Patent Description:
Quantifying a substance produced through a chemical reaction that occurs in biological specimens such as cells, cell aggregates, and pieces of tissue is a technique required for viability assay, functional assay and the like of biological specimens in fields such as medical and drug-discovery. One method for quantifying a chemical reaction product released from a biological specimen is electrochemical measurement. For example, the progress of stem cell differentiation is monitored using electrochemical measurement in Non-patent literature <NUM>.

Electrochemical measurement is a method in which an oxidization or reduction reaction, by removing electrons from a measurement target or supplying electrons to the measurement target through an electrode, is caused on the measurement target in an electrolytic solution in which two or more electrodes connected to an external power source are inserted and, at the same time, a current flowing between the electrodes is measured to determine whether an oxidation-reduction reaction has occurred, that is, to detect the presence or absence of the measurement target.

A typical electrochemical measurement device includes a working electrode which supplies and receives electrons to and from a measurement target to cause an oxidation-reduction reaction, a counter electrode which is connected to the working electrode through an external power source and compensates for electron transfer occurring at the working electrode, an electrolytic solution which enables transfer of electrons through ions in a measurement system and makes the measurement system a closed circuit, and a reference electrode for providing a reference for voltage.

In Non-patent literature <NUM>, alkaline phosphatase (ALP), which is an undifferentiation marker which exists in the cell membranes of embryonic stem (ES) cells is indirectly measured by electrochemical measurement on an embryoid body (EB) which is an aggregate of ES cells produced from ES cells of a mouse.

The reaction in which a stem cell whose function is yet to be determined changes to a somatic cell whose function is determined is commonly referred to as differentiation and a substance that indicates that differentiation has not occurred is referred to as an undifferentiation marker.

ALP is an undifferentiation marker and also has the property of hydrolyzing a phosphoric ester compound under alkaline conditions. For example, ALP acts as an enzyme in a reaction that changes p-aminophenol phosphate (PAPP), which is a phosphoric ester compound, into p-aminophenol (PAP). PAP produced by the enzymatic reaction is a substance that is electrochemically active and is oxidized to p-quinone imine (PQI) by application of a voltage to a working electrode using the reference electrode as a reference. Specifically, the presence of ALP is detected as a current value in electrochemical measurement through two reactions, namely an enzymatic reaction and an oxidation-reduction reaction.

In Non-patent literature <NUM>, a multielectrode amperometric device in which <NUM> × <NUM> = <NUM> working electrodes each having φ40-µm are provided in an array with a pitch of <NUM> is used for measurement. The device two-dimensionally images reactions in biological specimens of several to several hundred micrometers over time by using electrode current values acquired from the <NUM> electrodes.

Prior art relating to or similar to those disclosed in the non-patent literature <NUM> or those described as background art is known from non-patent literatures <NUM> to <NUM> and patent literatures <NUM> to <NUM>.

Non-patent literature <NUM> discloses a local redox cycling-based electrochemical (LRC-EC) chip device, in which ring-type interdigitated array electrodes are incorporated at n<NUM> measurement points with only 2n bonding pads for external connection and microwells are also fabricated at each measurement point to trap cell aggregates.

Non-patent literature <NUM> discloses a local redox cycling-based electrochemical (LRC-EC) system with microwells in each of which a nanocavity is formed.

Patent literature <NUM> discloses a device for measuring a blood glucose level with a high degree of accuracy, the device being configured to change at least one of i) a voltage value to be applied to a blood component measurement counter electrode and a blood component measurement working electrode in a second biological information measurement mode and ii) a voltage application time during which a voltage is applied to the blood component measurement counter electrode and the blood component measurement working electrode in the second biological information measurement mode based on biological information in a first biological information measurement mode.

Patent literatures <NUM> and <NUM>, the latter of which is an entry into the European phase of the former and thus is substantially the same as the former, each discloses an electrochemical measurement device which includes: a base; a specimen placement portion disposed on the base, for placing a biological specimen; a first electrode disposed on the base and surrounding the specimen placement portion; and a first insulating layer covering the first electrode. The first insulating layer has a plurality of openings, and the first electrode has a plurality of first electrode exposed portions which are portions of the first electrode that are exposed from the openings of the first insulating layer.

