JP2018151397A - Single molecule recognition method, device, and program - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a single molecule recognition method, a device, and a program which are standardized so as to enable single molecule recognition by trace amount current measurement by using gap electrodes without requiring procedures such as separation and purification even on a sample including unknown molecules.SOLUTION: A sample 50 is added with a standard substance 54 in which magnitude of a signal corresponding to a trace amount of current flowing between electrodes when passing through the electrodes is known and variation of the magnitude of the signal is within a predetermined variation range, and includes at least one kind or more of single molecules 52 being an identification object. The signal corresponding to the trace amount of current flowing when each of the standard substance 54 and the single molecules 52 being the identification object included in the sample 50 is passed through the electrodes is measured, and with reference to a signal showing a standard substance included in a plurality of measured signals, the kind of single molecules shown by another signal included in the plurality of signals is identified.SELECTED DRAWING: Figure 1

Description

The present invention relates to a single molecule identification method, apparatus, and program.

Conventionally, biomolecules such as amino acids constituting proteins, nucleotides constituting nucleic acids, and monosaccharides constituting sugar chains have been identified, particularly single molecules constituting biopolymers. For identification of single molecules, for example, there is an identification method using single molecule measurement using light, electricity or the like as a probe signal. In the identification method using single molecule measurement, a specific single molecule can be detected by modifying the target sample with a fluorescent molecule or a probe molecule having electric activity.

However, the above-described method for detecting a single molecule for modifying a probe molecule requires a chemical reagent and has a problem of modification efficiency. Furthermore, there is a problem that only specific chemical species can be detected, and it cannot be applied to single molecule identification for biological samples in which a wide variety of molecular species are mixed.

In addition, in order to distinguish a wide variety of single molecules, it is necessary to obtain a highly sensitive measurement signal and to standardize the measurement signal.

As a single molecule identification method capable of obtaining a highly sensitive measurement signal, single molecule identification is performed by measuring a tunnel current flowing through a single molecule using a nanogap electrode in which the distance between electrodes is fixed to 1 nm or less. Techniques have been proposed (see, for example, Patent Documents 1 to 5). In the measurement using the tunnel current, the electron energy state of a single molecule can be directly measured.

In addition, in connection with standardization of measurement signals, the internal standard substance, which is another substance not involved in the measurement of the test substance, is put in the reagent mixed in the sample, the internal standard substance is measured, and the amount of true sample collected An electrochemical measurement method has been proposed in which an accurate quantitative value of a test substance is obtained by correcting the error (see, for example, Patent Documents 6 and 7).

JP 2013-36865 A US Patent Application Publication No. 2012/0322055 US Patent Application Publication No. 2013/0001082 US Patent Application Publication No. 2012/0193237 US Patent Application Publication No. 2010/0025249 JP 2011-163934 A JP 2008-32529 A

In single-molecule identification based on a minute current measurement using a gap electrode, such as tunneling current measurement, the measurement result depends on the measurement method and state, and thus it is necessary to standardize the measurement signal. Therefore, it is possible to perform standardization indirectly by applying standardization as in Patent Documents 6 and 7 to single molecule identification by tunneling current measurement and defining relative conductance with the sample molecule itself as an internal standard substance. It is.

However, there is a problem that the measurement time becomes longer in order to sufficiently measure the internal standard substance. In addition, since the standardization as described above cannot be applied to a sample containing an unknown molecule, procedures such as separation and purification are required when measuring a sample containing an unknown molecule. Thus, in order to apply the conventional standardization method to the trace current measurement using the gap electrode device, there is a problem that it can be applied only under limited samples and conditions.

The present invention has been made in view of the above-mentioned problems, and a sample containing unknown molecules can be simply measured by measuring a minute current using a gap electrode without requiring a procedure such as separation and purification. It is an object to provide a single molecule identification method, apparatus, and program that are standardized so that molecular identification can be performed.

