in the control unit 30. .

  Next, the procedure of the automatic dispensing amount correction function executed in the present embodiment will be described.

(1) Quantitative dispensing of uncolored and colored water to a microplate with a calibrated pipette
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(2) Dispense colored water into wells in which uncolored water has been quantitatively dispensed with a pipette of a dispensing device (10, 20, 30, 40, 50, 100 μl)
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(3) Measure absorbance with a spectroscopic analyzer.
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(4) The ideal absorbance at each set dispensing amount is determined by calculation from the absorbances of uncolored water and colored water that have been quantitatively dispensed.
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(5) For each set dispensing volume, calculate the error between the absorbance at the actual dispensing volume and the ideal absorbance.
(Alternatively, the ratio of the actual absorbance value in each actual dispensing amount to the ideal absorbance is calculated.)
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(6) The above error data or data obtained by replacing the error amount with the rotation amount (movement amount) of piston driving means such as a stepping motor is stored in the control unit.
(Alternatively, the above ratio data or data obtained by replacing this data with the rotation amount (movement amount) of the biston drive means such as a stepping motor is stored in the control unit.)
↓
(7) Based on the data stored in the control unit, corrective dispensing is performed in consideration of the rotation amount (movement amount) of piston driving means such as a stepping motor at the time of dispensing.

  As described above in detail, according to the present embodiment, even when a variation in the size of the replaced seal member, aging of the piston drive portion, aging of the piston portion, or the like occurs, a target set dispensing amount is appropriately set. It is possible to calibrate the dispensing amount using a pipette by storing the amount of correction for the amount stored in the control unit and correcting the command signal for the set dispensing amount based on the correction amount. Since the dispensing accuracy can be maintained, the target amount can be dispensed without deteriorating the accuracy.

  The dispensing device of the above embodiment has an existing spectroscopic analysis device like the EIA method analysis device, even if an absorbance measurement function is added to a device having a dispensing function. Needless to say, it may be.

  In the above example, the ideal absorbance was calculated in a straight line from the measured absorbances of uncolored water and colored water only, but more accuracy can be obtained by obtaining the ideal absorbance line at several points according to the set dispensing volume. Can be increased. (For example, divide the amount into small, medium, and large areas and obtain 10 to 50 μl, 50 to 100 μl, 100 to 200 μl, etc.) Also, the ideal absorbance (reference data) should be obtained as a curve instead of a straight line. It may be.

  In addition, after correcting the dispensing amount, the absorbance may be measured again by the same procedure to improve the accuracy.

  The solution whose absorbance can be measured may be a colorless solution having absorption characteristics in the ultraviolet region or the like.

  Further, the colorant is not limited as long as the color density changes in proportion to the amount, even if it is not the exemplified food color.

  In addition, colored water with a calibrated handheld pipette, etc., quantitative dispensing of water, each set amount of colored water to be dispensed with a pipette of a dispensing device (including EIA method analyzer), and dispensing amount The type or the like may not be the numerical value given in the example.

  Further, the dispensing error amount (dispensing correction amount) in each set dispensing amount may be stored as data by increasing the number of samples, or the set dispensing amount from some stored data. An approximate curve of the dispensing error amount with respect to the dispensing amount may be obtained and obtained by calculation.

The perspective view which shows the outline | summary of the dispensing apparatus of one Embodiment which concerns on this invention. Sectional drawing which shows the outline of the pipette with which the dispensing apparatus of this embodiment is equipped. The side view which shows typically the light absorbency measuring apparatus with which the dispensing apparatus of this embodiment is equipped. Explanatory drawing showing the procedure of absorbance measurement for automatic correction of dispensing volume Other explanatory drawing showing the procedure of absorbance measurement for automatic correction of dispensing volume Diagram showing the relationship between dispense volume and absorbance Schematic diagram showing the outline of a conventional pipetting pipette

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Dispensing apparatus 12 ... Pipette 14 ... Microplate 16 ... Nozzle 18 ... Piston 20 ... Cylinder 22 ... Feed screw 24, 26 ... Gear 28 ... Stepping motor 30 ... Control part 40 ... Lens holding member 42 ... Mounting base 44 ... Light Fiber 46 ... Projection lens 48 ... Light receiving lens

Claims (2)

Prepare a solution capable of measuring absorbance, measure the absorbance of one or more quantitative solutions in which the solution is dispensed into a predetermined container, create reference data based on the measured absorbance,
Pipette the solution into the same type of container with a pipette that automatically dispenses to the desired set dispensing volume,
Measure the absorbance of the solution of the actual dispensing amount dispensed with respect to the set dispensing amount,
From the difference between the measured actual absorbance and the reference absorbance obtained from the reference data for the set dispense amount, a dispensing correction amount for the set dispense amount is obtained in advance.
An automatic dispensing amount correction method, wherein when a set dispensing amount is dispensed for a liquid sample by the pipette, the dispensing correction amount is corrected. In a dispensing device equipped with a pipette that sucks and discharges a liquid sample by controlling a driving means for moving the piston in the cylinder forward and backward by the control means,
Prepare a solution capable of measuring absorbance, measure the absorbance of one or more quantitative solutions in which the solution is dispensed into a predetermined container, create reference data based on the measured absorbance,
Pipette the solution into the same type of container with a pipette that automatically dispenses to the desired set dispensing volume,
Measure the absorbance of the solution of the actual dispensing amount dispensed with respect to the set dispensing amount,
From the difference between the measured actual absorbance and the reference absorbance obtained from the reference data for the set dispense amount, a dispensing correction amount for the set dispense amount is obtained in advance.
Correction data relating to the determined dispensing correction amount is stored in the control means,
When dispensing a set dispensing amount for a liquid sample with the pipette, the dispensing device controls the driving means based on the correction data to correct the dispensing correction amount.

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