Biomolecule evaluation method and biomolecule evaluation device

Abstract

To provide a method for evaluating a biomolecule, which does not require preparation of a substrate having reaction wells formed in advance, can evaluate the biomolecule in a reaction chamber of a fine size, and can perform a solution operation in a droplet, and to provide an apparatus for evaluating the biomolecule which can be used for the method.SOLUTION: The method for evaluating a biomolecule includes a preparation step of preparing a plurality of evaluation samples T each having at least one biomolecule 11 immobilized on a substrate 10 and having at least one predetermined enzyme 12 and at least one pipette 2 filled with an electrolytic solution 20 containing at least one kind of substrate 21 reacting with the predetermined enzyme 12, a droplet forming step of adhering a droplet 3 held at the tip of the pipette 2 to the evaluation sample 1 and forming a plurality of droplets 3 on the evaluation sample 1, and an evaluation information acquisition step of acquiring information on the number and / or position of droplets 3 in which a reaction between the predetermined enzyme 12 contained in the droplet 3 and the substrate 21 occurs.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating a biomolecule and an apparatus for evaluating a biomolecule. [Background technology]

[0002] Enzyme-linked immunosorbent assays (ELISAs) have traditionally been used at the laboratory level to measure biomolecules and diagnose diseases such as viral and bacterial infections and cancer. ELISAs use antigen-antibody and enzyme reactions to detect target proteins and other biological substances. In recent years, a single-molecule digital ELISA technique has been proposed as a technology aimed at improving the lower detection limit of ELISA. This technique confines the target substance and its associated enzyme-modified antibody in a microcontainer, and enables highly sensitive detection of the target substance's concentration by detecting the signal resulting from the enzyme reaction bound to a single molecule.

[0003] For example, Patent Document 1 discloses a technique for detecting a signal resulting from the enzymatic activity of a single enzyme molecule by using a microchamber in which a container (reaction well) is formed by the recesses, each having a volume of 1000 fL (femtoliter), by bonding a polydimethylsiloxane (PDMS) substrate having a large number of minute recesses formed by photolithography to a glass substrate, and then sealing a sample droplet containing a fluorescent dye in the container. In this type of technique, a signal resulting from the enzymatic activity of a single enzyme molecule is detected by sealing a sample droplet containing a fluorescent dye in the container. 2 It is known that a container portion of this size can be formed.

[0004] Patent Document 2 discloses a technique related to a scanning ion conductance microscope, although it is not related to the ELISA method. Specifically, Patent Document 2 describes a scanning ion conductance microscope that includes a pipette having an opening at its tip, a first pseudo reference electrode inserted into the pipette, a second pseudo reference electrode immersed in an electrolyte solution into which the pipette is inserted, a voltage source connected to the first pseudo reference electrode and the second pseudo reference electrode, an ammeter, and a recovery device that includes a pump connected to the base end of the pipette and that applies negative or positive pressure to the inside of the pipette, and in which the pipette is supported on a three-axis stage. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 3727026

[0006] [Patent Document 2] Japanese Patent Application Publication No. 2023-106932 Summary of the Invention [Problem to be solved by the invention]

[0007] However, when attempting to evaluate biomolecules using the technology of Patent Document 1, it is necessary to prepare a substrate with a large number of reaction wells formed by microfabrication in advance. Furthermore, special technology is required to control minute flow rates on the order of fL or less. Furthermore, the technology of Patent Document 1 makes it difficult to manipulate the solution in the droplets sealed in the reaction wells.

[0008] The technology in Patent Document 2 relates to a scanning probe microscope (SPM) that performs electrochemical measurements at the liquid-mediated contact point between a solution filled in a pipette and a sample, and is not intended to evaluate biomolecules using an enzyme reaction, nor is there any suggestion of this.

[0009] The present invention has been made in consideration of such problems, and aims to provide a method for evaluating biomolecules that does not require the preparation of a substrate with reaction wells formed in advance, can evaluate biomolecules in a micro-sized reaction chamber, and is capable of manipulating the solution within a droplet, as well as an apparatus for evaluating biomolecules that can be used for this method. [Means for solving the problem]

[0010] One aspect of the present invention is a preparation step of preparing an evaluation sample in which a large number of evaluation targets each having at least one biomolecule are immobilized on a substrate, some of the evaluation targets having at least one predetermined enzyme, and at least one pipette filled with an electrolyte solution containing at least one substrate that reacts in response to the predetermined enzyme; a droplet forming step of attaching the droplet held at the tip of the pipette to the evaluation sample to form a plurality of droplets on the evaluation sample; and an evaluation information acquisition step of acquiring the number and / or position information of the droplets in which a reaction between the predetermined enzyme contained in the evaluation target and the substrate has occurred. The present invention relates to a method for evaluating biomolecules.

[0011] Another aspect of the present invention is a stage on which an evaluation sample is placed, the evaluation sample having a plurality of evaluation targets each having at least one biomolecule immobilized on a substrate, some of the plurality of evaluation targets each having at least one predetermined enzyme; a pipette that is arranged perpendicular to the stage and is filled with an electrolyte solution containing at least one substrate that reacts with the predetermined enzyme; a circuit unit including a pair of electrodes immersed in the electrolyte in the pipette, a voltage unit that applies a voltage between the pair of electrodes, and a current measurement unit that measures a current flowing through the electrodes and the electrolyte; a position control unit that controls the position of the pipette and the position of the evaluation sample; a microscope for observing the evaluation sample on the mounting stage; an imaging unit that captures an image by the microscope, the position control unit reduces the distance between the tip of the pipette and the evaluation sample, detects contact between the droplet held at the tip of the pipette and the evaluation sample from a change in the value of the current flowing through the electrolyte measured by the current measurement unit, and, after the detection, is configured to be capable of performing at least a first operation of increasing the distance between the tip of the pipette and the evaluation sample by the position control unit; the position control unit repeatedly performs the first operation while changing the position of the pipette in the XY directions relative to the evaluation sample, thereby causing the droplet held at the tip of the pipette to adhere to the evaluation sample, forming a plurality of droplets on the evaluation sample, and then the imaging unit can capture an image of the evaluation sample. It is a device for evaluating biomolecules. [Effects of the Invention]

[0012] The biomolecule evaluation method has the above configuration. In the biomolecule evaluation method, droplets are formed directly on an evaluation sample on a substrate, where the evaluation target is immobilized. The formed droplets function as reaction chambers, and an enzyme and a substrate are reacted within the droplets. Therefore, the biomolecule evaluation method eliminates the need to prepare a substrate with a reaction well formed in advance. Furthermore, the biomolecule evaluation method does not require the formation of reaction wells on a substrate by microfabrication, and can use smaller droplet volumes as reaction chambers than in general digital ELISA methods, regardless of well size. Therefore, the biomolecule evaluation method allows biomolecules to be evaluated in reaction chambers of smaller sizes. Furthermore, the biomolecule evaluation method does not encapsulate droplets in reaction wells, but rather exposes the droplets formed on the evaluation sample. This allows new droplets to be formed by overlaying the same or different droplets on previously formed droplets. Therefore, the biomolecule evaluation method allows for operations such as adding solution to droplets or changing the solution in the droplets after the enzyme reaction.