In order to achieve the above object, the single molecule identification method of the present invention has a known signal magnitude corresponding to a minute current flowing between the electrodes when passing between the electrodes, and the fluctuation of the magnitude of the signal is known. A standard substance that falls within a predetermined variation range is added, and each of the standard substance and the single molecule to be discriminated included in a sample containing at least one type of single molecule to be discriminated is connected between the electrodes. Measuring a signal according to a small amount of current that flows when the signal passes through, and a signal indicating the standard substance included in the plurality of measured signals as a reference, the other signals included in the plurality of signals are Identifying the type of single molecule to be shown.

As a result, a stable signal indicating a standard substance can be obtained even when the measurement result depends on the measurement method and state, such as a minute current measurement using a gap electrode. It is also possible to standardize the contained sample so that single molecule identification can be performed by a minute current measurement using a gap electrode without requiring procedures such as separation and purification.

In addition, the standard substance may be a substance having the same shape that has electrical conductivity and does not bind to the single molecule to be identified. Thereby, a signal indicating a standard substance that can be easily distinguished from a signal indicating a single molecule to be identified can be obtained.

The standard substance may be a substance having the same posture with respect to the electrodes when passing between the electrodes. Thereby, the magnitude | size of the signal which shows the reference material for every measurement is equalize | homogenized.

Further, the shape of the standard substance can be a sphere. Thereby, the magnitude | size of the signal which shows the reference material for every measurement is equalized irrespective of the structure of an electrode.

The standard substance may be metal nanoparticles or fullerene.

Further, the concentration of the standard substance relative to the sample can be optimized so that the ratio of the signal indicating the standard substance to the plurality of signals falls within a predetermined ratio range. Thereby, it is possible to stably detect a signal indicating the standard substance and to prevent the signal indicating the standard substance from becoming noise.

Further, in the identifying step, based on the relative value of the plurality of signals with respect to the signal indicating the standard substance and the predetermined relationship between the type of the single molecule and the relative value of the signal, the single signal indicated by the other signal is displayed. The type of molecule can be identified.

Further, in the measuring step, for a sample to which a plurality of different standard substances corresponding to a plurality of different interelectrode distances are added, a signal corresponding to the minute current is measured for each of a plurality of states having different interelectrode distances. In the identifying step, for each state, a signal indicating a standard substance corresponding to the corresponding state included in a plurality of measured signals is compared with the other signals, and based on the comparison result in each state. Thus, the type of single molecule indicated by the other signal can be identified. Thereby, identification with higher accuracy can be performed.

Further, the single molecule identification device of the present invention has a known signal magnitude corresponding to a minute current flowing between the electrodes when passing between the electrodes, and the fluctuation of the magnitude of the signal is within a predetermined fluctuation range. And a sample containing at least one type of identification target single molecule and an electrode pair arranged so that a small amount of current flows when the sample passes between the electrodes, and the sample Each of the standard substance and the single molecule to be identified included in a measurement unit that measures a signal according to a minute current that flows when passing between the electrodes, and a plurality of signals measured by the measurement unit And an identification unit for identifying the type of a single molecule indicated by another signal included in the plurality of signals on the basis of a signal indicating the standard substance included in the reference signal.

In addition, the single molecule identification program of the present invention is a computer in which the magnitude of a signal according to a small amount of current flowing between electrodes when passing between the electrodes is known, and the fluctuation of the magnitude of the signal is predetermined. A reference material within a variation range is added, and at least one kind of identification target single molecule includes a pair of electrode pairs arranged so that a small amount of current flows when the sample passes between the electrodes. A measurement control unit that controls a measurement unit to measure a signal corresponding to a minute current that flows when each of the standard substance and the single molecule to be identified included in the sample passes between electrodes; and An identification unit for identifying a type of a single molecule indicated by another signal included in the plurality of signals based on a signal indicating the standard substance included in the plurality of signals measured by the measurement unit; Is a program for making the function Te.

According to the single molecule identification method, apparatus, and program according to the present invention, a stable signal from a standard substance can be obtained even in a minute current measurement using a gap electrode whose measurement result depends on the measurement method and state. Since it can be obtained, it is standardized so that samples containing unknown molecules can be identified by single-current measurement by measuring a minute current using a gap electrode without requiring procedures such as separation and purification. be able to.

