Measurement reagents and measurement methods in the electrical detection band method

Abstract

This invention provides a means for accurately determining the charge state of an object under test using the electrical detection zone method. [Solution] The sensor device has a structure in which a test substance 101 is suspended in a measuring reagent, a first chamber 110 having a first electrode 112 and a second chamber 120 having a second electrode 122 communicate through a pore 140, and while the first electrode and the second electrode are electrically conductive through the pore, the transient change in current between the two electrodes that occurs each time the test substance suspended in the measuring reagent passes through the pore from the first chamber to the second chamber is acquired as a pulse signal, and the positive or negative charge or amount of charge of the test substance is estimated based on the pulse width. The measuring reagent contains a chaotropic salt of 1 mM or more and 1 M or less.

Description

[Technical Field]

[0001] This invention relates to a reagent for measuring a test substance using the Electrical Detection Zone Method, a method for measuring a test substance using the Electrical Detection Zone Method, and a method for determining the results of a test substance. Here, the test substance refers to the fine particles that are to be measured in the Electrical Detection Zone Method. [Background technology]

[0002] The measurement technology of microparticles is an extremely important area in the fields of nanotechnology, biology, and materials science. A wide range of evaluation methods exist for characterizing microparticles, including particle size distribution, particle shape, charge state, surface area, thermal properties, crystallinity, and fluidity. In particular, in the fields of nanotechnology and biology, the evaluation of particle size distribution, particle shape, and charge state is a crucial element.

[0003] Typical methods for measuring particle size distribution include dynamic light scattering (DLS) and laser diffraction, while electron microscopy (SEM, TEM) and atomic force microscopy (AFM) are widely used for measuring particle shape. In recent years, particle size analysis using mass photometry (MP) based on interference scattering microscopy (iSCAT) has also been performed. Furthermore, electrophoretic light scattering (ELS) for zeta potential measurement is common for evaluating the charge state. The instruments used for these measurements are usually large and expensive, and in addition to the high costs of instrument calibration and maintenance of consumables, special pretreatment of the test material is required for each instrument. Therefore, a comprehensive interpretation that takes into account the actual state of the test material is required, and there have been limitations in evaluating particle characteristics.

[0004] The electrical detection zone method is a particle size measurement method based on the principle of electrically detecting the volume and number of particles by suspending the test material in an electrically conductive measurement buffer. Because this method can measure individual particles under physiological conditions, it has recently attracted attention in the fields of nanotechnology and biology. In the electrical detection zone method, a measurement buffer containing the test material is placed in one chamber on either side of a pore, and the other chamber is filled with only the measurement buffer. A voltage is applied while the upstream and downstream sides are electrically conductive through the pore. This technique uses electroosmotic flow (EOF) as the main driving force to measure the transient change in current generated each time a test material passes through the pore. With this method, it is possible to observe each test material passing through the pore in real time, so detailed and accurate data on particle size distribution and particle shape can be obtained. Furthermore, because it does not require an optical system for detection, the device can be miniaturized, enabling high-precision measurement at a low cost.

[0005] The electrical sensing method and its related technology, nanopore sensing, are strictly speaking different technologies because they measure different targets. The detection mechanism of nanopore sensing involves measuring the continuous fluctuations in ion flow that occur when linear molecules pass through pores of 1-2 nanometer size formed by proteins such as α-hemolysin, and analyzing the sequence information of the linear molecules based on the results. This technology is particularly applied to nucleic acid sequencing of negatively charged materials such as DNA and RNA, and Oxford Nanopore Technologies' DNA sequencing technology is also based on this principle. On the other hand, the electrical sensing method, also known as resistance pulse sensing or Coulter counting, is a technology that uses pressure and EOF as driving forces to pass the test material one particle at a time through submicron-sized pores. The detection mechanism of this technology involves measuring the individual pulses generated when the test material passes through submicron-sized pores and analyzing the information of each test material based on these pulses. The electrical detection zone method is particularly suitable for measuring substances suspended in liquids and is widely used for blood cell counting and measuring the number and particle size distribution of cells.

[0006] Until now, it has been difficult to evaluate the charge state of an object being tested using the electrical detection band method. The reason for this will be explained by comparing it with the nanopore measurement method.

[0007] DNA (ssDNA), the target of measurement in nanopore assays, exhibits a very strong negative charge with a zeta potential of approximately -30mV due to its phosphate groups. Therefore, a strong electrical response can be obtained even under physiological conditions, and it can be efficiently moved by electrophoretic force. On the other hand, when comparing erythrocytes and adeno-associated virus 2 (AAV2), which are measured using the electrophoretic detection band method, their zeta potentials are approximately -15mV and -11mV, respectively, indicating only a slight negative charge. Therefore, movement within pores by electrophoretic force is difficult.

[0008] Electrophoretic force is the force that causes charged particles to move due to an electric field, and its velocity is expressed by the Smoluchowski equation:

number

[0009] The primary driving force in the electrical detection band method is the EOF, which is the phenomenon of solvent movement due to the influence of an electric field. It functions as the main driving force when the sample passes through pores. The flow velocity of the EOF is expressed by the Smoluchowski equation as follows:

number

[0010] In nanopore measurement methods, in addition to protein nanopores such as α-hemolysin, DNA sequencing using solid-state nanopores, in which nano-sized pores are formed in solid inorganic materials, is also being attempted. As described in Non-Patent Literature 1, a technique has already been established to measure the intrinsic charge of nucleic acids by utilizing the strong negative charge of nucleic acids and passing them through pores using electrophoretic force. However, with the electrical detection zone method, it is difficult to control the passage of the test material through the pores, and until now, measuring the charge of each test material has been difficult and limited. According to Non-Patent Literature 2, despite having a small diameter of 14 nm, the measurement of the protein holohuman serum transferrin (hSTf) has been performed using the electrical detection zone method. The authors used high concentrations of lithium chloride (LiCl) of 3 M or higher to disrupt part of the protein structure, exposing new charge sites and enabling measurement. Furthermore, even when using potassium chloride (KCl), high salt concentrations of 2 M or higher are required, and it was difficult to control pore passage at concentrations of 1 M or lower, which have little effect on proteins. Traditionally, nanopore measurement methods and electrical detection band methods commonly use high-concentration electrolytes of 1 M or higher. While this did not pose a major problem for nanopore measurement methods targeting nucleic acids, when measuring proteins using electrical detection band methods, high salt concentrations caused protein denaturation, leading to structural changes and making it difficult to accurately determine the charge state of the test sample.

