JP5483134B2 - Method and apparatus for measuring volume of microdroplet - Google Patents

Description

  The present invention relates to a method for measuring the volume of a minute droplet suitable for accurately and accurately measuring the volume of a minute droplet, and an apparatus for performing the method.

  In recent years, due to the remarkable development of chip analysis methods and rapid multi-analyte analysis technology, high-throughput analysis devices in the environmental and bio fields are accelerating. Accordingly, the amount of sample solution is accelerating, and the need for applying a multi-sample solution to a minute region is increasing. In fact, the amount of sample handled by the sample dispensing apparatus in the environmental / bio field that has been developed in recent years has decreased from the conventional microliter level to the nanoliter level or picoliter level.

  In such a background, since the separation of analysis samples with high accuracy and accuracy greatly affects the reliability of analysis results, the importance of a method for accurately measuring a minute amount of sample solution is also increasing. In addition, from the viewpoint of traceability in this field, it is considered that calibration of the measurement volume of a sample dispensing apparatus that handles a small amount of sample solution will become increasingly important.

When the volume of the droplet is large, in the conventional method, a droplet of a liquid having a known specific gravity such as water is created, and then the weight of the droplet is measured to obtain the volume of the droplet. This method is widely used for calibration of instruments that dispense a relatively large volume (0.5 microliters or more) of liquid, such as a micropipette. FIG. 1 shows a specific procedure of this method. That is, the droplet weight w per one is obtained from the weight (W ₁ and W ₂ ) of the liquid container before and after dispensing and the number n of the dispensed droplets, and the droplet is determined from the specific gravity r of the liquid. Determine the volume V of. Alternatively, the weight W of the dispensed liquid is directly obtained, and the volume V of the droplet is obtained by the same method. This method can be performed relatively easily without using a special device other than a weighing instrument such as a balance. However, since the liquid evaporates during measurement, the exact weight of the droplet is measured. It is difficult to do this, and this method is not suitable particularly in the case of nanoliter or picoliter level droplets whose evaporation is negligible.

  As another method for obtaining the volume of a minute droplet, a droplet ejected from the ejection port by a pulse light using a light source such as a strobe, LED, laser, etc. There is a method of measuring by observing with a microscope while illuminating (see, for example, Patent Document 1).

  According to this method, it is possible to measure one volume of a minute droplet from a microscopic image. However, the liquid droplets are measured based on external dimensions from one direction orthogonal to the ejection direction or from two directions orthogonal to each other. Calculating the volume by approximating the cross-sectional shape of the droplet with an ellipse, the pulse width of the irradiation light cannot be reduced to ensure the amount of light required for microscopic imaging, and the evaporation of liquid cannot be ignored due to the small amount of droplets As a result, errors were likely to occur.

  Another way to determine the volume of a microdroplet is to attach a microdroplet to a lyophilic surface area delimited by a lyophobic surface and then use the optical volume to create a three-dimensional shape of the liquid droplet that extends throughout the area. There is a method of calculating a volume by measuring with a measuring device and integrating the three-dimensional shape (see, for example, Patent Document 2).

  According to this method, it is possible to directly measure the volume of the droplet formed in the lyophilic surface area without performing the shape approximation as described above. Since a relatively large liquid volume is required to cover, it is difficult to determine the volume of one minute droplet. Further, since the evaporation of the liquid cannot be ignored due to the small amount of droplets, an error is likely to occur. This also applies to a method (for example, refer to Patent Document 4) in which a minute droplet dropped on a substrate is imaged and the droplet volume is calculated from numerical values such as a contact angle and a contact diameter between the droplet and the substrate.

  In order to suppress the evaporation of the liquid as much as possible, a small amount of liquid droplets that drop the droplets on a petri dish etc. and then touch the droplets to regulate the size of the droplets and improve the measurement accuracy The volume measurement method is also reported (for example, see Patent Document 3). However, it is inevitable that the liquid evaporates into the air layer remaining between the petri dish and the lid. Rather, the evaporation of the liquid is promoted by the interfacial tension, and measurement errors are likely to occur. The same applies to a method (for example, refer to Patent Document 5) in which a minute droplet is sucked into a microtube and the volume of the droplet is calculated from the length and cross-sectional area of the microtube in which the droplet is held.