[0013] Therefore, according to the above-mentioned method for evaluating biomolecules, there is no need to prepare a substrate with reaction wells formed in advance, biomolecules can be evaluated in a micro-sized reaction chamber, and a method for evaluating biomolecules that allows for solution manipulation within a droplet can be provided.

[0014] The biomolecule evaluation device has the above-described configuration. The biomolecule evaluation device uses a position control unit to change the position of the pipette in the XY directions relative to the evaluation sample, while decreasing the distance between the tip of the pipette and the evaluation sample. The device detects contact between the droplet held at the tip of the pipette and the evaluation sample from changes in the current value flowing through the electrolyte. After this detection, the device repeatedly performs a first operation of increasing the distance between the tip of the pipette and the evaluation sample, thereby causing the droplet held at the tip of the pipette to adhere to the evaluation sample, forming multiple droplets on the evaluation sample, and then capturing an image of the evaluation sample using an imaging unit. Therefore, the biomolecule evaluation device can obtain an observation image containing information on the number and / or position of droplets in which a reaction between a predetermined enzyme contained in the evaluation target and a substrate has occurred.

[0015] Therefore, the biomolecule evaluation device can be used in the biomolecule evaluation method. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is an explanatory diagram for schematically explaining an example of a method for evaluating a biomolecule according to an embodiment. [Figure 2] FIG. 2 is an explanatory diagram for schematically explaining another example of the biomolecule evaluation method according to the embodiment. [Figure 3] FIG. 3 is an explanatory diagram for schematically explaining an example of an evaluation target in the evaluation sample. [Figure 4] FIG. 4 is an explanatory diagram for explaining the substrate concentration after the reaction when a general ELISA method is used. [Figure 5]FIG. 5 is an explanatory diagram for explaining the substrate concentration after the reaction in the biomolecule evaluation method according to the embodiment. [Figure 6] FIG. 6 is an explanatory diagram showing a schematic example of the configuration of a biomolecule evaluation device according to an embodiment. [Figure 7] FIG. 7 is an explanatory diagram schematically showing a flow when droplets are formed by a pipette in the biomolecule evaluation device according to the embodiment. [Figure 8] FIG. 8 is an explanatory diagram corresponding to FIG. 7, which schematically shows the relationship between time, pipette height, and current value. [Figure 9] FIG. 9 is an explanatory diagram schematically illustrating an example of an operation of scanning a pipette by the biomolecule evaluation device according to the embodiment to repeatedly form droplets on a substrate. [Figure 10] FIG. 10 shows a bright-field image, a fluorescent image, and a brightness-adjusted fluorescent image of the experimental evaluation sample and the comparative evaluation sample obtained in the experimental example. DETAILED DESCRIPTION OF THE INVENTION

[0017] Hereinafter, embodiments of the biomolecule evaluation method and biomolecule evaluation device according to the present invention will be described in detail with reference to the drawings. Note that the reference numerals in the drawings are mainly used when describing specific figures, and may be used in other cases as necessary. The biomolecule evaluation method and biomolecule evaluation device according to the present invention are not limited to the following examples. Furthermore, the lower and upper limits of the numerical ranges shown below can be arbitrarily combined (omitted below).

[0018] (Method for evaluating biomolecules) The biomolecule evaluation method according to this embodiment (hereinafter sometimes referred to as the present evaluation method) will be described mainly with reference to FIGS. 1 to 4 (also with reference to FIGS. 5 to 9 described later as appropriate).

[0019] This evaluation method includes a preparation step, a droplet formation step, and an evaluation information acquisition step.

[0020] The preparation step is a step of preparing an evaluation sample 1 and a pipette 2, as exemplified in FIGS. 1(a) and 2(a).

[0021] The evaluation sample 1 has a large number of evaluation objects T fixed on a substrate 10.

[0022] As illustrated in FIG. 3, the evaluation target T has at least one biomolecule 11. Examples of the evaluation target T include biological cells Tc and higher-order structures such as extracellular vesicles released by biological cells Tc. More specifically, examples of substances released by biological cells Tc include substances released by living biological cells Tc moving on the substrate 10. Examples of the biomolecule 11 include enzymes, proteins such as inflammatory cytokines and growth factors, nucleic acids such as RNA and DNA, and lipids.

[0023] In the evaluation sample 1, some of the multiple evaluation targets T have at least one predetermined enzyme 12, as illustrated in FIG. 3. That is, the evaluation sample 1 may include evaluation targets T (Te) having at least one predetermined enzyme 12 and evaluation targets T (Tn) not having the predetermined enzyme 12, as illustrated in FIGS. 1(a) and 2(a). The enzyme 12 may be bound to the evaluation target T via an antibody 13, as illustrated in FIG. 3, or may be bound directly to the evaluation target T without the antibody 13 (not shown), or may be inherently contained in the evaluation target T (not shown). Examples of the enzyme 12 include β-galactosidase and horseradish peroxidase.

[0024] 3 shows an example of an evaluation target T in which an enzyme 12 that emits fluorescence upon reaction with a substrate 21 is bound to one of several biomolecules 11 such as proteins present on the surface of a living cell Tc via an antibody 13. In FIG. 3, reference numeral 210 denotes the substrate after the reaction, reference numeral 14 denotes intracellular mRNA, and reference numeral 15 denotes proteins present in the membrane, etc.

[0025] Examples of the substrate 10 include a substrate such as a glass substrate provided in a container such as a glass bottom dish, and various electrodes such as transparent electrodes such as indium tin oxide electrodes and general opaque electrodes.

[0026] The object to be evaluated T may be immobilized on the substrate 10, for example, by being bound to an antibody (not shown) bound to the substrate 10, or may be directly immobilized on the substrate 10 by adsorption or the like.

[0027] As illustrated in Figures 1(a) and 2(a), the pipette 2 is filled with an electrolyte solution 20. The electrolyte solution 20 contains at least one substrate 21. From the viewpoints of affinity with cells and biomolecules, pH stability, and the like, it is preferable to use phosphate-buffered saline, cell culture medium, or the like as the electrolyte solution 20. The substrate 21 is one that can react with a predetermined enzyme 12 contained in the evaluation target T. From the viewpoint of reliably acquiring information on the number and / or position of droplets in which an enzyme reaction has occurred, it is preferable to use a fluorescent substrate that can emit colored fluorescence from non-fluorescent light upon reaction with the enzyme 12 as the substrate 21.

[0028] The concentration of the substrate 21 in the electrolyte solution 20 can be determined in consideration of the increase in signal intensity, and can be set to, for example, about 15.2 mM.

[0029] The electrolyte solution 20 may contain a substance (not shown) that can solubilize lipid membranes derived from cells.

[0030] In this case, when a droplet 3 is formed on the evaluation sample 1, the substance dissolves the lipid membrane derived from the cells of the evaluation subject T, making it possible to obtain evaluation information about the biomolecules 11 inside the cells, such as mRNA, DNA, and proteins.