[0011] Non-patent document 3 describes the use of α-hemolysin to create a 1.4 nm diameter nanopore, and then uses nanopore measurement to confirm the sequence of a linearized test protein. In the experiment, a buffer solution containing a high concentration of electrolytes, including 1.5-2 M guanidine hydrochloride and 1 M KCl, was used to pass the linearized protein through the pore. The purpose of guanidine hydrochloride in this study is not only to promote structural denaturation of the test protein, but also to promote EOF by electrostatically adsorbing guanidine ions onto α-hemolysin and positively charging the pore. As a result, the charge state of the test material could not be accurately determined.

[0012] Non-patent document 4 describes the measurement of protein microparticles using a 20 nm diameter micropore using the electrical detection band method. Here, avidin with an isoelectric point of 9.3 is used, and the relationship between electrolyte pH and zeta potential, as well as the pore passage results, are shown. Pulse measurement data is shown in the pH range of 2 to 10, but this does not mean that charge evaluation was performed. Furthermore, the only conditions under which charge evaluation is possible are under strongly acidic conditions of pH 2, which severely damages the protein, making it difficult to grasp the charge state without denaturing the protein.

[0013] Detailed analysis of the characteristics of test samples is expected to lead to applications in pharmaceutical quality control and clinical diagnosis. In recent years, gene therapy drugs in the form of microparticles, such as adeno-associated virus vectors (AAVs) and lipid nanoparticles (LNPs), have attracted attention. Non-patent document 5 proposes the application of the electrical detection band method to quality control of AAVs, measuring AAV particles using a 100 nm pore. The flexibility of the AAV capsid protein correlates with the rate of change in current value when passing through the pore, and the presence or absence of nucleic acids in the AAV is determined as pulse height, i.e., the size of the microparticles, and is not intended to determine electric charge.

[0014] Non-patent document 6 also attempts to determine the infectivity of the HIV virus using the electrical detection zone method. In this study, the flexibility of the HIV virus envelope is used as an indicator, which correlates with the rate of change of current value when passing through pores, similar to non-patent document 5. The membrane deformability of the HIV virus is determined as pulse height, i.e., the size of the microparticles, and is not intended to determine the electric charge.

[0015] Patent Document 1 provides an example of using chaotropic salts in nanopore measurement. Patent Document 1 discloses a technique for creating controlled pores using chaotropic salts on a synthetic polymer membrane, rather than using conventional protein-derived pores such as α-hemolysin or MspA. However, this technique uses chaotropic salts for pore creation, not for controlling nanopore passage.

[0016] In the electric detection strip method, Patent Document 2 shows a description of the electrophoretic force as a driving force for passing the test object through the pores. Here, three driving forces, namely electrophoresis, EOF, and pressure, are defined, and it is said that the test object is passed through the pores by adjusting at least one of these. However, as described in paragraph 0043 of Patent Document 2 as a note for Example 6 related to electrophoresis, EOF is dominant and pore passage by electrophoresis has not been achieved.

[0017] In the electric detection strip method, based on the characteristics of the pulse waveform obtained when the test object passes through the pores, a computer control program performs probability density estimation to derive the number and type of analytes, as shown in Patent Document 3. This technique was conventionally a method for analyzing the properties of a test object that simply passed through the pores by EOF.

Prior Art Documents

Patent Documents

[0018]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0019]

Non-Patent Document 1

Non-Patent Document 2

[0020] The present invention has been completed in view of the above problems, and in one embodiment, aims to provide a means for accurately determining the charge state of an object under test using the electrical detection band method. [Means for solving the problem]

[0021] As a result of diligent research by the inventors, it has been found that the above problems can be solved by effectively utilizing electrophoretic force while suppressing the influence of EOF. In other words, conventionally, in the electrical sensing zone method, the main driving force for the passage of the test substance through the pore has been considered to be EOF, but in reality, the direction of passage through the pore is determined by the combined vector of EOF and electrophoretic force. When the test substance passes through the pore due to EOF, even if the test substances have the same size and other conditions but different charges, the pulse shape will be almost the same, making it difficult to estimate the charge of each individual test substance. On the other hand, when electrophoretic force is used as the driving force, the manner of passage through the pore changes according to the amount of charge of the test substance, and by detecting this as a change in pulse shape such as pulse width and asymmetry of the pulse signal using the electrical sensing zone method, the amount of charge of each individual test substance can be measured. Therefore, if the influence of EOF can be suppressed, it will be possible to achieve accurate pore measurement of each individual particle using electrophoretic force and overcome the limitations of conventional methods.

[0022] In the electrophoretic detection zone method, the electrophoretic fault (EOF) is closely related to the state of the electrical double layer (EDL) formed within the pores. Counterions accumulate in accordance with the charge state of the pore surface, forming the EDL. Ions in the electrolyte exist in a solvated state, bound to multiple water molecules, and solvated ions near the interface shield the interface's charge. This shielding effect consists of an inner stern layer and an outer diffusion layer, which constitute the basic structure of the EDL. Ion flow in the EOF mainly occurs within the diffusion layer, and the strength of this flow depends on the thickness of the EDL. It is known that as the electrolyte concentration increases, the shielding effect strengthens, the EDL shrinks, and the EOF is suppressed. However, if the electrolyte concentration is too high, structural changes in the spectroscopy material and strong shielding of the surface charge occur, resulting in a decrease in the electrophoretic velocity of the spectroscopy material. Therefore, the present invention provides a technology that enables high-precision evaluation of the charge state of a spectroscopy material in the electrophoretic detection zone method by achieving both EOF suppression and appropriate control of electrophoretic velocity.

[0023] This invention was completed based on the above findings and is illustrated below.