The measurement in Non-patent literature <NUM> is performed in the following process, which is illustrated in <FIG> where the horizontal axis represents time.

In <FIG>, the time A from the start of the enzymatic reaction after the introduction of the EB to the application of the voltage is set by taking into consideration the time required for a start of diffusion of PAP from the surface of the EB and the PAP concentration distribution in the electrolytic solution to stabilize, and the time B from the voltage application to the current value acquisition is provided by taking into consideration the time required for changes in the PAP concentration distribution near the electrodes to be stabilized by a oxidation-reduction reaction which occurs on the electrodes.

In measurement of ALP activity in EB that follows such a process, uncertainty of the measurement can occur.

For example, in the case where a multiple EBs are concurrently measured, the EBs contact PAPP at different timings when the EBs are placed on the electrodes in an array with a pipette or the like in multiple batches. Accordingly, the time A from the start of an enzymatic reaction to voltage application differs from EB to EB and the PAP concentration distributions around the EBs and current values vary even if all of the EBs have activities (PAP release rates per unit time) similar to one another. That is, both of a difference due to different levels of ALP activities of the EBs and a difference due to the lengths of the time A from the start of the enzymatic reaction to the voltage application appear in measured current values.

Even if multiple EBs are concurrently introduced in a PAPP solution, it may be difficult to compare a result of measurement on a group of EBs performed at a certain time with a result of measurement on the group of EBs performed at a different time because of reasons such as a difference in liquid fluctuations due to, for example, a manner in which an operator has introduced the EBs.

An object of the present invention is to provide an electrochemical measurement method capable of making measurement conditions uniform among iterations of measurement and among samples on which measurement is performed at a time, thus enabling accurate measurement and accurate comparison between results of measurement in the iterations and results of measurement performed on the samples at a time.

In view of this object, the present invention provides an electrochemical measurement method specified in independent claim <NUM>. Preferred embodiments of the inventions are also described in the dependent claims.

The electrochemical measurement method according to the present invention eliminates a measurement target produced and diffused in an electrolytic solution, then produces and diffuses a measurement target again and performs measurement. Therefore, the electrochemical measurement method according to the present invention is capable of making conditions for production and diffusion of measurement targets uniform, i.e. capable of making measurement conditions uniform, thereby enabling accurate measurement.

Consequently, results of measurement can be accurately compared with one another among iterations of the measurement and among samples (biological specimens) on which measurement is performed at a time.

Results of a numerical analysis by a finite element method will be described first using measurement of ALP activity of mouse EBs as an example. COMSOL Multiphysics Ver. <NUM> was used as numerical analysis software. Analytical model forms and boundary conditions will be described below.

An analytic space of <NUM> × <NUM> × <NUM> was provided and electrodes (working electrodes) each having φ40-µm are disposed in an array on the bottom surface of the analytic space. The thickness of the electrodes was specified to be <NUM> as a sufficiently negligible value for the setting.

An origin was set at the center of the bottom surface of the analytic space of <NUM> × <NUM>, <NUM> × <NUM> = <NUM> electrodes were placed with a pitch of <NUM> so that the center of the entire electrode array coincides with the center of the bottom surface of the analytic space. A spherical object that models an EB of φ300 µm was placed so that the center of the EB was above the electrode in the fourth row and the fifth column near the center of the electrode array. The distance between the spherical object and the electrode located immediately below the spherical object was chosen to be <NUM> by taking into consideration the ease of cutting an analytic mesh.

A substrate pAPP with a concentration of <NUM> × <NUM>-<NUM> mol/L was set in the analytic space as the initial value of the concentration in the space and the four walls and ceiling of the analytic space were set as open boundaries where the concentration outside the analytic space was <NUM> × <NUM>-<NUM> mol/L. The surface of the EB (the surface of the spherical object) was set as a boundary through which PAP was released in accordance with the Michaelis-Menten equation (<NUM>) given below, depending on the concentration of PAPP near the surface. This was an enzymatic reaction model. [Equation <NUM>] <MAT>.