  From the viewpoint of droplet volume measurement methods that do not depend on the shape or optical characteristics of the liquid, a method for determining the volume of a droplet from the change in capacitance in the electric field by passing the droplet through a flow path that forms an electric field. (For example, patent document 6) is also proposed. However, since a relatively large droplet volume is required and the evaporation of the liquid cannot be ignored, it is difficult to adapt to a small amount of droplets.

  In the scientific field based on a technique of forming a minute droplet by applying a pulse based on electrostatic force, a liquid droplet containing particles is dropped from the nozzle onto the substrate while a pulse potential is applied between the nozzle and the substrate, There is a method (for example, Patent Document 7) for determining the volume of a droplet by integrating the amount of charge flowing during the dropping. However, since it is necessary to measure the amount of charge at the same time as the dispensing of the droplet, it can only be applied to a dispensing device equipped with a mechanism for applying a pulse potential to the droplet, and the applicable liquid also has particles. Since it is necessary to include, it is difficult to say that it is a general method for measuring the volume of a minute droplet.

  Dispense and dilute a dye solution with a known concentration in a small container such as a cuvette or microtiter plate filled with a certain amount of liquid such as water, and determine the dilution ratio from the change in absorbance before and after dilution. There is a method for obtaining the volume of the liquid droplets. In order to measure with an absorptiometer, it is necessary to increase the volume to a measurable capacity. However, in addition to increasing the error in absorbance measurement due to dilution, the evaporation of the liquid cannot be ignored. Therefore, it is difficult to adapt to a minute droplet.

  That is, in order to obtain the volume of a minute droplet with high accuracy, the conventional method cannot easily ignore the evaporation of the liquid, and an error is likely to occur. Therefore, there is a need for a method for measuring the volume of a minute droplet that is not related to the evaporation of the liquid. However, at present, such a method has not been developed, and the volume of a minute droplet is obtained by any of the above-described methods. It is the actual situation.

Japanese Patent Publication No. 29682880 JP 2004-177243 JP 2001-41799 A JP 2006-167534 A JP2003-14442 JP 2004-513710 A JP 2006-58188 A

  The present invention has been made in view of the circumstances as described above, and provides a method for accurately and accurately measuring the volume of a minute droplet regardless of the evaporation of the liquid, and an apparatus for carrying out the method. It is for the purpose.

  As a result of intensive studies to achieve the above object, the present inventors have dispensed droplets using a measurement liquid containing a labeled substance having a detectable physical property and a known concentration. It was found that the above-mentioned purpose of knowing the volume of the droplet can be achieved by a method of detecting the physical property of the labeling substance in the droplet. In addition, when detecting the physical properties of the labeling substance in the droplet, a substrate having a measurement region for placing the droplet and a reference region for correcting the concentration if necessary is used as the reference region. Based on the physical property values obtained in this way, the knowledge that the above object can be achieved by measuring the absolute amount of the labeling substance contained in the droplets in the measurement region was obtained.

  The present invention solves the problem by the technical idea as described above, but more specifically, is as follows. That is, in order to solve the above problems, the method for measuring a volume of a trace amount liquid according to the present invention dispenses a liquid by using a measurement liquid containing a labeling substance having a detectable physical property and having a known concentration. By detecting the physical properties of the labeling substance in the liquid, the volume of the trace liquid after dispensing is measured.

  In addition, according to another volumetric liquid volume measuring method according to the present invention, in the volumetric liquid volume measuring method, a measuring liquid containing a labeling substance is dispensed in a plurality of spaces formed on a substrate. A liquid containing only the labeling substance is placed in the space, and the physical property of the labeling substance in the dispensed measurement liquid is detected, the physical property of the liquid containing only the labeling substance is detected, and the measurement liquid after dispensing based on both detection values It is characterized by measuring the volume of.

  Further, in another volumetric liquid volume measuring method according to the present invention, in the volumetric liquid volume measuring method, a part of the plurality of spaces formed on the substrate is a space not including liquid, and the liquid of the labeling substance is It is used as a reference space when detecting physical properties.