[0031] Examples of substances capable of solubilizing cell-derived lipid membranes include surfactants such as dodecyl maltoside. These can be used alone or in combination. Specifically, commercially available surfactants such as "Triton-X" (registered trademark) can be used.

[0032] In the preparation step, at least one pipette 2 is prepared. As will be described later, when an additional step is performed, in addition to the pipette 2 (2a) used to form the droplet 3 as illustrated in Figures 1(a) and 2(a), a pipette 2 (2b) to be used in the additional step can be prepared as illustrated in Figures 1(c) and 2(c) (details will be described later). When multiple pipettes 2 are prepared, each pipette 2 may be filled with an electrolyte solution 20 containing the same substrate 21, or may be filled with an electrolyte solution 20 containing a different substrate 21.

[0033] The pipette 2 may be a double-barrel type as exemplified in Figure 7, or may be a single-barrel type (not shown). Specifically, a double-barrel pipette 2 has a partition wall 24 that divides the inside of the pipette 2 into two sections, as exemplified in Figure 7. The double-barrel pipette 2 has the advantage that distance control is easy even on an insulating substrate or in an insulating solution.

[0034] The droplet formation process is a process in which a droplet 3 held at the tip of a pipette 2 is attached to an evaluation sample 1 to form multiple droplets 3 on the evaluation sample 1, as illustrated in Figures 1(b) and 2(b).

[0035] In a typical ELISA method, as shown in Fig. 4, the volume of solution 9 on substrate 10 is relatively large, and therefore, after the enzyme 12 contained in the evaluation object T reacts with substrate 21, the reacted substrate 210 diffuses and takes time to reach a detectable concentration. In contrast, as shown in Fig. 5, when droplet 3 is formed on substrate 10 using pipette 2, the volume of droplet 3 can be significantly reduced, and the reacted substrate 210 after the reaction between enzyme 12 and substrate 21 does not diffuse out of droplet 3, so the concentration of the reacted substrate 210 within the closed droplet 3 can quickly reach a detectable concentration.

[0036] Specifically, the adhesion of the droplet 3 held at the tip of the pipette 2 to the evaluation sample can be achieved by shortening the distance between the tip of the pipette 2 and the evaluation sample 1 (substrate 10), bringing the droplet 3 held at the tip of the pipette 2 into contact with the evaluation sample 1 (substrate 10), and then increasing the distance between the tip of the pipette 2 and the evaluation sample 1 (substrate 10), as shown in Figure 7 (for details, see the description of the device for evaluating biomolecules below).

[0037] The formed droplets 3 may include droplets 3 that do not include the evaluation target T fixed on the substrate 10, as long as the formed droplets 3 include at least one droplet 3 that includes the evaluation target T fixed on the substrate 10. In other words, the formed droplets 3 may include each of the formed droplets 3 including the evaluation target T fixed on the substrate 10, as exemplified in FIG. 1(b), or may include droplets 3 that include the evaluation target T fixed on the substrate 10 and droplets 3 that do not include the evaluation target T fixed on the substrate 10, as exemplified in FIG. 2(b).

[0038] In addition, multiple droplets 3 can be formed by repeatedly performing the above operation while changing the XY position of the pipette 2 relative to the evaluation sample 1 (for details, see the explanation of the device for evaluating biomolecules below).

[0039] Note that "changing the XY position of pipette 2 relative to evaluation sample 1" includes not only cases where the XY position of pipette 2 is fixed and the XY position of evaluation sample 1 is changed, but also cases where the XY position of evaluation sample 1 is fixed and the XY position of pipette 2 is changed, and cases where both the XY position of evaluation sample 1 and the XY position of pipette 2 are changed.

[0040] In the droplet formation step, the droplet 3 can be selectively formed on the target evaluation object T.

[0041] In this case, evaluation information regarding a targeted evaluation target T among a large number of evaluation targets T on the substrate 10 can be obtained, and the biomolecules 11 can be evaluated for the targeted evaluation target T. This has the advantage of eliminating the need to form droplets 3 that do not contain the evaluation target T, thereby shortening the measurement time.

[0042] Using FIG. 1, a case where droplets 3 are selectively formed on targeted evaluation targets T will be described in more detail. Here, as shown in FIG. 1(a), it is assumed that four types of evaluation targets T (t1, t2, t3, t4) are fixed on a substrate 10 of an evaluation sample 1. For example, if all four types of evaluation targets T (t1, t2, t3, t4) are targeted evaluation targets T, as shown in FIG. 1(b), droplets 3 are formed by a pipette 2 so as to include each evaluation target T (t1, t2, t3, t4) on the substrate 10. In this case, droplets are selectively formed only on the targeted evaluation targets T, and therefore droplets 3 are not formed on portions of the substrate 10 where no evaluation targets T are present. Note that Figure 1(b) illustrates an example in which, after the first formation of droplets 3, an enzymatic reaction occurs in the droplets 3 formed on the evaluation objects T (t1, t2) (indicated by "+" in Figure 1), but no enzymatic reaction occurs in the droplets 3 formed on the evaluation objects T (t3, t4) (indicated by "-" in Figure 1).

[0043] To selectively form droplets 3 only on the target evaluation object T, for example, if the evaluation object T can be observed using a microscope 44 (for details, see the description of the device for evaluating biomolecules below), the pipette 2 can be moved to the position of the specific evaluation object T from the image of the acquired evaluation sample 1, and droplets 3 can be formed.

[0044] In the droplet forming step, the droplets 3 can also be formed comprehensively within a certain region of the evaluation sample 1.

[0045] In this case, even if it is not known in advance or even if it is unknown where the evaluation object T is fixed within a certain area on the substrate 10, evaluation information can be obtained for all evaluation objects T present within the certain area, and the biomolecules 11 can be evaluated for all evaluation objects T within the certain area.

[0046] To form droplets 3 comprehensively within a certain area on the evaluation sample 1, for example, a certain area on the substrate 10 can be determined from an image of the evaluation sample 1, and the pipette 2 can be moved up and down in the Z direction while changing the position of the pipette 2 in the XY directions relative to the evaluation sample 1 so as to cover the entire certain area.

[0047] Using FIG. 2, a case where droplets 3 are formed comprehensively within a fixed region on the evaluation sample 1 will be described in more detail. Here, as in the above, it is assumed that four types of evaluation targets T (t1, t2, t3, t4) are fixed on the substrate 10 of the evaluation sample 1 as shown in FIG. 2(a). The fixed region on the evaluation sample 1 is the entire surface of the substrate 10 shown in FIG. 2(a). In this case, as shown in FIG. 2(b), droplets 3 can be formed so as to cover the entire surface of the substrate 10 by sequentially forming a plurality of droplets 3 at a predetermined pitch over the entire surface of the substrate 10 using a pipette 2. In this case, to form droplets 3 comprehensively within a fixed region, droplets 3 containing the evaluation target T and droplets 3 not containing the evaluation target T are typically formed on the substrate 10. Note that Figure 2(b) illustrates an example in which, upon the formation of the first droplet 3, an enzymatic reaction occurs in the droplet 3 formed to contain the evaluation pair T (t1, t2) (indicated by "+" in Figure 2), while no enzymatic reaction occurs in the droplet 3 formed to contain the evaluation target T (t3, t4) or the droplet 3 that does not contain the evaluation target T (indicated by "-" in Figure 2).