It is the schematic which shows the structure of the single molecule identification device which concerns on 1st Embodiment. It is a block diagram which shows the functional structure of the control part in 1st Embodiment. It is a figure which shows a typical example of a conductance-time profile. It is a figure which shows an example of the relative conductance table in 1st Embodiment. It is a figure for demonstrating the single molecule identification in 1st Embodiment. It is a figure which shows an example of the histogram of conductance. It is a figure which shows an example of the histogram of conductance. It is a figure for demonstrating optimization of the density | concentration of a standard substance. It is a flowchart which shows the single molecule identification process in 1st Embodiment. It is the schematic which shows the structure of the single molecule identification device which concerns on 2nd Embodiment. It is a block diagram which shows the functional structure of the control part in 2nd Embodiment. It is a figure which shows an example of the conductance of each amino acid for every distance between electrodes. It is a figure which shows an example of the relative conductance table in 2nd Embodiment. It is a figure for demonstrating the single molecule identification in 2nd Embodiment. It is a flowchart which shows the single molecule identification process in 2nd Embodiment.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the following embodiment, a case where a tunnel current that flows when a single molecule passes between electrodes as a minute current is measured will be described as an example.

<First Embodiment>
As shown in FIG. 1, the single molecule identification device 10 according to the first embodiment includes a nanogap electrode pair 12, a measurement power source 18, an electrophoresis electrode pair 20, an electrophoresis power source 22, an ammeter 24, And the control unit 26. Each configuration will be described below.

The nano-gap electrode pair 12 is formed by a pair of two opposing electrodes each provided with an insulating film 14 when a single molecule 52 and a standard material 54 (details will be described later) contained in the sample 50 pass between the electrodes. They are arranged at a distance such that current flows. The specific manufacturing method of the nanogap electrode pair 12 is not particularly limited.

The measurement power supply 18 applies a voltage to the nanogap electrode pair 12. The magnitude of the voltage applied to the nanogap electrode pair 12 by the measurement power supply 18 is not particularly limited, and can be, for example, 0.25V to 0.75V. The specific configuration of the measurement power supply 18 is not particularly limited, and a known power supply device can be used as appropriate.

Electrophoresis electrode pair 20 is arranged so as to form an electric field in the moving direction of single molecule 52 and standard substance 54 contained in sample 50 (block arrow A in FIG. 1). When an electric field is formed between the electrodes of the electrode pair 20 for electrophoresis, the single molecule 52 and the standard substance 54 move in the direction of the electric field by electrophoresis. That is, the single molecule 52 and the standard substance 54 move so as to pass between the electrodes of the nanogap electrode pair 12.

The power supply 22 for electrophoresis applies a voltage to the electrode pair 20 for electrophoresis. The magnitude of the voltage applied to the electrophoresis electrode pair 20 by the electrophoresis power source 22 is not particularly limited, and the speed at which the single molecule 52 and the standard substance 54 pass between the electrodes of the nanogap electrode pair 12 is controlled. Can be set as appropriate. The specific configuration of the power supply 22 for electrophoresis is not particularly limited, and a known power supply device can be used as appropriate.

The ammeter 24 measures a tunnel current generated when the single molecule 52 and the standard material 54 pass between the electrodes of the nanogap electrode pair 12 to which a voltage is applied by the measurement power supply 18. The specific configuration of the ammeter 24 is not particularly limited, and a known current measuring device may be used as appropriate.

The control unit 26 controls each component of the single molecule identification device 10 and identifies the type of the single molecule 52 based on a signal corresponding to the measured tunnel current.

The control unit 26 can be configured by a computer including a CPU (Central Processing Unit), a RAM (Random Access Memory), and a ROM (Read Only Memory) in which a single molecule identification program described later is stored. The control unit 26 configured by this computer can be functionally represented by a configuration including an electrophoresis control unit 30, a measurement control unit 32, and an identification unit 34, as shown in FIG. Hereinafter, each part is explained in full detail.

The electrophoresis control unit 30 controls application of voltage by the electrophoresis power supply 22 so that the single molecule 52 and the standard substance 54 pass between the electrodes of the nanogap electrode pair 12.