In order to express an oxidation-reduction reaction of PAP, the PAP concentration was set to <NUM> on the electrodes during voltage application and a current value was calculated from the PAP concentration gradient. The current value is proportional to the concentration gradient in the direction perpendicular to the electrodes and follows equation (<NUM>). [Equation <NUM>] <MAT>.

Note that in order to evaluate the influence of the PAP concentration distribution in a visually clear manner, current values of seven electrodes (seven electrodes along the Y axis) in the same column on which the EB was placed among the <NUM> electrodes were used for evaluation.

First, according to the process illustrated in <FIG>, current values when the time A from the start of an enzymatic reaction to voltage application is <NUM> seconds and the time B from the voltage application to acquisition of the current values was <NUM> seconds and current values when the time A is <NUM> seconds and the time B is <NUM> seconds were calculated. The results are illustrated in <FIG>. As illustrated in <FIG>, the difference in time A from the start of the enzymatic reaction to the voltage application appeared as a difference in the current values. Note that in <FIG>, the position of the electrode located immediately below the EB is y = <NUM> and current values from the electrode located immediately below the EB and the six electrodes located on both side are plotted (the same applies to graphs in <FIG>, which will be described later).

The present invention performs a measurement target elimination step of eliminating a measurement target by providing a measurement target eliminating electrode in an electrolytic solution and applying a eliminating voltage of the same polarity as a measuring voltage between the measurement target eliminating electrode and a counter electrode to oxidize or reduce a measurement target, a measurement target diffusion step of diffusing a new measurement target after stopping the application of the eliminating voltage, and an electrochemical measurement step of measuring a current by applying the measuring voltage between a working electrode and the counter electrode after diffusing the new measurement target. Three forms of the measurement target eliminating electrodes (forms <NUM> to <NUM>) used in the numerical analysis will be described below.

Voltage application to the measurement target eliminating electrode is reproduced by setting analytic boundary conditions so that the PAP concentration during voltage application becomes zero as with the working electrodes.

P1 in <FIG> illustrates a process of producing the calculation results illustrated in <FIG> and P2 in <FIG> illustrates a process including the measurement target elimination step using a measurement target eliminating electrode, i.e. a process of the electrochemical measurement method according to the present invention.

It was assumed that the measurement target elimination step (PAP concentration distribution elimination step) was performed for <NUM> seconds by applying an eliminating voltage of the same polarity as a measuring voltage to the working electrodes and the measurement target eliminating electrode in a state <NUM> seconds after the start of an enzymatic reaction and in a state <NUM> seconds thereafter, then the application of the eliminating voltage was stopped and the measurement target diffusion step of causing an enzymatic reaction again for <NUM> seconds and diffusing a new measurement target was performed, thereafter the measuring voltage was applied to the working electrodes, and the electrochemical measurement step was performed. On this assumption, current values <NUM> seconds after were calculated.

<FIG>, <FIG> illustrate calculated current values for measurement target eliminating electrodes in form <NUM>, form <NUM> and form <NUM>, respectively.

Since the reaction (production and diffusion of the measurement target. the measurement target diffusion step) after the measurement target elimination step is invariable regardless of whether the period of time before the measurement target elimination step is <NUM> seconds or <NUM> seconds, it is desirable that the results for both of the case where the period of time before the measurement target elimination step is <NUM> seconds and the case where the period of time is <NUM> seconds be identical. The results for <NUM> seconds and <NUM> seconds for forms <NUM>, <NUM> and <NUM> are more sufficiently close to each other than the results illustrated in <FIG>.

When there is convection in an electrolytic solution, for example, the flow of the electrolytic solution can disturb the PAP concentration distribution formed by an EB and can influence measurement. Regarding this, results of calculation on effects of the electrochemical measurement method according to the present invention in the case where there is convection in an electrolytic solution will be described.

