Method for Measuring Discharge Amount of Sub-Dispenser

Abstract

The present invention involves: dissolving a dye in a volatile liquid to prepare a dye-containing volatile liquid that contains the dye at a predetermined concentration (step 11); suctioning the dye-containing volatile liquid by a dispenser and discharging the same into a measurement vessel (step 12); drying the dye-containing volatile liquid discharged into the measurement vessel (step 13); adding, after the drying, a predetermined amount of a low-volatile liquid having a volatility lower than that of the volatile liquid to the measurement vessel to dissolve the dye and prepare an assessment liquid (step 14); conducting an optical measurement on the assessment liquid (step 15); and determining a discharged amount that is the volume of the dye-containing volatile liquid discharged into the measurement vessel, on the basis of a measured value of the assessment liquid obtained by the optical measurement, the added amount of the low-volatile liquid, and the concentration of the dye in the dye-containing volatile liquid (step 16). As a result, the amount of a volatile liquid dispensed by a dispenser such as a micropipette can be accurately measured.

Description

Technical Field

[0001] The present invention relates to a method for measuring the discharge amount of a dispenser.

Background Art

[0002] In physicochemical experiments, piston-type dispensers such as micropipettes or automatic pipetting devices (hereinafter simply referred to as "dispensers") are widely used to accurately dispense liquid samples or reagents. Since the error in the dispensed amount by such a dispenser may greatly affect the subsequent experimental results, it is necessary to periodically check whether the dispenser is functioning accurately. Conventionally, for the inspection of micropipettes, measurement of the discharge amount by the gravimetric method has been widely adopted (see, for example, Non-Patent Document 1). In the gravimetric method, a standard liquid (for example, water) is aspirated and discharged using the micropipette to be inspected, and the mass of the discharged standard liquid is measured with an electronic balance. Then, based on the measured value at that time and the density of the standard liquid at the temperature during measurement, the volume of the standard liquid discharged from the micropipette is calculated. Thereby, it can be confirmed whether the micropipette can discharge the intended amount of the standard liquid (that is, the accuracy of dispensing by the micropipette).

[0003] However, when attempting to inspect a dispenser having a plurality of channels, such as a multi-channel type automatic pipetting device, by such a gravimetric method, it is necessary to perform suction, discharge, and mass measurement of the standard liquid one channel at a time as described above, which has a problem of being very time-consuming.

[0004] Therefore, when inspecting a dispenser having such a plurality of channels, discharge amount measurement using absorbance measurement is performed (see, for example, Non-Patent Document 2). Specifically, a standard liquid in which a dye is dissolved at a predetermined concentration is aspirated through each channel of an automatic pipetting device to be inspected, and the standard liquid aspirated through each channel is discharged into different wells of a microtiter plate. Thereafter, a certain amount of water is added to each well and mixed with the standard liquid, and then the absorbance of the liquid in each well is measured using a microplate reader. Then, by applying the measured value of the absorbance to a calibration curve prepared in advance, the concentration of the dye in the liquid in each well is obtained, and based on the concentration, the added amount of the water, and the concentration of the dye in the standard liquid, the discharge amount of the standard liquid through each channel is calculated.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] In the measurement of the discharge amount by the above conventional method, it is common to use water as a standard substance. However, in that case, in the dispensing of a liquid having significantly different characteristics from water, the accuracy of the dispensing confirmed by the above method may not be guaranteed. For example, when dispensing a highly volatile liquid such as acetonitrile or acetone with a dispenser, the internal pressure of the dispenser may change due to the vapor generated from the liquid after sucking the liquid, resulting in a change in the dispensed amount. Therefore, even a dispenser that has been confirmed to be able to dispense with sufficient accuracy by measurement using water as a standard substance may not be able to dispense a volatile liquid with the same accuracy. Therefore, in order to accurately dispense such a volatile liquid, it is necessary to measure the discharge amount using a liquid having the same characteristics as the liquid to be dispensed. However, in the gravimetric method, since the measured value of the electronic balance fluctuates due to the volatilization of the volatile liquid, it is difficult to correctly measure the mass of the volatile liquid discharged by the dispenser. Also, in the method using absorbance measurement as described above, the value of the discharged amount calculated varies depending on the degree of volatilization of the volatile liquid between the time when the volatile liquid added with a dye is discharged into the well and the time when water is added to each well. In particular, in the microdispensing in the sub-microliter to microliter order, such an error in the measured value due to volatilization becomes significant.