[0048] In the droplet formation step, the droplet 3 held at the tip of the pipette 2 can be attached to the evaluation sample 1 by a wetting phenomenon.

[0049] In this case, droplets 3 can be formed only at targeted positions more easily than when droplets are discharged from a pipette 2 using a pump or the like. Another advantage is that the size of droplets 3 can be adjusted by adjusting the wettability of the substrate 10.

[0050] The droplet 3 is preferably formed on the evaluation sample 1 in a volume on the order of attoliters (aL order) or femtoliters (fL order), and more preferably in a volume on the order of attoliters. In this case, the reduced droplet volume allows the concentration of the substrate 210 after the reaction in the droplet 3 to reach a detectable concentration more quickly, thereby shortening the time required for evaluation. It also makes it possible to extend the lower limit of detection. Another advantage is that spatial resolution is improved in comprehensive evaluations. The attoliter order refers to a range of 1 aL to 1000 aL. The femtoliter order refers to a range of 1 fL to 1000 fL.

[0051] The evaluation information acquisition step is a step of acquiring information on the number and / or position of droplets 3 in which a reaction between a predetermined enzyme 12 contained in the evaluation target T and a substrate 21 has occurred.

[0052] The number and positional information of the droplets 3 in which the enzyme reaction has occurred can be ascertained from the observation image obtained by, for example, capturing an image of the evaluation sample 1 using a microscope 44. Specifically, the number of droplets 3 in which the enzyme reaction has occurred in the observation image, and the number of droplets 3 in which the enzyme reaction has not occurred in the observation image can be counted. Furthermore, by ascertaining the positions on the substrate 10 in which the enzyme reaction has occurred in the observation image, positional information regarding the evaluation object T can be obtained.

[0053] The present evaluation method may include additional steps as necessary in addition to the above-described preparation step, droplet formation step, and evaluation information acquisition step.

[0054] The additional process can be, for example, a process of forming a new droplet 3 on top of a previously formed droplet 3, or a process of removing the previously formed droplet 3 and then forming a new droplet 3, and additionally obtaining information on the number and / or position of droplets 3 in which a reaction identical to or different from the previous reaction has occurred within the newly formed droplet 3.

[0055] In this case, evaluation information can be obtained not only from the evaluation sample 1 on which the droplets 3 were formed earlier, but also from the evaluation sample 1 on which new droplets 3 were subsequently formed. Therefore, in this case, it becomes possible to evaluate a plurality of items regarding the biomolecule 11.

[0056] Specifically, the additional step can be added after the evaluation information acquisition step. In the additional step, the droplet 2 may be formed by the same method as the previous droplet 2 formation method, or by a method different from the previous droplet 2 formation method. In the additional step, the substrate 21 contained in the electrolyte 20 in the pipette 2 may be the same as that used when the droplet 3 was previously formed, or may be different from that used when the droplet 3 was previously formed.

[0057] The case where an additional step is included will be described in more detail with continued reference to FIG. 1. Here, as illustrated in FIG. 1(c), in the additional step, droplets 3 are selectively formed on the target evaluation object T, as in FIG. 1(b). A different pipette 2 (2b) from the pipette 2 (2a) used in FIG. 1(b) is used, and the substrates 21 in the electrolyte 20 in both pipettes 2 (2a, 2b) are different from each other. In this case, as shown in FIG. 1(c), the pipette 2 is scanned to form another droplet 3 on top of each droplet 3 on the evaluation sample 1 shown in FIG. 1(b). Then, the electrolyte 20 of the droplet 3 formed on top of the previously formed droplet 3 mixes with the previously formed droplet 3, forming a new droplet 3. It is also possible to form a new droplet 3 after removing the previously formed droplet 3 by evaporation, such as drying, or cleaning. In Figure 1(c), the case where an enzymatic reaction occurs upon the formation of the first droplet 3 and also occurs upon the formation of the second droplet 3 is indicated by "++", the case where an enzymatic reaction does not occur upon the formation of the first droplet 3 and also occurs upon the formation of the second droplet 3 is indicated by "-+", the case where an enzymatic reaction occurs upon the formation of the first droplet 3 and also does not occur upon the formation of the second droplet 3 is indicated by "+-", and the case where an enzymatic reaction does not occur upon the formation of the first droplet 3 and also does not occur upon the formation of the second droplet 3 is indicated by "--".

[0058] Further, the case where an additional step is included will be described in more detail with continued reference to FIG. 2 described above. Here, as illustrated in FIG. 2(c), in the additional step, droplets 3 are formed comprehensively within a certain region of the evaluation sample 1, as in FIG. 2(b). Furthermore, a pipette 2 (2b) different from the pipette 2 (2a) used in FIG. 2(b) is used, and the substrates 21 in the electrolyte 20 in both pipettes 2 (2a, 2b) are different from each other. In this case, the pipette is scanned in the same manner as in FIG. 2(b) when each droplet 3 of the evaluation sample 1 is present, or after each droplet 3 is removed, as shown in FIG. 2(b), to form new droplets 3 so as to cover the entire surface of the substrate 10, as shown in FIG. 2(c). The indication of whether or not an enzyme reaction occurs upon the formation of the first and second droplets in FIG. 2(c) is as described above in the explanation of FIG. 1(c).

[0059] When the additional process described above is performed, a new droplet 2 can be formed on the evaluation sample 1 on which the droplet 2 was previously formed and observed, so that additional evaluation information can be obtained from the evaluation sample 1 on which the droplet 2 was additionally formed.

[0060] In the above-described evaluation method, a droplet 3 is formed directly on an evaluation sample 1 on which an evaluation target T is immobilized on a substrate 10. The formed droplet 3 functions as a reaction chamber, and an enzyme 12 and a substrate 21 react within the droplet 3. Therefore, this evaluation method eliminates the need to prepare a substrate with a reaction well formed thereon in advance. Furthermore, this evaluation method does not require the formation of a reaction well on a substrate by microfabrication. The volume of the droplet 3 can be reduced compared to that of a typical digital ELISA method (e.g., reducing the reaction volume from the fL order to the aL order) regardless of the well size, making it possible to use the droplet 3 as a reaction chamber. Therefore, this evaluation method allows the evaluation of biomolecules 11 in a reaction chamber of a smaller size. Furthermore, this evaluation method does not encapsulate the droplet 3 within a reaction well. Instead, the droplet 3 formed on the evaluation sample 1 is exposed. Therefore, a new droplet 3 can be formed by forming the same droplet 3 or a different droplet 3 on top of a previously formed droplet 3. Therefore, this evaluation method allows the addition of a solution to the droplet 3 or changing the solution in the droplet 3 after the enzyme reaction.

[0061] The configurations and explanations described in the section "(Biomolecule Evaluation Apparatus)" below can be incorporated into the explanation of this evaluation method, either alone or in any combination.