Calculations were performed for the following three cases (cases <NUM> to <NUM>).

<FIG> illustrates results of the calculations of current values in cases <NUM> to <NUM>. It can be seen that when the measurement target elimination step is not performed, the results are distorted by convection as in case <NUM> and the peak current value is at a position deviated from where the peak should be (the position in which the EB is located), whereas in case <NUM> in which the measurement target elimination step is performed, a current value close to the current value in case <NUM> which should be acquired can be acquired, thus influences of convection and liquid fluctuations can be eliminated.

The results of the numerical analysis performed have been described above. By performing the measurement target elimination step as described above in electrochemical measurement in which working electrodes that supply and receive electrons to and from a measurement target to cause an oxidation-reduction reaction and a counter electrode connected to the working electrodes through an external power source are provided in an electrolytic solution containing the measurement target and a measuring voltage is applied between the working electrodes and the counter electrode to measure a current flowing between the working electrodes and the counter electrode in proportion to the amount of the measurement target, the measurement target that exists at least in a range in the electrolytic solution that influences the measurement are entirely eliminated by being oxidized or reduced and the process of production and diffusion thereof is initialized and the state in the electrolytic solution is reset. Accordingly, conditions of production and diffusion of the measurement target can be made uniform among iterations of measurement and among samples measured at a time and therefore measurement conditions can be made uniform by controlling and keeping the duration of the subsequent measurement target diffusion step constant.

Further, by performing the measurement target elimination step in this way, influences of liquid fluctuations and convection of electrolytic solution can be avoided and, in addition, measurement can be performed at a timing desired by a measurer after introduction of samples (biometric specimens) that produce a measurement target.

Note that though the measurement target elimination step is performed by applying an eliminating voltage to both of a measurement target eliminating electrode and working electrodes in the method described above, the measurement target elimination step may be performed by applying the eliminating voltage only to the measurement target eliminating electrode, for example.

Forms of measurement target eliminating electrodes are not limited to those described above. For example, forms as illustrated in <FIG> may be employed. In <FIG>, each measurement target eliminating electrode <NUM> is in the form of a ring, provided in the same plane as working electrodes <NUM> and disposed around a working electrode <NUM> with φ40 µm. Each ring-like measurement target eliminating electrode <NUM> has an outer diameter of φ120 µm, for example, and an inner diameter of φ80 µm, for example.

<FIG> illustrates a measurement target eliminating electrode formed in a two-dimensional grid pattern. The measurement target eliminating electrode <NUM> is provided in the same plane as working electrodes <NUM>, and is disposed around each working electrode <NUM> to surround the working electrode <NUM>. The grid in this example is <NUM> wide with a pitch of <NUM>.

Measurement target eliminating electrodes may have form <NUM> (the three-dimensional grid) illustrated in <FIG>, <FIG> described above in combination with the forms illustrated in <FIG>. Note that when biological specimens that produce measurement targets are small, measurement target eliminating electrodes provided only on the same plane as working electrodes may achieve the effects.

Further, measurement target eliminating electrodes that have a three-dimensionally extending form different from the form <NUM> (the three-dimensional grid) may be fabricated. For example, holes or recesses for placing biological specimens inside them may be provided in a mass of gold fibers aggregated in a steel-wool form and the mass of the gold fibers may be supported or suspended above working electrodes in such a way that the gold fibers do not contact the working electrodes. Alternatively, a porous material that has an appropriate porosity and is plated with gold may be used.

A configuration of an electrochemical measurement device useful for the present invention, which is not covered by the appended claims, will be described next.