The measurement control unit 32 controls the ammeter 24 so as to measure the tunnel current flowing between the electrodes of the nanogap electrode pair 12. The measurement time of the tunnel current is not limited, but may be, for example, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour. Further, the measurement control unit 32 acquires the current value of the tunnel current measured by the ammeter 24, calculates conductance from the acquired current value, and creates a conductance-time profile. The conductance can be calculated by dividing the current value of the tunnel current by the voltage V applied to the nanogap electrode pair 12 when the tunnel current is measured. By using the conductance, a uniform reference profile can be obtained even when the voltage value applied between the nanogap electrode pair 12 differs for each measurement. In addition, when the voltage value applied between the nanogap electrode pair 12 is made constant for each measurement, the current value of the tunnel current and the conductance can be handled equally.

Alternatively, the measurement control unit 32 may acquire the tunnel current measured by the ammeter 24 after having been amplified using a current amplifier. By using a current amplifier, a weak tunnel current value can be amplified, so that the tunnel current can be measured with high sensitivity. As the current amplifier, for example, a commercially available variable high-speed current amplifier (manufactured by Femto, catalog number: DHPCA-100) can be used.

The discriminating unit 34 discriminates the type of single molecule indicated by other signals with reference to the signal indicating the standard substance 54 included in the plurality of signals appearing in the conductance-time profile created by the measurement control unit 32.

FIG. 3 shows a schematic example of a conductance-time profile. The plurality of signals appearing in the conductance-time profile is a portion having a peak value as shown in FIG. 3, and corresponds to one signal for each peak. Therefore, in the example of FIG. 3, it is shown that one signal exists in the portion indicated by A and four signals exist in the portion indicated by B.

In the example of FIG. 3, the signal of the portion indicated by A is a signal indicating the standard substance 54, and each signal included in the signal group of the portion indicated by B is a signal indicating the single molecule 52. In this case, if the conductance of the signal indicating the standard substance 54 and the relative conductance of each type of the single molecule 52 to be identified with respect to the conductance of the signal indicating the standard substance 54 are known, the type of the single molecule indicated by each signal is identified. can do.

Specifically, the relative conductance of the single molecule 52 to be identified with respect to the intrinsic conductance of the standard substance 54 is stored in advance in the relative conductance table 36. FIG. 4 shows an example of the relative conductance table 36. Then, as shown in FIG. 5, the identification unit 34 conducts signals other than the signal indicating the standard substance 54 in the obtained conductance-time profile, and the identification target single molecule stored in the relative conductance table 36. The relative conductance of 52 is compared, and the type of the single molecule having the same relative conductance is identified as the type of the single molecule indicated by the signal. Note that the fact that the relative conductances match does not only mean that the relative conductances completely match, but also includes the case where the difference between the two becomes equal to or less than a predetermined threshold.

Here, as described above, what standard substance 54 is preferably used for identifying the type of the single molecule 52 indicated by each signal using the relative conductance value will be described.

In the measurement of a minute current using a gap electrode such as a tunnel current, the distance between the electrode and a molecule passing between the electrodes affects the magnitude of the measured minute current. Therefore, in the case of a molecule in which the posture with respect to the electrodes when passing between the electrodes is not uniform, the conductance (signal magnitude) varies for each measurement. For example, as shown in FIG. 6, when a histogram of conductance obtained by a plurality of measurements is created, a substance having a large variance in the histogram is not suitable for use as the standard substance 54. Therefore, a material having a small variation in conductance for each measurement is used as the standard material 54. For example, as shown in FIG. 7, a substance having a small variance in the conductance histogram obtained by a plurality of measurements is suitable for use as the standard substance 54.

As described above, in order to reduce the variation in conductance for each measurement, a material having the same posture with respect to the electrodes when passing between the electrodes is used as the standard material 54. For example, a material that uniquely determines the posture when passing between electrodes due to the relationship between the electrodes and the shape of the material, a material that can be controlled so that the posture when passing between the electrodes is the same by electrophoresis, etc. Is mentioned. Moreover, if the shape of the substance is spherical, the posture when passing between the electrodes is the same without considering the relationship with the electrodes or performing control such as electrophoresis.