[0062] (Device for evaluating biomolecules) The biomolecule evaluation device according to this embodiment (hereinafter sometimes referred to as the present evaluation device) will be described mainly with reference to FIGS. 6 to 9 (also with reference to the above-mentioned FIGS. 1 to 5 as appropriate).

[0063] As shown in FIG. 6, the evaluation device includes a mounting stage 41, a pipette 2, a circuit section 42, a position control section 43, a microscope 44, and an imaging section 46.

[0064] In the following description, the X and Z directions are the directions shown in Fig. 6, and the Y direction is the direction perpendicular to Fig. 6 (the plane of Fig. 6) and extending from the front to the back of Fig. 6 (the plane of Fig. 6). That is, in this embodiment, the Z axis is an axis along the vertical direction, the X axis intersects the Z axis at an angle of 90°, and the Y axis intersects the X and Z axes at an angle of 90°.

[0065] In this evaluation device 4, the mounting stage 41 is a stage on which the evaluation sample 1 is mounted. The evaluation sample 1 has a large number of evaluation targets 1, each having at least one biomolecule 11, immobilized on a substrate 10, and some of the large number of evaluation targets T have at least one predetermined enzyme 12. Details of the evaluation sample 1 are as described above in "(Biomolecule Evaluation Method)" and will not be described here.

[0066] The pipette 2 is positioned perpendicular to the mounting stage 41. In FIG. 6, the pipette 2 is supported by a support member 434 so that the tip of the pipette 2 faces vertically downward. Specifically, the pipette 2 has a tapered tip portion 211 and a tip opening 22 (see FIG. 7) at the tip of the tip portion 211. The diameter of the tip opening 22 can be formed to be approximately several tens of nanometers to several micrometers, for example. More specifically, the diameter of the tip opening 22 can be formed to be approximately 100 nm to 2 μm. A pipette 2 having a diameter of the tip opening 22 in the nano-order range of more than 0 nm and less than 1000 nm is sometimes called a nanopipette. The main body of the pipette 2, excluding the tip portion, can be formed in a cylindrical shape with the same outer diameter as the tubular body, such as a capillary tube, that will be used as the pipette material. In addition, FIGS. 6, 7, and 9 show an example using the double-barrel nanopipette described above.

[0067] The pipette 2 may be made of, for example, a capillary tube made of borosilicate glass or quartz glass, which is pulled by a laser puller to narrow the diameter of the tip opening 22 to the above-mentioned diameter. Note that the pipette 2 may be made of other materials besides glass, such as resin or ceramics.

[0068] The pipette 2 is filled with an electrolyte solution 20 containing at least one substrate 21 that reacts with a predetermined enzyme 12. Details of the electrolyte solution 20 are as described above in "(Method for evaluating biomolecules)" and will not be described here.

[0069] The circuit unit 42 includes a pair of electrodes 421, 422, a voltage unit 423, and a current measurement unit 424. The pair of electrodes 421, 422 are immersed in the electrolyte 20 in the pipette 2. FIGS. 7 and 9 show an example in which one electrode 421 is immersed in the electrolyte 20 filled in one hollow channel 25 of the double-barrel pipette 2, and the other electrode 422 is immersed in the electrolyte 20 filled in the other hollow channel 422. In the double-barrel pipette 2, the electrolyte 20 filled in one hollow channel 421 and the electrolyte 20 filled in the other hollow channel 422 combine at the tip of the pipette 2, thereby forming a droplet 3 of the electrolyte 20 at the tip of the pipette 2. The electrodes 421, 422 may be, for example, Ag / AgCl electrodes (silver-silver chloride electrodes).

[0070] The voltage unit 423 is configured to apply a voltage between the pair of electrodes 421 and 422. In FIG. 6, the upper end of one electrode 421 is electrically connected to the negative electrode of the voltage unit 423 via a first wiring 425. The upper end of the other electrode 422 is electrically connected to the positive electrode of the voltage unit 423 via a second wiring 426. The current measurement unit 424 is configured to measure the current flowing through the electrodes 421 and 422 and the electrolyte solution 20. The current measurement unit 424 can also have a function to amplify the current. In FIG. 6, the current measurement unit 424 is provided midway along the second wiring 426. The circuit unit 42 is grounded via a third wiring 427, which is electrically connected to the negative electrode of the voltage unit 423. According to the circuit unit 42, when a voltage is applied between the pair of electrodes 421 and 422 from the voltage unit 423, an ionic current can be generated, and the ionic current can be measured by the current measurement unit 424.

[0071] The position control unit 43 is configured to be able to control the position of the pipette 2 and the position of the evaluation sample 1. In other words, the position control unit 43 is configured to be able to position the pipette 2 and the evaluation sample 1 in the X, Y, and Z directions.

[0072] The position control unit 43 may be configured to determine the position of the pipette 2 and the position of the evaluation sample 1 by moving both the pipette 2 and the evaluation sample 1, or may be configured to determine the position of the pipette 2 and the position of the evaluation sample 1 by moving either the pipette 2 or the evaluation sample 1.

[0073] Specifically, the position control unit 43 may be configured, for example, to be able to move the evaluation sample 1 in the X and Y directions and to be able to move the pipette 2 in the Z direction, or to be able to move the pipette 2 in the X and Y directions and to be able to move the evaluation sample 1 in the Z direction, or to be able to move the pipette 2 in the X, Y, and Z directions and to be able to move the evaluation sample 1 in the X, Y, and Z directions. Alternatively, the position control unit 43 may be configured to immobilize the evaluation sample 1 and to be able to move the pipette 2 in the X, Y, and Z directions, or to be able to immobilize the pipette 2 and to be able to move the evaluation sample 1 in the X, Y, and Z directions.

[0074] 6 shows an example in which the position control unit 43 is configured to be able to move the evaluation sample 1 in the X and Y directions and move the pipette 2 in the Z direction. In this case, the lighter pipette 2 is moved in only one direction, the Z direction, compared to the evaluation sample 1, which is generally placed in a container such as a glass-bottom dish. Therefore, the pipette 2 can be moved quickly in the Z direction, and the increased speed in the Z direction can improve the operating speed of the evaluation device 1. Note that a separate manual X, Y, and Z position control device may be incorporated into the Z-direction pipette to improve maneuverability.

[0075] Specifically, in FIG. 6, an XY coarse movement actuator 431 is provided on a microscope 44. The XY coarse movement actuator 431 is a driving device with a maximum range of motion on the order of several tens of mm, capable of controlling movement in the X and Y directions in micrometer increments. For example, an XY stepping motor can be used as the XY coarse movement actuator 431. An XY manual manipulator (not shown) can also be used instead of or in combination with the XY coarse movement actuator 431. An XY piezo stage 410 is provided on the XY coarse movement actuator 431 as a mounting stage 41 on which the evaluation sample 1 is mounted. The XY piezo stage 410 is a stage that uses piezo elements with a maximum range of motion on the order of several micrometers to several tens of micrometers, capable of controlling movement in the X and Y directions with a resolution of about 1 nm.