[0007] The present invention has been made in view of the above points, and an object thereof is to enable accurate measurement of the dispensed amount of a volatile liquid by a dispenser.

Means for Solving the Problems

[0008] A method for measuring the discharge amount of a dispenser according to the present invention, which has been made to solve the above problems, is as follows: By dissolving a dye in a volatile liquid, a volatile liquid containing the dye at a predetermined concentration is prepared. The dispenser sucks the volatile liquid containing the dye and discharges it into a measurement container. Dry the volatile liquid containing the dye discharged into the measurement container, After the drying, add a predetermined amount of a low-volatile liquid having a lower volatility than the volatile liquid to the measurement container to dissolve the dye, thereby preparing an evaluation liquid, Perform optical measurement on the evaluation liquid, Based on the measurement value of the evaluation liquid obtained by the optical measurement, the added amount of the low-volatile liquid, and the concentration of the dye in the volatile liquid containing the dye, the discharge amount, which is the volume of the volatile liquid containing the dye discharged into the measurement container, is determined.

Advantages of the Invention

[0009] According to the method for measuring the discharge amount of the dispenser according to the present invention, it is possible to accurately measure the dispensed amount of the volatile liquid by the dispenser.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Embodiments for Carrying Out the Invention

[0011] Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. FIG. 1 is a flowchart showing the execution procedure of the calibration method of the dispenser according to the present embodiment. In the calibration method according to the present embodiment, the volatile liquid is sucked and discharged by a micropipette at a predetermined set volume, the discharge amount at that time is measured, and the accuracy of the discharge amount by the micropipette is evaluated by comparing the discharge amount with the set volume. Further, if necessary, the micropipette is adjusted so that the volatile liquid can be dispensed with sufficient accuracy.

[0012] The volatile liquid in the present invention means a substance having a boiling point at 1 atm within the range of 50 to 95°C and being in a liquid state at normal temperature (20°C ± 15°C), or a liquid containing 10% or more of such a substance. Examples of substances having a boiling point at 1 atm within the range of 50 to 95°C and being in a liquid state at normal temperature (20°C ± 15°C) include, for example, acetonitrile, methanol, ethanol, acetone, toluene, isopropanol, hexane, butanol, cyclohexane, ethylene glycol, benzene, chloroform, acetaldehyde, triethylamine, phenol, naphthalene, formaldehyde, tetrahydrofuran, or ethyl acetate.

[0013] In the method according to this embodiment, first, a liquid containing the dye at a predetermined concentration is prepared by adding and dissolving a dye in a volatile liquid (step 11). Hereinafter, this liquid is referred to as a "volatile liquid containing dye". Examples of the dye include, for example, tartrazine or Coomassie Brilliant Blue (CBB), etc., but the dye in the present invention is not limited thereto. In addition, as the dye, it is desirable to use one having a viscosity close to that of the volatile liquid used for measuring the discharge amount. Specifically, it is desirable to use one having a viscosity within ±30% (preferably ±15%) of the volatile liquid, but it is not limited thereto.

[0014] Using the micropipette to be evaluated, the volatile liquid containing dye is aspirated in a predetermined set volume and discharged into a measurement container (step 12). As the measurement container, for example, a cuvette for spectroscopic measurement, a microtiter plate, etc. can be used, but any container that can be used for optical measurement may be used.

[0015] Subsequently, the volatile liquid containing dye discharged into the measurement container is completely dried (step 13). The drying method is not particularly limited, but in order to eliminate the possibility of the dye sublimating by heating, it is desirable to perform natural drying.

[0016] Next, add a predetermined amount of water to the measurement container and dissolve the dye in the water (step 14). The aqueous solution of the dye thus obtained is hereinafter referred to as the "evaluation liquid".

[0017] Set the measurement container in a spectrophotometer and measure the absorbance of the evaluation liquid at the absorption wavelength of the dye (step 15). When a microtiter plate is used as the measurement container, a microplate reader is used as the spectrophotometer.

[0018] Also, prepare a plurality of calibration curve preparation liquids, which are aqueous solutions containing the same dye as the dye contained in the evaluation liquid at different concentrations (step 21), and measure the absorbance of each calibration curve preparation liquid in the same manner (step 22). In step 15 described above, the plurality of calibration curve preparation liquids may be set in the spectrophotometer together with the evaluation liquid, and the absorbance measurement of the evaluation liquid and the absorbance measurements of the plurality of calibration curve preparation liquids may be performed continuously.