<FIG> schematically illustrates a configuration of the electrochemical measurement device. The electrochemical measurement device includes an electrolytic solution well <NUM> designed to contain an electrolytic solution <NUM> and a biological specimen <NUM> that produces a measurement target in the electrolytic solution <NUM>. Working electrodes <NUM>, measurement target eliminating electrodes <NUM>, a counter electrode <NUM> and a reference electrode <NUM> are provided in the electrolytic solution well <NUM>. While the working electrodes <NUM> and the measurement target eliminating electrodes <NUM> are schematically depicted in <FIG>, many working electrodes <NUM> are arranged in an array with a predetermined pitch as described previously and the measurement target eliminating electrodes <NUM> have any of the configurations of the measurement target eliminating electrode <NUM> illustrated in <FIG>, the measurement target eliminating electrode <NUM> illustrated in <FIG>, <FIG>, the measurement target eliminating electrode <NUM> illustrated in <FIG>, and the measurement target eliminating electrode <NUM> illustrated in <FIG>, or have a configuration in which any of the measurement target eliminating electrodes <NUM>, <NUM>, and <NUM> are used in combination with the measurement target eliminating electrode <NUM>. Reference numeral <NUM> in <FIG> indicates a salt bridge.

The working electrodes <NUM>, the measurement target eliminating electrodes <NUM>, the counter electrode <NUM>, and the reference electrode <NUM> in this example are connected to a potentiostat <NUM> as illustrated in <FIG>. The potentiostat <NUM> includes a variable power source <NUM>, a voltmeter <NUM> and an ammeter <NUM>. A measuring voltage is applied between the working electrodes <NUM> and the counter electrode <NUM> by the potentiostat <NUM> and an interelectrode current that flows between the working electrode <NUM> and the counter electrode <NUM> in proportion to the amount of a measurement target while the measuring voltage is being applied is measured by the potentiostat <NUM>.

Further, when the measuring voltage is not applied between the working electrode <NUM> and the counter electrode <NUM>, an eliminating voltage that has the same polarity as the measuring voltage is applied between the measurement target eliminating electrodes <NUM> and the counter electrode <NUM> by the potentiostat <NUM>. Application of the measuring voltage to the working electrode <NUM> is accomplished by turning on a switch <NUM> and turning off switches <NUM> and <NUM>; application of the eliminating voltage to the measurement target eliminating electrodes <NUM> is accomplished by turning on the switches <NUM> and <NUM> and turning off the switch <NUM>. Note that the eliminating voltage may also be applied to the working electrode <NUM> by turning on the switch <NUM>.

Though the eliminating voltage is applied from the potentiostat <NUM> to the measurement target eliminating electrodes <NUM> in <FIG>, application of the eliminating voltage is not so limited and the eliminating voltage may be applied using a power source separate from the potentiostat <NUM>.

A configuration of a transducer useful for the present invention but is not covered by the appended claims, which is used for electrochemical measurement of a measurement target generated from a biological specimen, will be described next with reference to <FIG> and <FIG>.

The transducer is called Bio-LSI chip, in which an electrolytic solution well <NUM> that can contain an electrolytic solution <NUM> and a biological specimen immersed in the electrolytic solution <NUM> is mounted on an LSI chip <NUM>. A hole <NUM> is formed in the center of the electrolytic solution well <NUM> and the LSI chip <NUM> is disposed on the bottom end of the hole <NUM> in such a way that the LSI chip <NUM> covers the hole <NUM>.

The LSI chip <NUM> and the electrolytic solution well <NUM> is mounted and fixed on a substrate <NUM> and a pattern <NUM> of many conductors for connection with an external device that controls the transducer is formed on the substrate <NUM>. Reference numeral <NUM> in <FIG> indicates bonding wires that interconnect the LSI chip <NUM> and the pattern <NUM> of conductors.

A sensor region <NUM> is formed on the top surface of the LSI chip <NUM>. In <FIG>, the sensor region <NUM> is indicated by hatching and is defined in the position of the hole <NUM> in the bottom surface of the electrolytic solution well <NUM>. While details are omitted from the figure, <NUM> ×<NUM> = <NUM> working electrodes (first electrodes) of φ40 µm are formed in an array with a pitch of <NUM> in the sensor region <NUM> in this example. Further, measurement target eliminating electrodes (second electrodes) are formed in such a way that each of the measurement target eliminating electrodes is positioned in the same plane as the working electrodes and around each working electrode. The measurement target eliminating electrodes have any of the configurations of the measurement target eliminating electrodes <NUM>, <NUM> and <NUM> illustrated in <FIG>, <FIG>, respectively.