[0076] 6, a Z manual manipulator 432 is provided on a microscope 44, and a Z coarse movement actuator 433 is provided on the Z coarse movement actuator 433. The Z coarse movement actuator 433 is a driving device with a maximum range of motion of several tens of mm and capable of controlling movement in the Z direction in micrometer units. For example, a Z stepping motor can be used as the Z coarse movement actuator 433. A Z piezo stage 434 is provided on the Z coarse movement actuator 433. The Z piezo stage 434 is a stage using a piezo element with a maximum range of motion of several micrometers to several tens of micrometers and capable of controlling movement in the Z direction with a resolution of about 1 nm. The base end of a support member 435 that supports the pipette 2 so that the tip of the pipette 2 faces vertically downward is connected to the Z piezo stage 434.

[0077] 6, the position control unit 43 can position the pipette 2 and the evaluation sample 1 in the X, Y, and Z directions by controlling the XY coarse movement actuator 431, the XY piezo stage 410, the Z coarse movement actuator, and the Z piezo stage 434. In this way, the evaluation device 4 is configured so that the position of the pipette 2 in the X and Y directions relative to the evaluation sample 1 can be changed by the position control unit 43, and the pipette 2 can be moved closer to or farther away from the evaluation sample 1 in the Z direction (approaching or moving away in the Z direction).

[0078] The microscope 44 is used to observe the evaluation sample 1 on the mounting stage 41. FIG. 6 shows an example in which the microscope 44 is an inverted microscope. Note that an opening (not shown) is formed in the center of the XY coarse movement actuator 431 and the XY piezo stage 410 to enable observation using the inverted microscope. The Z-direction positions of the XY coarse movement actuator 431 and the XY piezo stage 410 are set so that the focal length of the inverted microscope is the same as the position of the mounted evaluation sample 1. FIG. 6 also shows an example in which the inverted microscope is installed on a vibration isolation table 45 to avoid the effects of vibration.

[0079] The imaging unit 46 is for capturing an image by the microscope 44. Specifically, a CCD camera or the like can be used as the imaging unit 46. The captured image can be imported into a personal computer (not shown) or the like as needed.

[0080] As illustrated in Figures 5 to 9, this evaluation device 4 is configured to be able to perform at least a first operation in which the position control unit 43 reduces the distance between the tip of the pipette 2 and the evaluation sample 1, detects contact between the droplet 3 held at the tip of the pipette 2 and the evaluation sample 1 from a change in the current value flowing through the electrolyte 20 measured by the current measurement unit 424, and, after this detection, increases the distance between the tip of the pipette 2 and the evaluation sample 1 using the position control unit 43.

[0081] Specifically, as shown in FIG. 7i), the position control unit 43 is scanned to decrease the distance between the tip of the pipette 2 and the substrate 10 of the evaluation sample 1. Here, the tip of the pipette 2 is moved closer in the vertical Z direction to the substrate 10 of the evaluation sample 1, which is positioned at predetermined coordinates in the X and Y directions. As a result, as shown in FIG. 7i), the height of the pipette 2 relative to the upper surface of the substrate 10 of the evaluation sample 1 gradually decreases. In this case, the droplet 3 held at the tip of the pipette 2 and the substrate 10 of the evaluation sample 1 have not yet come into contact, and therefore, as shown in FIG. 8i), the value of the current flowing through the electrolyte 20 measured by the current measurement unit 424 does not change.

[0082] As shown in FIG. 7ii), when the droplet 3 held at the tip of the pipette 2 comes into contact with the substrate 10 of the evaluation sample 1, the droplet 3 at the tip of the pipette 2 connects with the surface of the substrate 10 of the evaluation sample 1 and the electrolyte solution 20 in the pipette 2, causing the shape of the droplet 3 held at the tip of the pipette 2 to change. Specifically, the droplet 3 may contact the substrate 10 of the evaluation sample 1 in a manner that includes the evaluation target T fixed on the substrate 10, or in a manner that does not include the evaluation target T fixed on the substrate 3. In either case, the change in the shape of the droplet 3 causes a fluctuation in the current value flowing through the electrolyte solution 20 measured by the current measurement unit 424, as shown in FIG. 8ii). Specifically, the current value flowing through the electrolyte solution 20 increases. From this change in the current value flowing through the electrolyte 20, it is possible to detect contact between the droplet 3 held at the tip of the pipette 2 and the substrate 10 of the evaluation sample 1. Note that the current value may decrease depending on the measurement sample and the stopping position, but even in this case, a change in the current value can be observed, making it possible to detect contact.

[0083] After this detection, as shown in FIG. 7iii), the position control unit 43 is scanned to increase the distance between the tip of the pipette 2 and the substrate 10 of the evaluation sample 1. Here, the tip of the pipette 2 is moved away from the substrate 10 of the evaluation sample 1 in the direction opposite to the vertical Z direction (upward). As a result, as shown in FIG. 7iii), the height of the pipette 2 relative to the upper surface of the substrate 10 of the evaluation sample 1 gradually increases, and the droplet 3 held at the tip of the pipette 2 separates from the tip of the pipette 2 and adheres to the substrate 10 of the evaluation sample 1, forming a droplet 3 on the substrate 10 of the evaluation sample 1. In this case, as shown in FIG. 8iii), the current value flowing through the electrolyte solution 20 measured by the current measurement unit 424 fluctuates again. Specifically, the current value flowing through the electrolyte solution 20 momentarily increases, and then the droplet 3 separates from the pipette 2, so that the current value flowing through the electrolyte solution 20 returns to the state it was before the droplet 3 came into contact with the substrate 10 of the evaluation sample 1. Furthermore, the electrolyte solution 20 is supplied from inside the pipette 2 to the outside of the tip opening 22 of the pipette 2, and a droplet 3 is reformed at the tip of the pipette 2, returning to a steady state. The droplet 3 formed on the substrate 20 in this manner can function as a microscopic reaction chamber, as described above. In reality, as shown in FIG. 7ii), even when the droplet 3 held at the tip of the pipette 2 is connected to the upper surface of the substrate 10 of the evaluation sample 1 and the electrolyte solution 20 in the pipette 2, the substrate 21 can react with the enzyme 12, and the droplet 3 in this state can also function as a reaction chamber.

[0084] The evaluation device 4 is configured to repeatedly perform the first operation described above while changing the position of the pipette 2 in the X and Y directions relative to the evaluation sample 1 using the position control unit 43, thereby causing the droplet 3 held at the tip of the pipette 2 to adhere to the evaluation sample 1 and forming multiple droplets 3 on the evaluation sample 1. Note that "changing the position of the pipette 2 in the X and Y directions relative to the evaluation sample 1" includes not only the case where the position of the pipette 2 in the X and Y directions is fixed and the position of the evaluation sample 1 in the X and Y directions is changed, as described above, but also the case where the position of the evaluation sample 1 in the X and Y directions is fixed and the position of the pipette 2 in the X and Y directions is changed, or the case where both the position of the evaluation sample 1 in the X and Y directions and the position of the pipette 2 in the X and Y directions are changed, although these are not shown in the figures.