[0019] Then, based on the measurement results of the plurality of calibration curve preparation liquids, a calibration curve showing the relationship between the concentration and absorbance of the dye is created. Further, based on the concentration of the dye in the volatile liquid containing the dye and the amount of water added in step 14, the concentration of the dye in the calibration curve is converted into the volume of the volatile liquid containing the dye (i.e., the theoretical value of the discharge amount of the volatile liquid containing the dye by the micropipette). Thereby, a calibration curve showing the relationship between the absorbance and the discharge amount (theoretical value) is obtained (step 23).

[0020] Subsequently, the discharge amount of the volatile liquid containing the dye by the micropipette is specified by applying the absorbance of the evaluation liquid measured in step 15 to the calibration curve created in step 23 (step 16).

[0021] Here, in step 23, a calibration curve showing the relationship between the absorbance and the concentration of the dye was created, and this calibration curve was converted into a calibration curve showing the relationship between the absorbance and the discharge amount. However, the present invention is not limited to this. In step 23, only a calibration curve showing the relationship between the absorbance and the concentration of the dye may be created. In this case, in step 16, the concentration of the dye in the evaluation liquid is specified based on the calibration curve and the absorbance of the evaluation liquid measured in step 15, and based on that value, the concentration of the dye in the volatile liquid containing the dye, and the amount of water added in step 14, the discharge amount of the volatile liquid containing the dye is calculated.

[0022] As described above, in the method according to the present embodiment, by including the step of drying the volatile liquid containing the dye discharged by the dispenser, it is possible to eliminate fluctuations in the measured values due to the volatility of the volatile liquid. Therefore, according to the method of the present embodiment, even for a small amount of volatile liquid, its discharge amount can always be accurately measured.

[0023] Subsequently, the set volume of the micropipette in step 12 is compared with the discharge amount obtained in step 16, and it is determined whether the difference between the two is equal to or greater than a predetermined threshold value (step 17). And when the difference between the two is smaller than the threshold value (that is, when the answer is No in step 17), the series of operations is terminated as it is.

[0024] On the other hand, when the difference between the two is equal to or greater than a predetermined threshold value (that is, when the answer is Yes in step 17), the micropipette is adjusted so that the difference becomes smaller (step 18). Specifically, while finely adjusting a dial or the like provided on the micropipette, steps 12 to 18 are repeatedly executed, and when the answer becomes No in step 17, the series of operations is terminated.

[0025] As described above, the embodiments for carrying out the present invention have been described. However, the present invention is not limited to the above embodiments, and appropriate modifications are allowed within the scope of the gist of the present invention.

[0026] For example, in the above embodiment, the discharge amount of the micropipette was measured. However, instead of this, the discharge amount of other dispensers, for example, an automatic dispenser, may be measured. In addition, the dispenser includes a single-channel type having only one set of a cylinder, a piston operating in the cylinder, and a tip attachment portion provided at the tip of the cylinder, and a multi-channel type having a plurality of sets of the cylinder, piston, and tip attachment portion. However, the present invention can be applied to any of them. Further, the dispenser includes a manual type in which the piston is operated by a user and an automatic type driven by a motor, an air displacement type in which a certain amount of air exists between the piston and the liquid, and a forced displacement type in which the piston directly contacts the liquid, and various types such as a fixed-volume type in which the dispensed amount (set volume) is constant and a variable-volume type in which the dispensed amount can be changed. However, the discharge amount measurement method and the calibration method according to the present invention can be applied to any of them. When the calibration method according to the present invention is applied to an automatic dispenser, in step 18, the adjustment of the dispenser is performed by changing the relationship between the set volume and the driving amount of the motor stored in the dispenser or a computer for controlling the dispenser.

[0027] Also, in the above embodiment, the absorbance of the evaluation liquid was measured in step 15. However, the "optical measurement" in the present invention is not limited to absorbance measurement, and may be any measurement such as fluorescence measurement or transmittance measurement. When fluorescence measurement is performed as the optical measurement, a dye that emits fluorescence is used as the dye. Also, in this case, in the description of steps 15 to 16 and steps 22 to 23 in the above embodiment, "absorbance" is replaced with "fluorescence intensity".

[0028] In the above embodiment, water is used as the low-volatile liquid in the present invention (that is, water is added to the evaluation container in step 14 to dissolve the dye), but other low-volatile liquids may be used instead of water. The low-volatile liquid in the present invention means a substance whose boiling point at 1 atm is higher than 95°C and which is in a liquid state at room temperature (20°C ± 15°C). Examples of such low-volatile liquids include, in addition to water, dimethyl sulfoxide, glycerin, or phenol.