In addition, since the signal indicating the standard substance 54 is used as a reference, it is preferably a signal that can be clearly distinguished from the signal indicating the single molecule 52 to be identified. Therefore, the standard substance 54 is preferably a substance that has electrical conductivity and does not bind to a single molecule to be identified. In order to obtain a stable signal as a reference, it is preferable that the standard substances contained in the sample 50 have the same shape. Further, as shown in FIG. 3, since it is desirable that the difference from the signal indicating the single molecule 52 is large, a substance having a large intrinsic conductance is preferable as compared with the single molecule 52 to be identified.

In consideration of the above conditions, metal nanoparticles or fullerene can be used as the standard material 54. Examples of the metal nanoparticles include gold nanoparticles, silver nanoparticles, copper nanoparticles, and alumina nanoparticles. In addition, when the size of the identification target single molecule 52 is about 0.5 to 2 nm, it is suitable to use fullerene as the standard substance 54. Further, when the size of the single molecule 52 to be identified is 2 nm or more, it is suitable to use gold nanoparticles as the standard substance 54.

Next, a single molecule identification method performed using the single molecule identification apparatus 10 according to the first embodiment will be described.

First, at least one or more types of identification target single molecules 52 are dissolved in a solution. The solution is not particularly limited. For example, ultrapure water can be used. Ultrapure water can be produced, for example, by using Milli-Q Integral 3 (device name) (Milli-Q Integral 3/5/10/15 (catalog number)) manufactured by Millipore. Although the density | concentration of the single molecule 52 in a solution is not specifically limited, For example, it is 0.01-1.0 micromol.

Next, the standard substance 54 as described above is added to the solution in which the single molecule 52 is dissolved. The concentration of the standard substance 54 in the solution is optimized so that the ratio of the signal indicating the standard substance 54 to a plurality of signals appearing in the conductance-time profile falls within a predetermined range. As shown in FIG. 8, when the concentration of the standard substance 54 is low, the signal indicating the standard substance 54 (A in FIG. 8) in the conductance-time profile decreases, so that the signal indicating the standard substance 54 is stabilized. Cannot be detected. On the other hand, when the concentration of the standard substance 54 is high, a signal indicating the standard substance 54 in the conductance-time profile increases, and the signal becomes noise. Therefore, the predetermined range is determined so as to obtain an optimum number of signals in consideration of the balance between the stability of identification and the reduction of noise.

Then, the nanogap electrode pair 12 is disposed in the sample 50, and a voltage is applied to the nanogap electrode pair 12 by the measurement power supply 18, and a voltage is applied to the electrophoresis electrode pair 20 by the electrophoresis power supply 22. To do. Then, the single molecule identification process shown in FIG. 9 is performed by the single molecule identification device 10 by the CPU of the computer constituting the control unit 26 reading and executing the single molecule identification program stored in the ROM.

In step S10 of the single molecule identification process shown in FIG. 9, the measurement control unit 32 controls the ammeter 24, and the tunnel generated when the single molecule 52 and the standard substance 54 pass between the electrodes of the nanogap electrode pair 12. The current is measured for a predetermined time.

Next, in step S12, the measurement control unit 32 acquires the current value of the measured tunnel current, calculates conductance for each measurement point, and creates a conductance-time profile as shown in FIG. 3, for example. Next, in step S <b> 14, the identification unit 34 acquires the relative conductance of the single molecule 52 to be identified from the relative conductance table 36.

Next, in step S16, the identification unit compares the conductance-time profile created in step S12 with the relative conductance obtained in step S14, and identifies the type of single molecule indicated by each signal. Next, in step S18, the identification unit 34 outputs the identification result and ends the single molecule identification process.

As described above, according to the single molecule identification apparatus and method according to the first embodiment, as a standard substance, a substance having a small variation in conductance in a conductance-time profile created from a tunnel current flowing between nanogap electrodes. Is used. Then, based on the conductance of the signal indicating the standard substance in the conductance-time profile, the type of single molecule indicated by the other signal is identified. As a result, it is possible to standardize samples containing unknown molecules so that single molecules can be identified by measuring a minute current using a gap electrode without requiring procedures such as separation and purification. .