  3. The method for measuring a volume of a minute droplet according to claim 1 or 2, wherein a substance having a large molecular weight is added to the measurement liquid to reduce the evaporation rate of the measurement liquid.

  Further, according to another method for measuring a volume of a minute liquid according to the present invention, a droplet dispensed using a light-absorbing substance, a fluorescent substance, a luminescent substance, or a radioisotope as the labeling substance in the method for measuring a volume of a minute liquid. Using a densitometer, a fluorescence scanner, a luminometer, or autoradiography, image a substrate equipped with, and read the physical properties of absorbance, fluorescence intensity, emission intensity, or radiation intensity obtained from the labeling substance in the measurement liquid as an image The volume of the liquid for measurement is calculated by comparing with the physical property value of only the labeling substance.

  Moreover, the volume measuring device for a trace amount liquid according to the present invention forms a plurality of spaces on a substrate, dispenses a measuring liquid containing a labeling substance in a part of the space, and in another part of the space. Put the labeled substance only liquid, detect the physical properties of the labeled substance in the dispensed measurement liquid, detect the physical properties of the labeled substance only liquid, and the volume of the measured liquid after dispensing based on both detection values Is measured.

  Further, in the volumetric measurement device for other trace liquid according to the present invention, in the volumetric measurement apparatus for the trace liquid, the space is a plurality of through holes penetrating up and down a partition wall formed separately from the substrate, The groove formed around the substrate side opening in the space is characterized by using a jig provided with a sealing O-ring in contact with the substrate.

  Another volume measuring device for a small amount of liquid according to the present invention is the volume measuring device for a small amount of liquid, wherein a part of the space is a space that does not contain liquid, and is used for reference when detecting the physical properties of the liquid of the labeling substance. It is used as a space.

  According to another aspect of the present invention, there is provided a trace liquid volume measuring apparatus, wherein the substrate is formed of a conductive material and an electrochemically active substance is used as a labeling substance.

  Further, another volumetric liquid volume measuring device according to the present invention includes a lid for sealing the space of the partition wall in the volumetric liquid volume measuring device, and the space for containing only the labeling substance is filled with the labeling substance. Then, a jig that seals with the lid and defines the optical path length is used.

  According to another aspect of the invention, the volume measuring device for a small amount of liquid according to the present invention is such that the groove formed around the substrate side opening and the lid side opening in the space is in contact with the substrate and the lid, respectively. The O-ring is provided, and the substrate, the partition, and the lid are compressed and integrated and fixed by a fixing member that sandwiches the partition.

  According to another aspect of the invention, the fixing member is a screw that fixes the substrate and the lid with the partition wall interposed therebetween. And

  According to another aspect of the present invention, there is provided a volumetric device for measuring a small amount of liquid. In the volumetric device for a small amount of liquid, the fixing member can be slid and housed with the substrate and the lid sandwiched therebetween. It is a container provided with a space.

  According to another aspect of the present invention, there is provided a volumetric device for measuring a small amount of liquid, wherein the partition wall is formed of an elastic material and a regulating member having a height lower than that of the partition is provided in a free state. The partition of the elastic material is compressed when the space is sealed by the lid, and the height is regulated by closely attaching the lid to the upper surface of the defining member, thereby defining the optical path length.

  In addition, according to another aspect of the present invention, there is provided a micro liquid volume measuring device, wherein the substrate is provided with an electrode for measuring physical properties of the liquid in the space, and is made of an electrochemically active substance. It is characterized by the fact that the nature of a substance can be measured.

  According to another aspect of the present invention, there is provided a volumetric device for measuring a small amount of liquid, wherein the electrode is formed by separating a working electrode and a counter electrode.

  Since the present invention is configured as described above, it is possible to accurately and accurately realize the measurement of the volume of a minute droplet regardless of the evaporation of the liquid.

It is a figure explaining the conventional method. It is a figure explaining a partition. It is a figure explaining the area | region for a reference on the board | substrate for a measurement. It is a figure explaining the method to prescribe | regulate the optical path length of the area | region for reference. It is a figure explaining another method which prescribes | regulates the optical path length of the area | region for reference. It is a figure explaining another method which prescribes | regulates the optical path length of the area | region for reference. It is a figure explaining another method which prescribes | regulates the optical path length of the area | region for reference. It is a figure explaining the method to calculate the absolute amount of a labeling substance at the time of using a light-absorbing substance as a labeling substance. It is a figure explaining the method to measure the absolute amount of a labeling substance at the time of using an electrochemically active substance as a labeling substance.