Example

[0029] Using the discharge amount measurement method according to the present invention, the discharge amount in each channel (48 channels) of a multi-channel type automatic dispenser was measured.

[0030] 1. Preparation of volatile liquid containing dye By adding 0.5 g of tartrazine as a dye to 25 mL of water and dissolving it, a 20 g / L aqueous tartrazine solution was prepared. Further, the aqueous tartrazine solution and acetonitrile were mixed at a ratio of 3:7. The resulting mixture (tartrazine concentration: 6000 mg / L) is hereinafter referred to as "volatile liquid containing dye".

[0031] 2. Preparation of liquid for calibration curve creation By mixing a 20 g / L aqueous tartrazine solution and water at a ratio of 3:7, a 6 g / L aqueous tartrazine solution was prepared. Further, the aqueous tartrazine solution was diluted 30 to 360 times with water to prepare six types of aqueous solutions with different concentrations of tartrazine (hereinafter referred to as "dye"). These six types of aqueous solutions are hereinafter referred to as "liquid for calibration curve creation".

[0032] 3. Measurement of absorbance and calculation of discharge amount (1) Using a multi-channel type automatic dispenser, the volatile liquid containing dye was simultaneously dispensed into each well of a microtiter plate for absorbance measurement from each channel, and then dried. (2) 100 μL of water was added to each of the wells after drying, and the mixture was stirred with a shaker. The aqueous solution of the dye thus obtained is hereinafter referred to as the "evaluation liquid". (3) 100 μL of each calibration curve preparation liquid was dispensed into two separate wells each, different from the wells containing the evaluation liquid. (4) 100 μL of water was dispensed into another two wells as blanks. (5) The microtiter plate was set in a microplate reader, and the absorbance at the absorption wavelength (426 nm) of the dye was measured. (6) The average value of the measured values of the blanks was subtracted from the measured values (absorbance) of each well obtained by the absorbance measurement. (7) Based on the dye concentration (mg / L) in each calibration curve preparation liquid and the value obtained in (6) above, a calibration curve (Figure 2) showing the relationship between absorbance and dye concentration was created by the least squares method. (8) By converting the dye concentration (mg / L) into the theoretical discharge volume (μL) using the following formula, the calibration curve was converted into a calibration curve (Figure 3) showing the relationship between absorbance and theoretical discharge volume. Theoretical discharge volume (μL) = {dye concentration (mg / L) × liquid volume in the well (100 μL)} / dye concentration in the volatile liquid containing the dye (6000 mg / L) (9) From the measured value (absorbance) of the evaluation liquid in the absorbance measurement, the theoretical discharge volume of the volatile liquid containing the dye for each channel was calculated using the calibration curve in (8).

[0033] Table 1 shows the measured values (absorbances N1 and N2) for each of the above calibration curve preparation liquids, the inverse regression values and the fidelity calculated from the calibration curve (Figure 2) created based on the measured values.

Table 1

[0034] Table 2 shows the measured absorbance values of the liquid for evaluation in each well of the microtiter plate (in the table, "Ch number" indicates the channel number of the automatic pipetting device that discharged the volatile liquid containing the dye into each well).

Table 2

[0035] Table 3 shows the results of converting the measured absorbance values in Table 2 into the theoretical dispensed volume using the calibration curve in Figure 3. The numerical values in each cell of this table correspond to the theoretical discharge volume of the volatile liquid containing the dye by each channel of the automatic pipetting device (i.e., the discharge volume measured by the method according to the present invention).

Table 3

[0036] [Aspect] Those skilled in the art will understand that the above-described plurality of exemplary embodiments are specific examples of the following aspects.

[0037] (Item 1) The method for measuring the discharge volume of a dispenser according to one aspect of the present invention is By dissolving a dye in a volatile liquid, a volatile liquid containing the dye with a predetermined concentration is prepared, The dispenser sucks the volatile liquid containing the dye and discharges it into a measurement container, The volatile liquid containing the dye discharged into the measurement container is dried, After the drying, a predetermined amount of a low-volatile liquid having a lower volatility than the volatile liquid is added to the measurement container to dissolve the dye, thereby preparing a liquid for evaluation, Optical measurement is performed on the liquid for evaluation, Based on the measured value of the liquid for evaluation by the optical measurement, the added amount of the low-volatile liquid, and the concentration of the dye in the volatile liquid containing the dye, the discharge volume, which is the volume of the volatile liquid containing the dye discharged into the measurement container, is determined.