[0024] [Aspect 1] A measuring reagent, wherein the measuring reagent is A sensor device is used that has a structure in which a first chamber having a first electrode and a second chamber having a second electrode communicate through a pore, By introducing a measurement reagent containing the test material in suspension into the first chamber and a measurement reagent into the second chamber, and applying a bias voltage to the first electrode and the second electrode while the first electrode and the second electrode are electrically conductive through the pore, the transient current change between the first electrode and the second electrode that occurs each time the test material passes through the pore from the first chamber to the second chamber is acquired as a pulse signal. This is for estimating the positive or negative charge or the amount of charge of the object being tested based on the pulse width of the pulse signal. If the object being examined has a negative charge, The number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode; Alternatively, if the object being examined has a positive charge, The measuring reagent is characterized in that the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode. [Aspect 2] If the object being examined has a negative charge, The number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode is at least twice the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode; Alternatively, if the object being examined has a positive charge, The measuring reagent according to embodiment 1, characterized in that the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode is at least twice the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode. [Aspect 3] A measuring reagent, wherein the measuring reagent is A sensor device is used that has a structure in which a first chamber having a first electrode and a second chamber having a second electrode communicate through a pore, By introducing a measurement reagent containing two or more test materials suspended in the first chamber and a measurement reagent into the second chamber, and applying a bias voltage to the first and second electrodes while the first and second electrodes are electrically conductive through the pores, the transient current change between the first and second electrodes that occurs as the two or more test materials pass from the first chamber to the second chamber is acquired as a pulse signal. A measuring reagent for classifying two or more types of test subjects by the pulse width of the pulse signal, If all two or more of the aforementioned test subjects have a negative charge, The number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode; Alternatively, if all two or more of the above-mentioned objects have a positive charge, The measuring reagent is characterized in that the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode. [Aspect 4] If all two or more of the aforementioned test subjects have a negative charge, The number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode is at least twice the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode; Alternatively, if all two or more of the above-mentioned objects have a positive charge, The measuring reagent according to embodiment 3, characterized in that the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode is twice or more the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode. [Aspect 5] The measurement reagent according to any one of embodiments 1 to 4, characterized in that the measurement reagent contains a chaotropic salt of 1 mM or more and 1 M or less. [Aspect 6] A method for estimating the amount of charge possessed by a test object using the measuring reagent described in embodiment 1, 2, or 5. [Aspect 7] A method for classifying two or more test subjects using the measuring reagent described in embodiment 3, 4, or 5. [Effects of the Invention]

[0025] According to one embodiment of the present invention, a means can be provided for accurately determining the charge state of an object under test using the electrical detection band method. If it becomes possible to evaluate the charge of each object under test, it can be applied to quality control of pharmaceuticals, industrial products, and other items. [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1(a) shows an example of the structure of a pore sensor that can be used in the detection or concentration estimation method of the present invention. Figure 1(b) shows an electron microscope image of pore 140 of the pore sensor used for pulse waveform measurement. [Figure 2] This figure shows the pulsed transient change of the ion current flowing between the electrodes of the pore sensor shown in Figure 1. [Figure 3] This figure shows the pore passage method using the conventional electrical detection zone method. [Figure 4] This figure shows the state of passage through pores using an electrically detected zone method, which is one embodiment of the present invention. [Figure 5]This figure shows the pulses obtained when measuring the electrophoretic force of two types of beads with the same particle size but different charges in Example 1. [Figure 6] This figure shows the results of determining charged beads using the electrical detection band method in Example 1. The figure shows the results of machine learning that distinguished two types of beads with the same particle size but different charges as they passed through the pores. [Figure 7] This figure shows the pulses obtained when beads with the same particle size but two different isoelectric points were immobilized on them, measured by electrophoretic force under basic conditions, in Example 4. [Figure 8] This figure shows the results of determining charged beads using the electrical detection band method in Example 4. The figure shows the results of machine learning that distinguished between beads with the same particle size but different isoelectric points, each containing two types of protein immobilized on a single phase, as they passed through the pores. [Figure 9] This figure shows the pulses obtained when beads with the same particle size but different isoelectric points were immobilized on them, measured by electrophoretic force under acidic conditions, in Example 5. [Figure 10] This figure shows the results of determining charged beads using the electrical detection band method in Example 5. The figure shows the results of machine learning that distinguished between beads with the same particle size but different isoelectric points, each containing two types of protein immobilized on a single phase, as they passed through the pores. [Figure 11] This graph compares the number of pulses of the test object passing through the pores when ELP or EOF was used as the driving force in Example 2. [Modes for carrying out the invention]

[0027] Next, embodiments of the present invention will be described in detail with reference to the drawings. The present invention is not limited to the following embodiments, and it should be understood that appropriate design changes, improvements, etc., can be made based on the ordinary knowledge of those skilled in the art, without departing from the spirit of the invention.

[0028] (1. Test object) The test subject is not limited in type as long as it can be measured by the electrical detection band method, and may include, for example, antigens, antibodies, glycans, or genes with a specific base sequence in a biological sample. The test subject may also be a microparticle containing genes. In a preferred embodiment of the present invention, the test subject is a genetic material-containing particle such as AAV or LNP. The particle size of such genetic material-containing particles (measured by dynamic light scattering (DLS)) is typically 10 nm to 2 μm.

[0029] (2. Configuration of the pore sensor) Figure 1(a) shows an example of the structure of an electrical detection zone method apparatus (pore sensor) that can be used in some embodiments of the present invention. The pore sensor 100 has a cross-sectional structure in which two chambers 110 and 120 are separated by a partition wall 141 and connected via pores 140 provided in the partition wall 141. Electrodes 112 and 122 are installed in the two chambers, respectively. A sample containing the test substance suspended in a measurement reagent is introduced into chamber 110 from the inlet 111, and the measurement reagent is introduced into chamber 120 from the inlet 121. A bias voltage is applied to the two electrodes by a voltage source 152. These electrodes are connected to an amplifier 150, an ammeter 151, and a voltage source 152. For example, when a bias voltage is applied between electrode 112 and electrode 122, an ion current flows through the pores 140.

[0030] Figure 1(b) shows an electron microscope image of pore 140 used for measuring pulse waveforms in the experiment described later. The diameter of the pore is selected to be larger than the diameter of the antibody-modified particles to be measured. Note that Figure 1 is merely one example of a sensor used for measurement in this invention. The sensor used in this invention has one pore, two chambers connected to both sides of the pore, and one pair of electrodes installed in each of the two chambers. The shape and material of the chambers, pore, and inlet are not particularly limited and can be appropriately selected as needed.