  The present invention solves the problem of accurately and accurately measuring the volume of a minute droplet regardless of the evaporation of the liquid by using a measuring liquid containing a labeling substance having a known physical property and having a detectable physical property. This is realized by a method of dispensing droplets and detecting physical properties of a labeling substance in the dispensed droplets. In addition, when detecting the physical properties of the labeling substance in the droplet, a substrate having a measurement region for placing the droplet and a reference region for correcting the concentration if necessary is used as the reference region. This is realized by measuring the absolute amount of the labeling substance contained in the droplets in the measurement region by using the physical property values obtained in this way as a reference.

  The point of measurement in the present invention is that the concentration of the labeling substance contained in the liquid to be dispensed and the label contained in the dispensed droplet even if the liquid of the liquid dispensed in the stage from dispensing to measurement evaporates. The droplet volume can be accurately calculated from the absolute amount of the substance. Examples of labeling substances include light-absorbing substances, fluorescent substances, luminescent substances, radioisotopes, and electrochemically active substances. The detectable physical properties of labeling substances include absorbance, turbidity, fluorescence intensity, and luminescence. Intensity, radiation intensity, charge amount (number of electrons) and the like can be mentioned.

  The liquid droplets for measurement may decrease in liquid volume as the liquid evaporates, or the liquid volume may be increased by supplying liquid from the outside in order to facilitate physical property measurement. In order to slow down the evaporation rate, alcohol, ether, sugar, peptide, protein or the like having a large molecular weight may be added.

  The measurement substrate is preferably made of a material that does not affect the physical properties of the labeling substance. For example, when using a light-absorbing substance as a labeling substance, it is desirable to use a substrate made of a material such as glass or quartz that does not absorb light. When using an electrochemically active substance as a labeling substance, an electrically conductive metal is used. It is desirable to use a substrate made of a material such as platinum.

  FIG. 2 is an example of the measurement substrate 1 having a space partitioned by partition walls 2 so that physical property values between the droplets do not interfere with each other during measurement. Each droplet is dispensed into this partitioned space. Examples of the material of the partition wall 2 include colored quartz / glass, resin, and metal. For example, when using a light-absorbing substance, a fluorescent substance, or a luminescent substance as a labeling substance, a droplet is applied to a substrate 1 having a partition wall 2 made of a material that does not transmit measurement light so that the measurement light does not interfere with each other. It is desirable to dispense.

  The measurement substrate 1 and the partition wall 2 may be fabricated as a single unit, or both may be fabricated separately. When both are produced and combined separately, it is desirable to provide resin packing such as O-rings 4 and 6 in the grooves around the upper and lower ends of the space 3 as shown in FIG. When the partition 2 is resin, it does not restrict to this. By separating the two, liquid droplets can be dispensed on the substrate 1 having various surface properties. For example, the volume of the droplet that is actually dispensed depends on the surface tension between the measurement substrate surface, the discharge port surface of the dispensing apparatus, and the measurement liquid. When the dispensing accuracy of the dispensing device is measured from the droplet volume, it is possible to obtain the dispensing accuracy corresponding to the type of measurement substrate.

  FIG. 3 shows an example of the measurement substrate 1 having a reference region for correcting the density. For example, when a light-absorbing substance is used as a labeling substance, at least two reference regions with a defined optical path length are provided, at least one (region R) is filled with a measurement liquid, and at least one other ( By setting the region B) as a blank, the relationship between the absorbance and the absolute amount of the labeling substance can be known. By using this relationship, since the absolute amount of the labeling substance in the droplet can be calculated, the volume of the droplet can be calculated. The same method can be applied when a fluorescent substance, a luminescent substance, or a radioisotope is used as a labeling substance.