[0085] Furthermore, the evaluation device 4 is configured to be able to form a plurality of droplets 3 on the evaluation sample 1, and then capture an image of the evaluation sample 1 using the imaging unit 46. This allows the evaluation device 4 to obtain an observation image including information on the number and / or position of droplets 3 in which a reaction between a predetermined enzyme 12 contained in the evaluation object T and a substrate 21 has occurred.

[0086] According to the present evaluation device 4 described above, the position control unit 43 changes the position of the pipette 2 in the XY directions relative to the evaluation sample 1, while shortening the distance between the tip of the pipette 2 and the evaluation sample 1, and detects contact between the droplet 3 held at the tip of the pipette 2 and the evaluation sample 1 from changes in the value of the current flowing through the electrolyte 20. After this detection, the first operation of increasing the distance between the tip of the pipette 2 and the evaluation sample 1 is repeatedly performed, thereby attaching the droplet 3 held at the tip of the pipette 2 to the evaluation sample 1, forming multiple droplets 3 on the evaluation sample 1, and then capturing an image of the evaluation sample 1 with the imaging unit 46. Therefore, according to the present evaluation device 4, an observation image including information on the number and / or position of droplets 3 in which a reaction between a predetermined enzyme 12 contained in the evaluation object T and a substrate 21 has occurred in the droplet 3 can be obtained, and this can be used in the present evaluation method described above.

[0087] The evaluation device 4 can be configured to selectively form droplets 3 on, for example, a targeted evaluation target T in the evaluation sample 1 through the first operation by the position control unit 43 described above. In this case, evaluation information on the targeted evaluation target T among the multiple evaluation targets T on the substrate 10 can be obtained, and the biomolecules 11 can be evaluated for the targeted evaluation target T.

[0088] In order to selectively form droplets 3 only on the target evaluation object T, for example, a specific evaluation object T can be mechanically or manually determined from an image of the evaluation sample 1 based on brightness, shape, etc., and the relative distance between the previously set or detected position of the pipette 2 and the specific evaluation object T can be calculated from the image, and the position control unit 43 can be controlled based on this to bring the pipette 2 close to the substrate 10 again.

[0089] The evaluation device 4 can be configured to, for example, comprehensively form droplets 3 within a certain area of ​​the evaluation sample 1 through the first operation by the position control unit 43 described above. In this case, there is an advantage that the device system and protocol can be simplified because no optical observation is required in advance.

[0090] In the above case, the fixed area where the droplets 3 are formed can be a predetermined area on the substrate 10 where all of the evaluation targets T are present, or a predetermined area on the substrate 10 where some of all of the evaluation targets T are present. Note that, to comprehensively form droplets 3 within a fixed area on the evaluation sample 1, for example, a fixed area on the substrate 10 can be determined from an image of the evaluation sample 1, and the position control unit 43 can move the pipette 2 up and down in the Z direction while changing the position of the pipette 2 in the X and Y directions relative to the evaluation sample 1 so as to cover the entire fixed area.

[0091] It should be noted that the various configurations and explanations given in the above-mentioned "(Biomolecule Evaluation Method)" can be incorporated into the description of this evaluation device, either alone or in any combination.

[0092] (Experimental example) Biotinylated β-galactosidase (hereinafter sometimes referred to as β-Gal) was conjugated to streptavidin-bound beads (Tamagawa Seiki Co., Ltd., FG beads, average particle size 180 nm). β-Gal is an enzyme commonly used in ELISA. Next, phosphate-buffered saline (hereinafter referred to as PBS solution) containing the beads was dropped onto a glass-bottom dish coated with biotinylated polylysine and allowed to stand at 4°C, thereby immobilizing the beads on the glass substrate of the glass-bottom dish. After immobilization, 2 mL of mineral oil was added to the glass-bottom dish, and the PBS solution was removed from the glass-bottom dish. This prepared an experimental sample. The prepared experimental sample simulated an evaluation sample in which multiple evaluation targets, each containing at least one biomolecule, were immobilized on a substrate, some of which contained at least one specific enzyme.

[0093] A pipette (made of borosilicate glass, double-barreled, approximately 1 μm in diameter) was prepared, containing a PBS solution containing 15.2 mM fluorodeoxyglucose (FDG). FDG is a fluorescent substrate that changes from non-fluorescent to green fluorescent upon reaction with β-Gal.

[0094] The prepared experimental sample and pipette were then set in a biomolecule evaluation device (hereinafter sometimes simply referred to as evaluation device) having the configuration shown in FIG.

[0095] Next, the evaluation device was operated to repeatedly deposit the droplets held at the tip of the pipette onto the experimental sample, thereby forming multiple droplets on the experimental sample. In this experimental example, droplets were formed comprehensively within the square area enclosed by the white dotted line on the experimental sample as shown in Figure 10 (specifically, droplets were patterned to fill the square area enclosed by the white dotted line), and adjacent droplets were made to overlap each other.

[0096] Next, an image of the experimental evaluation sample was captured using the imaging unit of the evaluation device. The fluorescence intensity (excitation light 470 nm) after the enzyme reaction was confirmed for the obtained observation image. The results are shown in Figure 10.

[0097] In a control experiment, a comparative evaluation sample was imaged in the same manner as above, except that biotinylated bovine serum albumin (hereinafter referred to as BSA) was bound to beads with streptavidin bound to the surface. The fluorescence intensity (excitation light 470 nm) of the obtained observation image was measured. Note that BSA is a protein with no enzymatic activity. The results are shown in Figure 10.

[0098] In Figure 10, the top three images are the results for the experimental evaluation sample (using β-Gal), and the bottom three images are the results for the comparative evaluation sample (using BSA). As noted in Figure 10, the two images on the left are bright-field images, the two images in the middle are fluorescent images, and the two images on the right are fluorescent images with brightness adjustment.

[0099] As shown in Figure 10, it can be seen that fluorescent droplets were detected in the image of the experimental evaluation sample. From this result, it is possible to count the number of fluorescent droplets and identify the positions of fluorescent droplets in the evaluation area using the image of the experimental evaluation sample or a processed image obtained by performing image processing such as binarization on the image of the experimental evaluation sample. Furthermore, by forming the same or different droplets on top of fluorescent or non-fluorescent droplets as necessary and capturing images in the same manner as above, it is possible to count the number of fluorescent droplets and identify the positions of fluorescent droplets in the evaluation area for the additionally captured images.

[0100] As described above, the evaluation method and evaluation device according to the experimental example do not require the preparation of a substrate with reaction wells formed in advance, and it is possible to evaluate biomolecules in a micro-sized reaction chamber and manipulate the solution within the droplet.

[0101] The present invention is not limited to the above-described embodiments and experimental examples, and various modifications are possible without departing from the spirit and scope of the present invention. Furthermore, the configurations shown in the above-described embodiments and experimental examples can be combined in any manner.