[0031] As shown in Figure 1(a), when particles of the test material 101 present in the chamber 110 pass through the pore 140, the ion current is temporarily obstructed, and after the test material 101 passes through the chamber 120, the ion current returns to normal. Therefore, each time a test material 101 passes through the pore 140, the ion current flowing between the electrodes in Figure 1 shows a pulsed transient change as illustrated in Figure 2. In the example in Figure 1, this is measured by an ammeter 151. In Figure 2, the pulse signal 210 is the current value 202 at each time 201. The vertical axis 202 may also be a voltage value. The baseline 200 is the current value when there is no pulse (or the average over a certain period if there is noise), and the peak current 208 is the difference between the baseline 200 and the current value with the largest current phenomenon in the pulse waveform (or the average over a certain period if there is noise).

[0032] (3.Measurement method) Figure 3 shows the pore passage mode using the conventional electrical detection band method. As mentioned above, in the conventional electrical detection band method, EOF is the main driving force, exceeding the driving force by electrophoresis (ELP) (F EOF >F ELP Therefore, if the object under test is present in the cis chamber on one side of the pore (upper in the diagram), regardless of the charge state of the object under test, if the bias voltage of the transformer chamber on the other side of the pore (lower in the diagram) is negative, the object under test will pass through the pore from the cis chamber to the transformer chamber (from top to bottom in the diagram) due to the EOF, thereby generating a pulse signal (Figures 3(a), 3(d)). On the other hand, if the bias voltage is positive, the EOF will move from the transformer chamber to the cis chamber (from bottom to top in the diagram), so the object under test will not pass through the pore, and no pulse signal will be generated (Figures 3(b), 3(c)). In this example, it is assumed that the inside of the pore is negatively charged and that the EDL formed inside the pore consists of positive ions.

[0033] On the other hand, in one embodiment of the present invention, the driving force due to EOF is suppressed by making the EDL thinner. As a result, the driving force due to ELP exceeds the driving force due to EOF (F EOF <F ELPThis results in the following state. Therefore, by applying a bias voltage opposite to the charge state of the object being tested, which is located in the cis chamber of the pore (upper in the diagram), the object passes through the pore due to the effect of ELP, and a pulse signal is generated (Figures 4(a) to 4(d)). This makes it possible to accurately determine the charge state of each individual particle being tested.

[0034] However, in reality, the simultaneous presence of driving forces due to ELP and EOF complicates the motion of the object under test, and not all objects exhibit the ideal motion shown in Figure 4. Therefore, in one embodiment of the present invention, when the object under test has a negative charge, if the number of pulse signals detected when a bias voltage is applied so that the potential of the electrodes in the transformer chamber is higher than the potential of the electrodes in the sys chamber is greater than the number of pulse signals detected when a bias voltage is applied so that the potential of the electrodes in the transformer chamber is lower than the potential of the electrodes in the sys chamber, preferably 10 times or more, more preferably 20 times or more, the charge state of the object under test is evaluated as being accurately determined when the object under test has a positive charge, and the number of pulse signals detected when a bias voltage is applied so that the potential of the electrodes in the transformer chamber is lower than the potential of the electrodes in the sys chamber is greater than the number of pulse signals detected when a bias voltage is applied so that the potential of the electrodes in the transformer chamber is higher than the potential of the electrodes in the sys chamber.

[0035] Furthermore, the charge amount of a test substance can be estimated based on the pulse width, which is recorded as the passage time through the pores of the test substance. The charge amount of a test substance is determined by its isoelectric point and the pH of the measurement reagent. When a test substance is passed through pores using ELP, a test substance with a large charge amount can pass through the pores quickly, while a test substance with a small charge amount takes longer to pass through. Therefore, if the test substance is a mixture of multiple types with different isoelectric points, it is possible to classify the test substances based on the pulse width.

[0036] (4. Measurement reagents) In one embodiment, the present invention provides a measuring reagent for suspending a test substance. This measuring reagent works to suppress the driving force caused by EOF.

[0037] Therefore, in one embodiment, the present invention provides a measuring reagent. The measuring reagent uses a sensor device having a structure in which a first chamber having a first electrode and a second chamber having a second electrode communicate through pores. A measurement reagent containing the test material is introduced into the first chamber, and a measurement reagent is introduced into the second chamber. With the first electrode and the second electrode electrically conductive through the pores, a bias voltage is applied to the first electrode and the second electrode. This acquires a pulse signal of the transient current change between the first electrode and the second electrode that occurs each time the test material passes through the pores from the first chamber to the second chamber. Based on the pulse width of the pulse signal, the positive or negative charge or the amount of charge of the test material can be estimated.

[0038] Furthermore, if the object under test has a negative charge, the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode, preferably twice as many, more preferably ten times as many, and even more preferably twenty times as many. Alternatively, if the object under test has a positive charge, the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode, preferably twice as much, more preferably ten times as much, and even more preferably twenty times as much.

[0039] In another embodiment, the present invention provides a measuring reagent. The measuring reagent uses a sensor device having a structure in which a first chamber having a first electrode and a second chamber having a second electrode communicate through pores. The method involves introducing a measurement reagent containing two or more test materials suspended in a first chamber, introducing a measurement reagent into a second chamber, and applying a bias voltage to the first and second electrodes while the first and second electrodes are electrically conductive through the pores. This allows for the acquisition of a pulse signal of the transient current change between the first and second electrodes that occurs as the two or more test materials pass from the first chamber to the second chamber, thereby classifying the two or more test materials based on the pulse width of the pulse signal.

[0040] If all two or more of the aforementioned test subjects have a negative charge, The number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode, preferably twice as many, more preferably ten times as many, and even more preferably twenty times as many. Alternatively, if all two or more of the above-mentioned objects have a positive charge, The present invention is characterized in that the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode, preferably twice as much, more preferably ten times as much, and even more preferably twenty times as much.