  FIG. 4 is an example of a jig for defining the optical path length in the reference region. By covering the partition wall 2, the optical path length of the measurement liquid filled in the reference region is defined by the height of the partition wall. In order to improve the adhesiveness between the partition wall 2 and the lid, it is desirable to fit them through a resin packing such as an O-ring as shown in FIG. In the case where both the measurement substrate 1 and the partition wall 2 are separated, it is also desirable to match them through packing. However, the present invention is not limited to this when the partition wall 2 is made of a resin having good adhesion to the measurement substrate 1.

  FIG. 5 is another example of a jig for defining the optical path length in the reference region. When the partition wall is made of a soft material that can be easily compressed, such as a resin, a frame 7 is provided as a defining member for defining the optical path length of the reference region made of a hard material. Defines the optical path length of the reference region. The height of the frame 7 is configured to be lower than the partition wall 2. The measurement substrate 1 and the frame 7 may be manufactured as a single body, or may be separately manufactured and combined as in this example.

  FIG. 6 is another example of a jig for defining the optical path length in the reference region. The packing in FIG. 4 is compressed and fixed by applying pressure to the vertical direction of the measurement substrate 1 in order to define the optical path length of the reference region by the height of the partition wall. In this example, the bolt 9 and the nut 10 are used as four screws, but other fixing methods may be used.

  FIG. 7 is another example of a jig for defining the optical path length in the reference region. In this example, the container 11 is slid from the side and inserted into the container 11 as an opening frame with a prescribed height, but other fixing methods may be used.

  The absolute amount of the labeling substance contained in the dispensed droplet can be determined as follows. When the labeling substance is a light-absorbing substance, fluorescent substance, luminescent substance, or radioisotope, the measurement substrate carrying the dispensed droplet is imaged with a densitometer, fluorescent scanner, luminometer, autoradiography, etc. The volume of each droplet is calculated by reading the physical property values of the absorbance, fluorescence intensity, emission intensity, and radiation intensity obtained from the substance as an image and performing image processing in comparison with the physical property values in the reference region.

と表される。一方、微小体積における吸光物質のモル数ｄｍは、

であるから、式(1)と式(2)より、

と表すことができる。ここで、実際に画像処理により得られる情報は、微小面積としてのイメージ化装置の画素（ピクセル）、その画素における照射光や透過光の強度としての輝度である。照射光および透過光の強度に相当する輝度をそれぞれｋ_０、ｋ_ｓとすると、以下のように表すことができる。

参照用領域から得られる情報により、式(3)のεおよび式(4)のｋ_０を求めることができる。測定用液体を満たした領域Ｒの光路長をｌ_Ｒ、透過光をＩ_Ｒ、輝度をｋ_Ｒとすると、

と表すことができる。また、照射光の輝度ｋ_０はブランク領域Ｂにおける輝度ｋ_Ｂと等しいとすることができる。

式(3)を式(4)、式(5)、式(6)によって表すと、

したがって、液滴の体積Ｖは以下のように表すことができる。

この式により、液体の蒸発に無関係に液滴の体積Ｖを計算することができる。 FIG. 8 shows a calculation method using a light absorbing substance as an example. A droplet dispensed on the measurement substrate is shown in FIG. 8-a, an image obtained by imaging the measurement substrate is shown in FIG. 8-b, and a view of the dispensed droplet is shown in FIG. 8-c. . The volume of the droplet is V, the number of moles of the light-absorbing substance in the droplet, the concentration, and the molar extinction coefficient are m, c, and ε. The minute area in the image area S of the droplet in b is s (= dS), the minute volume of the droplet with this as the bottom is dV (= dS × l _s ), and the number of moles of the light-absorbing substance in this minute volume is dm. And Further, it is assumed that a transmitted light intensity I _s irradiation light intensity I ₀ is transmitted through the minute volume. In this case, the absorbance As in the small area _s is

It is expressed. On the other hand, the number of moles dm of the light-absorbing substance in the minute volume is

Therefore, from Equation (1) and Equation (2),

It can be expressed as. Here, the information actually obtained by image processing is a pixel (pixel) of the imaging device as a minute area, and luminance as intensity of irradiation light or transmitted light in the pixel. If the luminances corresponding to the intensity of the irradiation light and the transmitted light are k ₀ and k _s , respectively, they can be expressed as follows.