The LSI chip <NUM> includes functions such as the function of applying a voltage to each of the working electrodes and the measurement target eliminating electrodes, the function of detecting a reaction at each working electrode as a current value and amplifying the current value, and the function of switching. The working electrodes and the measurement target eliminating electrodes are formed by a liftoff method, for example.

While the transducer illustrated in <FIG> and <FIG> includes the measurement target eliminating electrodes in the same plane as the working electrodes, the transducer may further include measurement target eliminating electrodes having a three-dimensional grid structure as illustrated in <FIG>, <FIG> described above.

<FIG> illustrates a specific configuration of a measurement target eliminating electrode (third electrode) which has a three-dimensional grid structure and is to be provided in a transducer, and <FIG> illustrates a transducer as illustrated in <FIG> and <FIG> to which the measurement target eliminating electrode having the three-dimensional grid structure is added.

The measurement target eliminating electrode <NUM> having the three-dimensional grid structure in this example is made up of three metal plates <NUM> and a total of <NUM> spacers <NUM>. The metal plates <NUM> are made of copper or nickel and is approximately <NUM> thick. A mesh 131a is formed in each of the metal plates <NUM> as illustrated in <FIG> by processing such as photolithography and etching. The line/space (L/S) of the mesh 131a is <NUM>/<NUM>. The spacers <NUM> are disposed at the four corners of the metal plate <NUM> in which the mesh 131a is formed and the three such metal plates <NUM> are stacked with the spacers <NUM> between them to form the measurement target eliminating electrode <NUM> having a three-dimensional grid structure as illustrated in <FIG>. Each spacer <NUM> is <NUM> thick and, with the spacers <NUM>, the metal plates <NUM> in which the meshes 131a are formed are stacked with a gap of <NUM> between them. The spacers <NUM> and the metal plates <NUM> are fixed to one another by spot welding or otherwise and after being stacked, plated with gold to complete the measurement target eliminating electrode <NUM> having the three-dimensional grid structure.

The measurement target eliminating electrode <NUM> having the three-dimensional grid structure is placed in the hole <NUM> and disposed above the sensor region <NUM> as illustrated in <FIG>. Reference numeral <NUM> in <FIG> indicates a conductor to be connected to an external power source for applying an eliminating voltage to the measurement target eliminating electrode <NUM>. Note that a configuration is also possible in which an eliminating voltage is applied from the sensor region <NUM> of the LSI chip <NUM> to the measurement target eliminating electrode <NUM>.

Claim 1:
An electrochemical measurement method for measuring alkaline phosphatase, ALP, activity of mouse embryoid bodies, EBs, in which a working electrode (<NUM>) that supplies and receives electrons to and from a measurement target to cause an oxidation-reduction reaction and a counter electrode (<NUM>) connected to the working electrode (<NUM>) through an external power source (<NUM>) are provided in an electrolytic solution (<NUM>) containing the mouse EBs, a substrate for the ALP and the measurement target, wherein the measurement target is the product of the enzymatic activity of the ALP with the substrate, and a measuring voltage is applied between the working electrode (<NUM>) and the counter electrode (<NUM>) to measure a current that flows between the working electrode (<NUM>) and the counter electrode (<NUM>) in proportion to the amount of the measurement target,
wherein a measurement target eliminating electrode (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>) is provided in the electrolytic solution (<NUM>), and
the electrochemical measurement method performs the following steps:
a measurement target eliminating step of eliminating the measurement target by applying an eliminating voltage, which has the same polarity as the measuring voltage, between the measurement target eliminating electrode (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>) and the counter electrode (<NUM>) to oxidize or reduce the measurement target;
a measurement target diffusion step of diffusing a new measurement target after stopping the application of the eliminating voltage; and
an electrochemical measurement step of measuring the current by applying the measuring voltage between the working electrode (<NUM>) and the counter electrode (<NUM>) after the new measurement target is diffused.