[0041] As long as it is a measurement reagent that satisfies the above conditions, its components are not limited. As an example, by using a chaotropic salt or a normal salt as an electrolyte, ELP can be realized in the electrical detection strip method. The chaotropic salt or normal salt to be used can be selected for both anions and cations and can be used as counterions according to the charge state of the interface. It is also possible to use it in combination with other salts such as kosmotropic salts. A chaotropic salt is a salt containing ions with weak hydration that disrupt the structure and interactions of biomolecules. Representative chaotropic anions include nitrate ions (NO3 - ), iodide ions (I - ), thiocyanate ions (SCN - ), etc., and representative chaotropic cations include lithium ions (Li + ). On the other hand, a kosmotropic salt is a salt containing ions with strong hydration that stabilize the structure of biomolecules. Kosmotropic ions have a strong interaction with water molecules and tend to maintain order in the solution. Representative kosmotropic anions include sulfate ions (SO4 2- ) and phosphate ions (HPO4 2- ), and representative kosmotropic cations include sodium ions (Na + ) and potassium ions (K +) and others are included. Examples of chaotropic salts or ordinary salts usable in the present invention include: guanidine hydrochloride, guanidine thiocyanate, guanidine nitrate, urea, thiourea, sodium thiocyanate, potassium thiocyanate, lithium thiocyanate, ammonium thiocyanate, magnesium thiocyanate, calcium thiocyanate, potassium iodide, sodium iodide, lithium iodide, ammonium iodide, potassium bromide, sodium bromide, lithium bromide, ammonium bromide, calcium bromide, barium bromide, barium iodide, strontium iodide, strontium thiocyanate, potassium chloride, sodium chloride, lithium chloride, ammonium chloride, magnesium chloride, calcium chloride, sodium sulfate, potassium sulfate, lithium sulfate, ammonium sulfate, magnesium sulfate, calcium sulfate, sodium nitrate Examples include potassium nitrate, ammonium nitrate, sodium borate, potassium borate, sodium carbonate, potassium carbonate, lithium carbonate, magnesium carbonate, calcium carbonate, sodium metasilicate, potassium metasilicate, sodium ascorbate, potassium ascorbate, sodium oxalate, potassium oxalate, sodium citrate, potassium citrate, sodium tartrate, potassium tartrate, sodium fumarate, potassium fumarate, sodium phosphate, potassium phosphate, lithium phosphate, ammonium phosphate, sodium sulfite, potassium sulfite, magnesium sulfite, calcium sulfite, etc. More desirable options include guanidine hydrochloride, guanidine thiocyanate, guanidine nitrate, urea, and thiourea.

[0042] In EDL (Electrolyte-Derived Laminate), the layer closest to the pores is the Stern Layer, composed of ions with opposite charges that electrostatically bind to the pore surface. This layer is further classified into the Inner Helmholtz Plane (IHP) and the Outer Helmholtz Plane (OHP). The IHP refers to the position where unhydrated ions and molecules that are in direct contact with the pore surface are adsorbed. Because these ions are very close to the pore surface, they exert a linear shielding effect and strongly neutralize the charge on the pore surface. The OHP is the plane where ions solvated by solvent molecules (mainly water molecules) are located. Because these ions cannot approach the pore surface due to solvation, they are located further away than the IHP. Solvation refers to the state in which ions are surrounded by water molecules in solution, and in this state, the movement and adsorption of ions may be restricted.

[0043] When chaotropic salts are present in solution, chaotropic ions weaken their interaction with hydration water, reducing the number of water molecules held by the originally hydrated ions. As a result, the ion's hydration shell thins, altering the arrangement of surrounding water molecules. This causes chaotropic ions to non-specifically adsorb directly onto IHP, and other ions on OHP also lose hydration water and non-specifically adsorb onto IHP. This leads to more effective shielding of the charge on the pore surface and compression of the diffusion layer. By utilizing this phenomenon, it becomes possible to control the EOF by optimizing charge shielding and lowering the zeta potential.

[0044] The effect of adding chaotropic salts is effective not only in suppressing EOF but also in suppressing the zeta potential of the anatomical sample, enabling control of ELP. Specifically, by changing the pH of the measurement buffer, the charge state of the anatomical sample, which has an isoelectric point, can be made identical to or opposite to the charge state of the pores, thereby individually changing the zeta potential using chaotropic anions and chaotropic cations. Equation 1 shows that the electrophoretic rate can be controlled by controlling the zeta potential of the anatomical sample, and Equation 2 shows that EOF can be controlled by controlling the zeta potential of the pores.

[0045] Under low-concentration electrolyte conditions, the number of counterions is small and the electric field at the interface is strong, solvated counterions cannot approach the IHP and remain on the OHP. This thickens the diffusion layer, resulting in increased EOF. On the other hand, as an effect of chaotropic salts, adding a small amount of chaotropic salt to a low-concentration electrolyte solution weakens the interaction between solvated counterions and water molecules, destabilizing the hydration shell. This changes the behavior of the counterions, increasing the proportion of counterions adsorbed onto the IHP. As a result, it becomes possible to suppress EOF even under low-concentration electrolyte conditions.

[0046] The amount of chaotropic salt contained in the measuring reagent can be set according to the actual measuring device and the properties of the test sample, but is typically between 1 mM and 1 M. Preferably, the amount of chaotropic salt contained in the measuring reagent is between 10 mM and 500 mM.

[0047] Furthermore, the charge state of the test sample can be altered by changing the pH of the measurement reagent (measurement buffer). Specifically, when a pH 4 measurement reagent is used for a test sample with an isoelectric point around 6, the test sample becomes positively charged. Similarly, when a pH 8 measurement reagent is used, the test sample becomes negatively charged. Also, when pore passage using ELP is performed with a pH 2.7 measurement reagent, the amount of positive charge in the test sample increases compared to when a pH 4 measurement reagent is used, and the pore passage time, i.e., the pulse width, is shortened. Note that if a charge bias with the same sign as the charge of the test sample is applied to the trans chamber, the direction of EOF will be from the cis chamber to the trans chamber, but by using the measurement reagent, ELP becomes dominant and EOF is suppressed. As a result, the number of pulses passing through the pores decreases.

[0048] The measurement results obtained using the electrical detection band method can be classified using pulse features obtained for each particle that passes through. Machine learning can also be used for classification. [Examples]

[0049] The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way to the following examples.