<Second Embodiment>
Next, a second embodiment will be described. In addition, about the part same as the single molecule identification device 10 which concerns on 1st Embodiment, the same code | symbol is attached | subjected and detailed description is abbreviate | omitted.

As shown in FIG. 10, the single molecule identification device 210 according to the second embodiment includes nanogap electrode pairs 12A, 12B, and 12C, a measurement power supply 18, an electrophoresis electrode pair 20, an electrophoresis power supply 22, An ammeter 24 and a control unit 226 are included.

The configuration of each of the nanogap electrode pair 12A, 12B, and 12C is the same as that of the nanogap electrode pair 12 in the first embodiment. Each of the nanogap electrode pairs 12A, 12B, and 12C is laminated via the insulating film 14 so that the centers between the electrodes are aligned on the same axis. That is, a single passage through which the single molecule 52 and the standard substance 54 pass is formed between the electrodes of the nanogap electrode pair 12A, 12B, and 12C. The distance between the electrodes of the nanogap electrode pair 12A is d1, the distance between the electrodes of the nanogap electrode pair 12B is d2, and the distance between the electrodes of the nanogap electrode pair 12C is d3. In the example of FIG. 10, d1> d2> d3. For example, d1 = 1.0 nm, d2 = 0.7 nm, and d3 = 0.5 nm.

As shown in FIG. 11, the control unit 226 can be represented by a configuration including an electrophoresis control unit 30, a measurement control unit 232, and an identification unit 234.

The measurement control unit 232 controls the ammeter 24 so as to measure the tunnel currents generated between the electrodes of the nanogap electrode pairs 12A, 12B, and 12C. Further, the measurement control unit 232 acquires the current value of the tunnel current for each inter-electrode distance measured by the ammeter 24, calculates conductance, and creates a conductance-time profile for each inter-electrode distance.

The identification unit 234 compares a signal indicating the standard substance 54 corresponding to the inter-electrode distance included in the plurality of signals appearing in the conductance-time profile for each inter-electrode distance with other signals. And the identification part 234 identifies the kind of single molecule which another signal shows based on the comparison result for every distance between electrodes.

Here, FIG. 12 shows the relative conductance for each inter-electrode distance d for a plurality of types of single molecules (amino acids in the example of FIG. 12). Here, the relative conductance is the conductance of each amino acid when the conductance of amino acids in FIG. In the example of FIG. 12, the inter-electrode distance d is d1 = 1.0 nm, d2 = 0.7 nm, and d3 = 0.4 nm. As shown in FIG. 12, when the inter-electrode distance d is 0.4 nm, the relative conductances of His, Thr, Tyr, and Trp are approximated. Similarly, when the inter-electrode distance d is 0.7 nm, the relative conductances of Cys and Pro, and Tyr and Trp are approximated. Similarly, when the interelectrode distance d is 1.0 nm, the relative conductances of Cys, Pro, and Phe are approximated. Thus, when the relative conductance is approximate, the identification accuracy when identifying the type of a single molecule may be reduced.

Therefore, a single molecule that can be identified for each inter-electrode distance is determined in advance. For each interelectrode distance, a standard material 54 corresponding to the interelectrode distance is selected. The relative conductance of the single molecule 52 to be identified that can be identified by the interelectrode distance with respect to the intrinsic conductance of the standard material 54 corresponding to the interelectrode distance is stored in advance in the relative conductance table 236. FIG. 13 shows an example of the relative conductance table 236.

FIG. 14 schematically shows identification processing by the identification unit 234. As shown in FIG. 14, the identification unit 234 displays the conductance of a signal other than the signal indicating the standard material 54 corresponding to the distance between the electrodes and the relative conductance, which appear in the obtained conductance-time profile for each distance between the electrodes. The type of single molecule indicated by each signal is identified by comparing the relative conductance of the single molecule 52 to be identified, which can be identified by the distance between the electrodes, stored in the table 236. For a signal that cannot be identified from the conductance-time profile of the distance between the electrodes (the signal indicated by “X” in the figure), the identification unit 234 uses a single molecule in the conductance-time profile of the distance between the electrodes. Identify the type.

Next, a single molecule identification method performed using the single molecule identification apparatus 210 according to the second embodiment will be described.