From the information obtained from the reference region, ε in Expression (3) and k ₀ in Expression (4) can be obtained. If the optical path length of the region R filled with the measurement liquid is l _R , the transmitted light is I _R , and the luminance is k _R ,

It can be expressed as. Further, the luminance k _{0 of the} irradiation light can be made equal to the luminance k _B in the blank region B.

When Expression (3) is expressed by Expression (4), Expression (5), and Expression (6),

Therefore, the volume V of the droplet can be expressed as follows.

By this formula, the droplet volume V can be calculated regardless of the evaporation of the liquid.

  When the labeling substance is an electrochemically active substance, a droplet is dispensed onto a measurement substrate equipped with an electrode, and the amount of electrons (number of electrons) that flows by oxidizing or reducing the electrochemically active substance in the droplet Calculate the volume of each drop.

と表される。電気化学活性物質が酸化還元反応によりｎ個の電子が移動するとすると、液滴中の電気化学活性物質のモル数はファラデー定数Ｆを用いて

と表すことができる。したがって、液滴の体積Ｖは以下のように表すことができる。

液体の蒸発が多少起こったとしても、もともとの液体における電気化学活性物質の濃度ｃを用いて、液滴の体積Ｖを計算することができる。 FIG. 9 shows a measurement method using an electrochemically active substance as an example. A liquid droplet containing an electrochemically active substance c is dropped on the measurement substrate, and at least two working electrodes and a counter electrode are arranged inside the droplet, and flows when a voltage is applied between the electrodes. Measure the current value i. Measurement is performed until the current value becomes 0, and the number of moles m of the electrochemically active substance in the droplet can be calculated from the total charge amount Q calculated from the current value. A voltage higher than the redox potential of the electrochemically active substance is applied. An applied voltage is selected using an electrochemically active substance having an appropriate redox potential so that the redox reaction of the liquid itself does not interfere. When all of the electrochemically active substances in the liquid complete the oxidation-reduction reaction, no further reaction occurs, so the measured current value automatically becomes zero. Total charge Q is

It is expressed. When n number of electrons move due to the oxidation-reduction reaction of the electrochemically active substance, the number of moles of the electrochemically active substance in the droplet is calculated using the Faraday constant F.

It can be expressed as. Therefore, the volume V of the droplet can be expressed as follows.

Even if some evaporation of the liquid occurs, the volume V of the droplet can be calculated using the concentration c of the electrochemically active substance in the original liquid.

  In a small volume of bulk electrolysis, the diffusion region of the electrochemically active material is limited to a very small region, and a decrease in the diffusion rate of the electrochemically active material is avoided by subjecting the microdroplet to electrolysis without dilution. Therefore, there is an advantage that the time until completion of electrolysis is short.

  FIG. 9 shows an example of electrode arrangement. The working electrode may be fabricated on the measurement substrate as shown in (a) and the counter electrode may be arranged on another plane, or both the working electrode and the counter electrode may be placed as shown in (b) and (c). It may be fabricated on a measurement substrate. The shape of the electrodes may be completely separated from each other as shown in (b), or may be a comb shape as shown in (c). Other arrangements / shapes other than these may be used.

  Although the two-electrode system using the working electrode and the counter electrode has been described with reference to FIG. 9, a three-electrode system may be provided by further arranging a reference electrode inside the droplet for the purpose of controlling the redox potential in detail. In this case, a silver / silver chloride electrode or a calomel electrode can be used as the reference electrode.

DESCRIPTION OF SYMBOLS 1 Board | substrate 2 Partition 3 Space 4 O ring 5 Lid 6 O ring 7 Frame 8 Hole 9 Bolt 10 Nut 11 Container

Claims (15)