[0050] Example 1: Classification of latex beads by charge difference using the electrical detection band method [material] <Pore Module> Pore diameter 600nm, Cat#M-AS-600-A028-001 (Manufactured by Asahi Rubber Co., Ltd.) <Reagents> Guanidine hydrochloride (GHC): Cat#075-02431 (manufactured by Fujifilm Wako Pure Chemical Corporation) Polyoxyethylene (20) sorbitan monolaurate: Cat#162-21112 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid): Cat#GB70 (manufactured by Dojin Chemical Laboratories Co., Ltd.) <Beads (1)> COOH latex beads for chemical bonding (C300): Particle size 300nm, Cat#C300 (manufactured by Fujikura Chemicals Co., Ltd.) <Beads (2)> NIST Standard Beads (STD): Particle size 300nm, Cat#3300A (manufactured by Thermo Fisher Scientific) <Measuring Instruments> ADVANTEST nanoSCOUTER (manufactured by Advantest Corporation) <Analysis software> iPoa PC Software (manufactured by iPoa Corporation)

[0051] [Procedure] Bead measurement As measurement buffers, 40 mM GHC, 10 mM HEPES buffer (pH 7.6), and 0.05% Tween20 were prepared. Using these measurement buffers, 1 × 10⁻¹⁶ units were obtained from the stock solutions of C300 beads and STD beads. 10 A bead suspension was prepared to achieve a concentration of Particles / mL. 13 μL of measurement buffer was dispensed into the transformer chamber of the pore module, and 13 μL of the bead suspension was dispensed into the cis chamber. The pore module was set in the instrument, a voltage of +0.2V was applied to the transformer chamber, and measurements were taken for 5 minutes. After the measurement was complete, the pulse information was analyzed using analysis software, and the results of the sample identification were obtained using AI.

[0052] [result] As a result of the measurement, 5,300 pulses were obtained from the C300 beads and 2,326 pulses from the STD beads during a 5-minute measurement (Figure 5). Machine learning enabled the differentiation between the C300 bead pulses and the STD bead pulses with an accuracy of 99.6%, precision of 99.7%, and an F-score of 0.9963 (Figure 6).

[0053] We were able to distinguish between two types of beads with the same particle size but different charges using ELP. C300 beads have a large number of carboxyl groups on their surface as functional groups for binding protein components such as antibodies, while STD beads do not have any functional groups. Therefore, under pH 7.6 conditions with HEPES, there is a difference in the negative charge state of the two. When machine learning was performed using multiple pulse features obtained from the measurements, the feature that showed the largest difference was the peak width. The average value of all peak widths was 1.52 ± 0.23 ms for C300 beads and 2.21 ± 0.33 ms for STD beads. As a result, it was shown that strongly negatively charged samples pass through the pores quickly, while weakly negatively charged samples pass through the pores slowly, and the two could be distinguished using ELP in the electrical detection zone method.

[0054] Example 2: Confirmation of pore passage driving force using the electrical detection band method [material] <Pore Module> Pore diameter 600nm, Cat#M-AS-600-A028-001 (Manufactured by Asahi Rubber Co., Ltd.) <Reagents> Guanidine hydrochloride (GHC): Cat#075-02431 (manufactured by Fujifilm Wako Pure Chemical Corporation) Polyoxyethylene (20) sorbitan monolaurate: Cat#162-21112 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid): Cat#GB70 (manufactured by Dojin Chemical Laboratories Co., Ltd.) <Beads (1)> COOH latex beads for chemical bonding (C300): Particle size 300nm, Cat#C300 (manufactured by Fujikura Chemicals Co., Ltd.) <Beads (2)> NIST Standard Beads (STD): Particle size 300nm, Cat#3300A (manufactured by Thermo Fisher Scientific) <Measuring Instruments> ADVANTEST nanoSCOUTER (manufactured by Advantest Corporation) <Analysis software> iPoa PC Software (manufactured by iPoa Corporation)

[0055] [Procedure] Bead measurement As measurement buffers, 10 mM, 40 mM, and 200 mM GHC, 10 mM HEPES buffer (pH 7.6), and 0.05% Tween20 were prepared. Using these measurement buffers, 1 × 10⁻¹⁶ of C300 beads and STD beads stock solutions were extracted. 10 A bead suspension was prepared to a concentration of Particles / mL. 13 μL of measurement buffer was dispensed into the transformer chamber of the pore module, and 13 μL of the bead suspension was dispensed into the cis chamber. The pore module was set in the instrument, and a voltage of +0.2V or -0.2V was applied to the transformer chamber, and measurements were taken for 5 minutes.

[0056] [result] The measurement results are as follows: In a 5-minute measurement at +0.2V, the C300 beads with a GHC concentration of 10mM detected 7,340 pulses, the STD beads detected 6,254 pulses, the C300 beads with a GHC concentration of 40mM detected 4,424 pulses, the STD beads detected 2,493 pulses, the C300 beads with a GHC concentration of 200mM detected 1,052 pulses, and the STD beads detected 379 pulses. In a 5-minute measurement at -0.2V, the C300 beads with a GHC concentration of 10mM detected 10 pulses, the STD beads detected 14 pulses, the C300 beads with a GHC concentration of 40mM detected 79 pulses, the STD beads detected 45 pulses, the C300 beads with a GHC concentration of 200mM detected 8 pulses, and the STD beads detected 8 pulses (see Table 1 and Figure 11).

[0057] [Table 1]

[0058] Both C300 beads and STD beads have a negative charge, but their charge levels differ. When the electrode potential of the transformer chamber was set to +0.2V, which is higher than the electrode potential of the cis chamber, the number of detected pulse signals was found to be significantly different compared to when the electrode potential of the transformer chamber was set to -0.2V, which is lower than the electrode potential of the cis chamber. When the electrode potential of the transformer chamber was set to -0.2V, the pores have a negative charge, so the direction of EOF was from the cis chamber to the transformer chamber, but the number of detected pulses was small. On the other hand, when ELP was used as the driving force, more pulses were detected. Furthermore, the pulse width, which indicates the pore passage speed of the beads, differed between C300 beads and STD beads, suggesting that it reflected their respective charge states. From these results, it was confirmed that under the conditions using this measurement buffer, EOF is suppressed, ELP is dominant, and the charge state of the test object can be accurately grasped.