First, as in the first embodiment, at least one type of identification target single molecule 52 is dissolved in a solution. Next, the standard substance 54 as described above is added to the solution in which the single molecule 52 is dissolved. In the second embodiment, the standard materials 54 corresponding to the interelectrode distances (d1, d2, d3) of the nanogap electrode pairs 12A, 12B, 12C are respectively added.

Then, the nanogap electrode pair 12A, 12B, 12C is arranged in the sample 50, and a voltage is applied to each of the nanogap electrode pair 12A, 12B, 12C by the measurement power supply 18, and the electrophoresis power supply 22 A voltage is applied to the electrode pair 20 for electrophoresis. Then, the single molecule identification process shown in FIG. 15 is performed by the single molecule identification device 210 when the CPU of the computer constituting the control unit 226 reads and executes the single molecule identification program stored in the ROM.

In step S20 of the single molecule identification process shown in FIG. 15, the measurement control unit 232 controls the ammeter 24 so that one path formed between the electrodes of the nanogap electrode pairs 12A, 12B, and 12C is single. The tunnel current generated when the molecule 52 and the standard substance 54 pass is measured for a predetermined time.

Next, in step S22, the measurement control unit 232 acquires the current value of the measured tunnel current, calculates the conductance for each measurement point, and calculates, for example, the conductance-time profile as shown in FIG. Create each. Next, in step S24, the identification unit 234 sets 1 to the variable i.

Next, in step S26, the identification unit 234 determines from the relative conductance table 236 the relative conductance of the single molecule 52 corresponding to the interelectrode distance di, that is, the relative of the single molecule 52 to be identified that can be identified by the interelectrode distance di. Get conductance.

Next, in step S28, the identification unit 234 compares the conductance-time profile of the interelectrode distance di created in step S22 with the relative conductance acquired in step S26, and the single molecule indicated by each signal Identify the type.

Next, in step S30, the identification unit 234 determines whether or not the processing has been completed for all the inter-electrode distances di. If there is an unprocessed inter-electrode distance di, the process proceeds to step S32, i is incremented by 1, and the process returns to step S26. When the process is completed for all the inter-electrode distances di, the process proceeds to step S34, the identification unit 234 outputs the identification result, and the single molecule identification process is terminated.

As described above, according to the single molecule identification device and method according to the second embodiment, by using the conductance obtained from the tunnel current generated between nanogap electrodes having a plurality of interelectrode distances, In addition to the effects of the first embodiment, more accurate identification can be performed.

In the second embodiment, the nanogap electrode pairs 12A, 12B, and 12C are described as being stacked so that the centers between the electrodes are aligned on the same axis. However, the present invention is not limited to this. For example, each of the nanogap electrode pairs 12A, 12B, and 12C may be disposed on the same plane. In this case, the single molecule 52 and the standard substance 54 are provided in the respective electrodes of the nanogap electrode pair 12A, 12B, and 12C by providing an electrophoresis electrode corresponding to each of the nanogap electrode pair 12A, 12B, and 12C. What is necessary is just to control so that it may pass through between sequentially.

In the second embodiment, the case where a plurality of nanogap electrode pairs having different interelectrode distances is provided has been described. However, a mechanism for changing the interelectrode distance of one nanogap electrode pair may be provided. . For example, the distance between the electrodes can be changed by adjusting the geometrical arrangement of the force point, the fulcrum, and the action point using the principle of leverage. More specifically, by pushing up a part of the nanogap electrode pair with a piezo element, the electrode end portion serving as the action point can be moved to change the inter-electrode distance. In this case, the desired inter-electrode distance can be set based on the correspondence between the push-up distance of the piezo element and the inter-electrode distance.

In each of the above-described embodiments, the case where the tunnel current is measured has been described. However, the present invention can be applied as standardization of a single molecule identification method based on a minute current measurement using any gap electrode. Application of the present invention eliminates the need for pretreatment such as separation and purification prior to measurement, and enables highly accurate single molecule identification to be performed with high selectivity and over a wide range of experimental conditions. For example, when used for measurement of typical biopolymer nucleic acid base chains, higher accuracy and improved selectivity of gene sequencers and gene expression analysis methods are expected. It can also be applied to high-speed, high-sensitivity, low-cost allergen testing, disease diagnosis, etc. used in the fields of public health, safety, security, and environment.