[0038] (Item 2) The method for measuring the discharge amount of the dispenser according to Item 1 is preparing a plurality of calibration curve preparation liquids by dissolving the pigments in the low-volatile liquid at different concentrations, performing the optical measurement on each of the plurality of calibration curve preparation liquids, creating a calibration curve based on the measurement results of the plurality of calibration curve preparation liquids by the optical measurement, and the discharge amount may be obtained from the measurement value in the optical measurement of the evaluation liquid using the calibration curve.

[0039] According to the method for measuring the discharge amount of the dispenser according to Item 1 or Item 2, accurate measurement of the discharge amount can always be performed without being affected by the volatilization of the volatile liquid.

[0040] (Item 3) The method for measuring the discharge amount of the dispenser according to Item 1 or Item 2 is the dispenser may be a multi-channel type dispenser.

[0041] According to the method for measuring the discharge amount of the dispenser according to Item 3, since the discharge amount can be measured for each channel of the multi-channel type dispenser at once, the labor and time required for the discharge amount measurement can be reduced.

[0042] (Item 4) The calibration method of the dispenser according to one aspect of the present invention is by dissolving a pigment in a volatile liquid, preparing a volatile liquid containing the pigment at a predetermined concentration, aspirating the volatile liquid containing the pigment by a dispenser at a predetermined set volume and discharging it into a measurement container, drying the volatile liquid containing the pigment discharged into the measurement container, after the drying, adding a predetermined amount of a low-volatile liquid having a lower volatility than the volatile liquid to the measurement container to dissolve the pigment, thereby preparing an evaluation liquid, performing an optical measurement on the evaluation liquid, Based on the measured value of the liquid for evaluation by the optical measurement, the added amount of the low-volatile liquid, and the concentration of the pigment in the volatile liquid containing the pigment, the discharge amount, which is the volume of the volatile liquid containing the pigment discharged into the measurement container, is determined. The difference between the calculated discharge amount and the set volume is specified. The dispenser is adjusted so that the difference becomes smaller.

[0043] According to the calibration method of the dispenser described in claim 4, accurate calibration can always be performed without being affected by the evaporation of the volatile liquid.

Claims

1. By dissolving a pigment in a volatile liquid, a volatile liquid containing the pigment at a predetermined concentration is prepared. The volatile liquid containing the pigment is aspirated by a dispenser and discharged into a measurement container. The volatile liquid containing the pigment discharged into the measurement container is dried. After the drying, a predetermined amount of a low-volatility liquid having a lower volatility than the volatile liquid is added to the measurement container to dissolve the pigment, thereby preparing an evaluation liquid. Optical measurement is performed on the evaluation liquid. Based on the measured value of the evaluation liquid obtained by the optical measurement, the added amount of the low-volatility liquid, and the concentration of the pigment in the volatile liquid containing the pigment, the discharge amount, which is the volume of the volatile liquid containing the pigment discharged into the measurement container, is determined. A method for measuring the discharge amount of a dispenser.

2. A plurality of calibration curve preparation liquids are prepared by dissolving the pigment in the low-volatility liquid at different concentrations. Optical measurement is performed on each of the plurality of calibration curve preparation liquids. A calibration curve is created based on the measurement results of the plurality of calibration curve preparation liquids obtained by the optical measurement. Using the calibration curve, the discharge amount is determined from the measured value in the optical measurement of the evaluation liquid. The method for measuring the discharge amount of a dispenser according to Claim 1.

3. The method for measuring the discharge amount of a dispenser according to Claim 1, wherein the dispenser is a multi-channel type dispenser.

4. By dissolving a pigment in a volatile liquid, a volatile liquid containing the pigment at a predetermined concentration is prepared. The volatile liquid containing the pigment is aspirated by a dispenser at a predetermined set volume and discharged into a measurement container. The volatile liquid containing the pigment discharged into the measurement container is dried. After the drying, a predetermined amount of a low-volatility liquid having a lower volatility than the volatile liquid is added to the measurement container to dissolve the pigment, thereby preparing an evaluation liquid. Optical measurement is performed on the evaluation liquid. Based on the measured value of the evaluation liquid obtained by the optical measurement, the added amount of the low-volatility liquid, and the concentration of the pigment in the volatile liquid containing the pigment, the discharge amount, which is the volume of the volatile liquid containing the pigment discharged into the measurement container, is determined. The difference between the calculated discharge amount and the set volume is specified. The dispenser is adjusted so that the difference becomes smaller. A calibration method for a dispenser.

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