[0059] In this invention, if the driving force by ELP exceeds the driving force by EOF and the object under test can pass through the pore, it is possible to determine the charge state of the object under test. Example 2 above is an example of the present invention, and since the driving force by ELP significantly exceeds the driving force by EOF, a large difference was observed in the number of pulses between the driving conditions by ELP (Figures 11(a) to 11(c)) and the driving conditions by EOF during voltage reversal (Figures 11(d) to 11(f)). On the other hand, if the driving force by ELP exceeds the driving force by EOF, but the difference is small, the difference between the number of pulses under the driving conditions by ELP and the number of pulses during voltage reversal is not as significant as in the example in Table 1. However, even in such cases, if the number of pulses obtained under the driving conditions by ELP exceeds the number of pulses during voltage reversal, it is possible to determine the charge state of each object under test according to the present invention. Typically, if the number of pulses obtained under the driving conditions by ELP is twice or more the number of pulses during voltage reversal, it is possible to determine the charge state of each object under test with high accuracy.

[0060] Example 3: Protein immobilization onto latex beads [material] <Reagents> Water Soluble Carbodiimide (WSC): Cat#346-03632 (manufactured by Dojindo Laboratories Co., Ltd.) Bovine serum albumin (BSA): Cat#013-15143 (manufactured by Fujifilm Wako Pure Chemical Corporation) <Buffer> HEPES:Cat#GB70 (manufactured by Dojindo Laboratories Co., Ltd.) <Beads> COOH latex beads for chemical bonding (C300): Particle size 300nm, Cat#C300 (manufactured by Fujikura Chemicals Co., Ltd.) <Protein A> BSA:Cat#013-15143 (Manufactured by Fujifilm Wako Pure Chemical Corporation) <Protein B> Anti-PSA antibody: Clone#40 (Antibody B), IgG1κ (manufactured by Mikli Immunology Laboratories Co., Ltd.) <Protein C> Anti-PSA antibody: Clone#38 (Antibody A), IgG1κ (manufactured by Mikli Immunology Laboratories Co., Ltd.)

[0061] [Procedure] Protein immobilization onto latex beads <Buffer Preparation> A 10 mM HEPES buffer (pH 7.0) was prepared as a solid-phase buffer. A 10 mM HEPES buffer (pH 7.0) was prepared as a blocking solution with 1% BSA. A 20 mM HEPES buffer (pH 7.0) was prepared as a reagent storage solution with 0.1% BSA. <Protein Immobilization> 50 μL of 10% latex stock solution and 950 μL of solid-phase buffer were added to a microtube to prepare a 0.5 wt% latex dispersion. WSC was dissolved in sensitization buffer to a concentration of 1 mg / mL, and 300 μL of this dissolved solution was added to the latex dispersion. The mixture was mixed using a rotator at room temperature for 20 minutes. 300 μg of protein A was added, and the mixture was mixed using a rotator at room temperature for 1 hour. 15 μL of 10% Tween-20 was added to the sensitized solution and sonication was performed. The mixture was then centrifuged at 20,000 G for 20 minutes, and the supernatant was removed. <Blocking> After adding the blocking solution and redispersing by ultrasound, the mixture was mixed at room temperature to 37°C for 1 hour. The mixture was centrifuged again, and the supernatant was removed. Finally, it was dispersed in the reagent storage solution to obtain the C300-BSA bead solution. Protein B and protein C were immobilized onto beads using the same procedure to obtain the C300-40 bead solution and the C300-38 bead solution, respectively.

[0062] Example 4: Classification of protein solid-phase beads by charge difference using the electrical detection band method - 1 [material] <Pore Module> Pore diameter 1,200 nm, Cat#M-AS-1200-A028-001 (Manufactured by Asahi Rubber Co., Ltd.) <Reagents> Guanidine hydrochloride (GHC): Cat#075-02431 (manufactured by Fujifilm Wako Pure Chemical Corporation) Polyoxyethylene (20) sorbitan monolaurate: Cat#162-21112 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) HEPES:Cat#GB70 (manufactured by Dojindo Laboratories Co., Ltd.) <Beads> C300-BSA bead solution, C300-40 bead solution <Measuring Instruments> ADVANTEST nanoSCOUTER (manufactured by Advantest Corporation) <Analysis software> iPoa PC Software (manufactured by iPoa Corporation)

[0063] [Procedure] Bead measurement As measurement buffers, 50 mM GHC, 100 mM HEPES buffer (pH 7.6), and 0.05% Tween20 were prepared. Using these measurement buffers, C300-BSA bead solution and C300-40 bead solution were diluted 80-fold to prepare bead suspensions. 13 μL of the measurement buffer was dispensed into the transformer chamber of the pore module, and 13 μL of the bead suspension was dispensed into the cis chamber. The pore module was set in the instrument, and a voltage of +0.2V was applied to the transformer chamber, and measurements were performed for 5 minutes. After the measurement was completed, the pulse information was analyzed using analysis software, and the results of the AI-based identification of the test subject were obtained.

[0064] [result] As a result of the measurement, 6,346 pulses were obtained from the C300-BSA beads and 2,115 pulses from the C300-40 beads during a 5-minute measurement (Figure 7). Machine learning enabled the differentiation between the C300-BSA bead pulses and the C300-40 bead pulses with an accuracy of 93.9%, precision of 91.9%, and an F-score of 0.9402 (Figure 8).

[0065] Using C300 beads of the same particle size, each immobilized with two different proteins, measurements were performed using the electrical detection zone method under pH 7.6 citrate buffer, which is above the isoelectric point of the proteins. Machine learning was performed using multiple pulse features obtained from the measurements, and the feature that showed the largest difference was peak width. The average value of all peak widths was 2.38 ± 0.72 ms for C300-BSA beads and 3.51 ± 1.04 ms for C300-40 beads. From these results, it was shown that substances with a strong negative charge passed through the pores quickly, while substances with a weak negative charge passed through slowly. This is thought to be because the strength of the charge is reflected according to the isoelectric point of the antibody, since different antibodies are immobilized on the same bead. In other words, it was inferred that the isoelectric point of protein A is on the acidic side than that of protein B. The actual isoelectric points were pI4.9 for protein A and pI6.3 for protein B. This suggests that by using charge-known microparticles as a reference, it is possible to determine the charge of the test object using the ELP (Electroelectrical Detection Zone) method.