[0102] The features of the present invention are as follows. Section 1. a preparation step of preparing an evaluation sample in which a large number of evaluation targets each having at least one biomolecule are immobilized on a substrate, some of the evaluation targets having at least one predetermined enzyme, and at least one pipette filled with an electrolyte solution containing at least one substrate that reacts in response to the predetermined enzyme; a droplet forming step of attaching the droplet held at the tip of the pipette to the evaluation sample to form a plurality of droplets on the evaluation sample; and an evaluation information acquisition step of acquiring the number and / or position information of the droplets in which a reaction between the predetermined enzyme contained in the evaluation target and the substrate has occurred. Methods for evaluating biomolecules. Section 2. In the droplet forming step, Selectively forming the droplets on the target object, or The droplets are formed comprehensively within a certain area of ​​the evaluation sample. Item 1. A method for evaluating a biomolecule according to Item 1. Section 3. The droplets are formed on the evaluation sample in a volume on the order of attoliters or femtoliters. Item 3. A method for evaluating a biomolecule according to Item 1 or 2. Section 4. The method further includes an additional step of forming a new droplet by overlapping it with the previously formed droplet, or removing the previously formed droplet and then forming a new droplet, and additionally acquiring the number and / or position information of the droplets in which a reaction identical to or different from the previous reaction has occurred in the newly formed droplet. Item 4. The method for evaluating a biomolecule according to any one of items 1 to 3. Section 5. The electrolyte solution contains a substance capable of solubilizing a lipid membrane derived from a cell. Item 5. The method for evaluating a biomolecule according to any one of items 1 to 4. Section 6. The pipette is a double-barrel type. Item 6. The method for evaluating a biomolecule according to any one of items 1 to 5. Section 7. The droplet held at the tip of the pipette is allowed to adhere to the evaluation sample by a wetting phenomenon. Item 7. The method for evaluating a biomolecule according to any one of items 1 to 6. Section 8. a stage on which an evaluation sample is placed, the evaluation sample having a plurality of evaluation targets each having at least one biomolecule immobilized on a substrate, some of the plurality of evaluation targets each having at least one predetermined enzyme; a pipette that is arranged perpendicular to the stage and is filled with an electrolyte solution containing at least one substrate that reacts with the predetermined enzyme; a circuit unit including a pair of electrodes immersed in the electrolyte in the pipette, a voltage unit that applies a voltage between the pair of electrodes, and a current measurement unit that measures a current flowing through the electrodes and the electrolyte; a position control unit that controls the position of the pipette and the position of the evaluation sample; a microscope for observing the evaluation sample on the mounting stage; an imaging unit that captures an image by the microscope, the position control unit reduces the distance between the tip of the pipette and the evaluation sample, detects contact between the droplet held at the tip of the pipette and the evaluation sample from a change in the value of the current flowing through the electrolyte measured by the current measurement unit, and, after the detection, is configured to be capable of performing at least a first operation of increasing the distance between the tip of the pipette and the evaluation sample by the position control unit; the position control unit repeatedly performs the first operation while changing the position of the pipette in the XY directions relative to the evaluation sample, thereby causing the droplet held at the tip of the pipette to adhere to the evaluation sample, forming a plurality of droplets on the evaluation sample, and then the imaging unit can capture an image of the evaluation sample. Device for evaluation of biomolecules. Section 9. forming the droplets selectively on the target object; and / or The droplets are formed comprehensively within a certain area of ​​the evaluation sample. Item 9. An apparatus for evaluating biomolecules according to Item 8. [Explanation of symbols]

[0103] 1 Evaluation sample T Evaluation Target 10 Substrate 11 Biomolecules 12 Enzymes 2 pipettes 20 Electrolyte 21 Substrate 3 droplets 4. Devices for biomolecule evaluation 41 Mounting stage 42 circuit section 421, 422 Pair of electrodes 423 Voltage section 424 Current measurement unit 43 Position control section 44 Microscope 46 Imaging unit

Claims

1. a preparation step of preparing an evaluation sample in which a number of evaluation targets each having at least one biomolecule are immobilized on a substrate, some of the evaluation targets having at least one predetermined enzyme, and at least one pipette filled with an electrolyte solution containing at least one substrate that reacts in response to the predetermined enzyme; a droplet forming step of attaching the droplet held at the tip of the pipette to the evaluation sample to form a plurality of droplets on the evaluation sample; and an evaluation information acquisition step of acquiring the number and / or position information of the droplets in which a reaction between the predetermined enzyme contained in the evaluation target and the substrate has occurred. Methods for evaluating biomolecules.

2. In the droplet forming step, Selectively forming the droplets on the target object, or The droplets are formed comprehensively within a certain area of ​​the evaluation sample. The method for evaluating a biomolecule according to claim 1 .

3. The droplets are formed on the evaluation sample in a volume on the order of attoliters or femtoliters. The method for evaluating a biomolecule according to claim 1 or 2.

4. The method further includes an additional step of forming a new droplet by overlapping it with the previously formed droplet, or removing the previously formed droplet and then forming a new droplet, and additionally acquiring information on the number and / or position of the droplets in which a reaction identical to or different from the previous reaction has occurred in the newly formed droplet. The method for evaluating a biomolecule according to claim 1 or 2.

5. The electrolyte solution contains a substance capable of solubilizing a lipid membrane derived from a cell. The method for evaluating a biomolecule according to claim 1 or 2.

6. The pipette is a double-barrel type. The method for evaluating a biomolecule according to claim 1 or 2.

7. The droplet held at the tip of the pipette is allowed to adhere to the evaluation sample by a wetting phenomenon. The method for evaluating a biomolecule according to claim 1 or 2.

8. a stage on which an evaluation sample is placed, the evaluation sample having a plurality of evaluation targets each having at least one biomolecule immobilized on a substrate, some of the plurality of evaluation targets each having at least one predetermined enzyme; a pipette arranged perpendicular to the stage and filled with an electrolyte solution containing at least one substrate that reacts with the predetermined enzyme; a circuit unit including a pair of electrodes immersed in the electrolyte in the pipette, a voltage unit that applies a voltage between the pair of electrodes, and a current measurement unit that measures a current flowing through the electrodes and the electrolyte; a position control unit that controls the position of the pipette and the position of the evaluation sample; a microscope for observing the evaluation sample on the mounting stage; an imaging unit that captures an image by the microscope, the position control unit is configured to be capable of performing at least a first operation of decreasing the distance between the tip of the pipette and the evaluation sample, detecting contact between the droplet held at the tip of the pipette and the evaluation sample from a change in the value of the current flowing through the electrolyte measured by the current measurement unit, and, after the detection, increasing the distance between the tip of the pipette and the evaluation sample by the position control unit; the position control unit is configured to repeatedly perform the first operation while changing the position of the pipette in the XY directions relative to the evaluation sample, thereby causing the droplet held at the tip of the pipette to adhere to the evaluation sample, and after forming a plurality of the droplets on the evaluation sample, to be able to capture an image of the evaluation sample by the imaging unit. Device for evaluation of biomolecules.

9. forming the droplets selectively on the target object; and / or The droplets are formed comprehensively within a certain area of ​​the evaluation sample. The device for evaluating a biomolecule according to claim 8.

Images