A plurality of spaces formed on the measurement substrate are dispensed with a liquid drop for measurement containing a labeling substance having a known physical property and having a detectable physical property. A space filled with a substance-only liquid and a blank space,
While detecting the physical property in the space filled with the measurement liquid or the liquid of only the labeling substance and the physical property in the blank space, it detects the physical property of the dispensed droplet of the measurement liquid,
The relationship between the detected value of the physical property in the space filled with the liquid for measurement or the liquid of only the labeled material and the detected value of the physical property in the blank space is obtained from the detected value of the physical property of the labeled material and the absolute amount of the labeled material ,
Based on the obtained relationship, the volume of the dispensed liquid droplet of the measurement liquid is measured from the detected value of the physical property of the dispensed liquid droplet of the measurement liquid. Volume measurement method.   2. The method for measuring a volume of a minute droplet according to claim 1, wherein a substance having a large molecular weight is added to the measurement liquid to reduce the evaporation rate of the measurement liquid. Using a light-absorbing substance, a fluorescent substance, a luminescent substance, or a radioisotope as the labeling substance,
The measurement substrate carrying the dispensed droplets is imaged using a densitometer, fluorescence scanner, luminometer or autoradiography,
3. The method for measuring the volume of a microdroplet according to claim 1 or 2, wherein a physical property value of absorbance, fluorescence intensity, emission intensity, or radiation intensity obtained from the labeling substance is detected as an image. . A plurality of spaces formed on the measurement substrate are dispensed with a liquid drop for measurement containing a labeling substance having a known physical property and having a detectable physical property. A space filled with a substance-only liquid and a blank space,
While detecting the physical property in the space filled with the measurement liquid or the liquid of only the labeling substance and the physical property in the blank space, it detects the physical property of the dispensed droplet of the measurement liquid,
The relationship between the detected value of the physical property in the space filled with the liquid for measurement or the liquid of only the labeled material and the detected value of the physical property in the blank space is obtained from the detected value of the physical property of the labeled material and the absolute amount of the labeled material ,
Based on the obtained relationship, the volume of the dispensed liquid droplet of the measurement liquid is measured from the detected value of the physical property of the dispensed liquid droplet of the measurement liquid. Volume measuring device.   The volume measuring device for a minute droplet according to claim 4, wherein the space is isolated by a partition wall formed integrally with the measurement substrate. The space is a plurality of through holes penetrating up and down a partition formed separately from the measurement substrate,
5. The apparatus for measuring a volume of a minute droplet according to claim 4, wherein the partition wall is isolated by being in close contact with the measurement substrate.   The volume measurement of a microdroplet according to claim 6, wherein a groove is formed around the measurement substrate side opening in the partition wall, and a sealing O-ring in contact with the measurement substrate is provided in the groove. apparatus.   The volume measuring apparatus for a microdroplet according to claim 5 or 6, further comprising a lid for sealing the space of the partition wall and using a jig for defining an optical path length.   A groove is formed around the measurement substrate side opening and the lid side opening in the partition wall, and a sealing O-ring that contacts the measurement substrate and the cover is provided in the groove, and the measurement substrate, the partition wall, The O-ring is compressed and integrated and fixed by a fixing member that holds the partition between the lid, the height is determined by closely attaching the lid to the partition, and the optical path length is defined. The device for measuring a volume of a minute droplet according to claim 8.   The partition wall is formed of an elastic material, and a regulating member having a height lower than that of the partition wall is provided in a free state, and the measurement substrate, the partition wall, and the lid are sandwiched between the measurement substrate, the fixing member, and the lid. 9. The volume of a microdroplet according to claim 8, wherein the partition wall is compressed and integrated and fixed, and the optical path length is defined by defining the height by tightly attaching the lid to the upper surface of the defining member. measuring device.   The volume measuring device for a microdroplet according to claim 9 or 10, wherein the fixing member is a screw that fixes the measurement substrate and the lid with the partition interposed therebetween.   11. The microdroplet according to claim 9, wherein the fixing member is a container having a space in which the measurement substrate and the partition wall can be slid and accommodated while being sandwiched. Volume measuring device.   5. The apparatus for measuring a volume of a minute droplet according to claim 4, wherein the measurement substrate is formed of a conductive material, and an electrochemically active substance is used as the labeling substance.   14. The apparatus for measuring a volume of a minute droplet according to claim 13, wherein the measurement substrate includes an electrode for measuring an electron transfer reaction with the electrochemically active substance in the space.   15. The apparatus for measuring a volume of a minute droplet according to claim 14, wherein the electrode is formed by separating a working electrode and a counter electrode.

Images