[0066] Example 5: Classification of protein solid-phase beads by charge difference using the electrical detection zone method - 2 [material] <Pore Module> Pore diameter 1,200 nm, Cat# M-AS-1200-A028-001 (Asahi Rubber) <Reagents> Guanidine hydrochloride (GHC): Cat#075-02431 (manufactured by Fujifilm Wako Pure Chemical Corporation) Polyoxyethylene (20) sorbitan monolaurate: Cat#162-21112 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Trisodium citrate: Cat#191-01785 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Citric acid: Cat#030-05525 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) <Beads> C300-38 bead solution, C300-40 bead solution COOH latex beads for chemical bonding (C300): Particle size 300nm, Cat#C300 (Fujikura Chemical Co., Ltd.) <Measuring Instruments> ADVANTEST nanoSCOUTER (manufactured by Advantest Corporation) <Analysis software> iPoa PC Software (manufactured by iPoa Corporation)

[0067] [Procedure] Bead measurement As measurement buffers, 200 mM GHC, 50 mM citrate buffer (pH 2.6), and 0.05% Tween20 were prepared. Using these measurement buffers, C300-40 bead solution and C300-38 bead solution were diluted 40-fold to prepare bead suspensions. 13 μL of the measurement buffer was dispensed into the transformer chamber of the pore module, and 13 μL of the bead suspension was dispensed into the cis chamber. The pore module was set in the instrument, and a voltage of -0.2V was applied to the transformer chamber, and measurements were performed for 5 minutes. After the measurement was completed, the pulse information was analyzed using analysis software, and the results of the AI-based object identification were obtained.

[0068] [result] As a result of the measurement, 14,496 pulses were obtained from the C300-38 bead and 8,111 pulses from the C300-40 bead during a 5-minute measurement (Figure 9). As a result of machine learning, the C300-38 bead pulses and the C300-40 bead pulses could be distinguished with an accuracy of 97.4%, precision of 98.4%, and an F-score of 0.9734 (Figure 10).

[0069] Using C300 beads of the same particle size, each immobilized with two different proteins, measurements were performed using the electrical detection zone method under pH 2.6 citrate buffer, below the isoelectric point of the proteins. Machine learning was performed using multiple pulse features obtained from the measurements, and the feature that showed the largest difference was peak width. The average value of all peak widths was 1.51 ± 0.46 ms for C300-38 beads and 2.08 ± 0.57 ms for C300-40 beads. From these results, it was shown that substances with a strong positive charge passed through the pores quickly, while substances with a weak positive charge passed through slowly. This is thought to be because the strength of the charge is reflected according to the isoelectric point of the antibody, since different antibodies are immobilized on the same bead. In other words, it was inferred that the isoelectric point of protein C is on the basic side than that of protein B. The actual isoelectric points were pI6.3 for protein B and pI6.9 for protein C, suggesting that it is possible to determine the charge of the test substance using the ELP (Electroelectric Detection Point) method by using microparticles with known charges as reference samples. [Industrial applicability]

[0070] According to the present invention, it can be used for measuring test subjects where it is necessary to measure the charge state of the test subject. For example, it can be used in pharmaceutical development and quality control to measure charged particles in drugs. Specifically, it can non-destructively measure the presence or absence of genetic material encapsulation in nucleic acid drugs such as AAV and LNP, and simultaneously confirm the particle size distribution and number of particles. [Explanation of Symbols]

[0071] 100 pore sensor 101 Test object 110 Chamber 111 Inlet 112 Electrode 120 chambers 121 Inlet 122 electrodes 140 pores 141 Bulkhead 150 Amplifier 151 Ammeter 152 Voltage source 200 baselines 201 Time (horizontal axis) 202 Current Value (Vertical Axis) 208 Peak current 210 pulse signal

Claims

1. A measuring reagent, wherein the measuring reagent is A sensor device having a structure in which a first chamber having a first electrode and a second chamber having a second electrode communicate through a pore is used. By introducing a measurement reagent containing the test material into the first chamber and a measurement reagent into the second chamber, and with the first electrode and the second electrode electrically conductive through the pores, a bias voltage is applied to the first electrode and the second electrode, thereby acquiring the transient current change between the first electrode and the second electrode that occurs each time the test material passes through the pores from the first chamber to the second chamber as a pulse signal. This is for estimating the positive or negative charge or the amount of charge of the object being tested based on the pulse width of the pulse signal. If the object being tested has a negative charge, The number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode; Alternatively, if the object being examined has a positive charge, The measuring reagent is characterized in that the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode.

2. If the object being tested has a negative charge, The number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode is at least twice the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode; Alternatively, if the object being examined has a positive charge, The measuring reagent according to claim 1, characterized in that the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode is twice or more the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode.

3. A measuring reagent, wherein the measuring reagent is A sensor device having a structure in which a first chamber having a first electrode and a second chamber having a second electrode communicate through a pore is used. By introducing a measurement reagent containing two or more test materials suspended in the first chamber and a measurement reagent into the second chamber, and applying a bias voltage to the first and second electrodes while the first and second electrodes are electrically conductive through the pores, the transient current change between the first and second electrodes that occurs as the two or more test materials pass from the first chamber to the second chamber is acquired as a pulse signal. A measuring reagent for classifying two or more types of test subjects by the pulse width of the pulse signal, If all two or more of the above-mentioned test subjects have a negative charge, The number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode; Alternatively, if all two or more of the above-mentioned objects have a positive charge, The measuring reagent is characterized in that the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode is greater than the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode.

4. If all two or more of the above-mentioned test subjects have a negative charge, The number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode is at least twice the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode; Alternatively, if all two or more of the above-mentioned objects have a positive charge, The measuring reagent according to claim 3, characterized in that the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is lower than the potential of the first electrode is at least twice the number of pulse signals detected when the bias voltage is applied such that the potential of the second electrode is higher than the potential of the first electrode.

5. The measurement reagent according to claim 1 or 3, characterized in that the measurement reagent contains a chaotropic salt of 1 mM or more and 1 M or less.

6. A method for estimating the amount of charge possessed by a test object using the measuring reagent described in claim 1.

7. A method for classifying two or more test subjects using the measuring reagent described in claim 3.

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