The present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and the technical means disclosed in different embodiments are appropriately combined. The obtained embodiments are also included in the technical scope of the present invention.

In the specification of the present application, the program has been described as an embodiment in which the program is installed in advance. However, the program stored in an external storage device or recording medium is read as needed, and is downloaded and executed via the Internet. You may make it do. In addition, the program can be provided by being stored in a computer-readable recording medium.

DESCRIPTION OF SYMBOLS 10,210 Single molecule identification apparatus 12 Nano gap electrode pair 24 Ammeter 26,226 Control part 32,232 Measurement control part 34,234 Identification part 36,236 Relative conductance table 52 Single molecule 54 Standard substance

Claims (10)

A standard substance in which the magnitude of the signal according to the minute current flowing between the electrodes when passing between the electrodes is known and the fluctuation of the signal is within a predetermined fluctuation range is added, and at least A step of measuring a signal corresponding to a minute current that flows when each of the standard substance and the single molecule to be discriminated included in a sample containing one or more types of single molecules to be discriminated passes between the electrodes; When,
Identifying the type of a single molecule indicated by another signal included in the plurality of signals based on a signal indicating the standard substance included in the plurality of signals measured; and
A single molecule identification method comprising: The single molecule identification method according to claim 1, wherein the standard substance is a substance having the same shape that has electrical conductivity and does not bind to the single molecule to be identified. The single molecule identification method according to claim 1 or 2, wherein the standard substance is a substance having the same posture with respect to the electrodes when passing between the electrodes. The single molecule identification method according to claim 3, wherein the standard substance has a spherical shape. The single molecule identification method according to claim 4, wherein the standard substance is metal nanoparticles or fullerene. The concentration of the standard substance with respect to the sample is optimized so that the ratio of the signal indicating the standard substance to the plurality of signals falls within a predetermined ratio range. Single molecule identification method. In the identifying step, based on the relative value of the plurality of signals with respect to the signal indicating the standard substance and the predetermined relationship between the type of the single molecule and the relative value of the signal, the single molecule indicated by the other signal The single molecule identification method according to any one of claims 1 to 6, wherein the type is identified. In the measuring step, for a sample to which a plurality of different standard substances corresponding to different inter-electrode distances are added, a signal corresponding to the trace current is measured for each of a plurality of states having different inter-electrode distances,
In the identifying step, for each state, a signal indicating a standard substance corresponding to a corresponding state included in a plurality of measured signals is compared with the other signals, and based on a comparison result in each state. The single molecule identification method according to any one of claims 1 to 7, wherein a type of a single molecule indicated by the other signal is identified. A standard substance in which the magnitude of the signal according to the minute current flowing between the electrodes when passing between the electrodes is known and the fluctuation of the signal is within a predetermined fluctuation range is added, and at least A sample containing one or more types of single molecules to be identified passes through the electrodes, and a pair of electrodes arranged so that a small amount of current flows;
Each of the standard substance contained in the sample and the single molecule to be identified measures a signal according to a minute current that flows when passing between the electrodes,
An identification unit for identifying a type of a single molecule indicated by another signal included in the plurality of signals with reference to a signal indicating the standard substance included in the plurality of signals measured by the measurement unit;
Single molecule identification device comprising: Computer
A standard substance in which the magnitude of the signal according to the minute current flowing between the electrodes when passing between the electrodes is known and the fluctuation of the signal is within a predetermined fluctuation range is added, and at least When a sample containing one or more types of single molecules to be identified passes between the electrodes, the standard substance contained in the sample and the reference material included in the sample are arranged between the electrodes of the electrode pair arranged so that a minute amount of current flows. A measurement control unit that controls the measurement unit to measure a signal corresponding to a minute current that flows when each single molecule to be identified passes, and the standard included in the plurality of signals measured by the measurement unit A single molecule identification program for functioning as an identification unit for identifying a type of a single molecule indicated by another signal included in the plurality of signals on the basis of a signal indicating a substance.

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