JP2012181162A - Potential difference measurement device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve a problem in a potential difference measurement device that it has been found that a certain time period (response time) is required to generate a potential value conforming to a Nernst formula, and this response time tend to elongate with repeated use of the device.SOLUTION: The potential difference measurement device includes a sample solution holding section; a measurement electrode which is brought into contact with the sample solution to be introduced into the sample solution holding section, and on which molecules each having a hydrophilic group and molecules each having an electrochemical active material are aligned and coupled with each other; a reference electrode to be brought into contact with the sample solution to be introduced into the sample solution holding section; and a potentiometer for measuring a potential difference between the measurement electrode and the reference electrode.

Description

  The present invention relates to a measuring apparatus and a measuring method capable of measuring a biological substance / environmental substance with high accuracy, high sensitivity and high throughput by performing electrical measurement.

  Blood tests are widely used as a means of understanding health conditions and early detection of diseases. In blood tests, depending on required accuracy, speed, cost, etc., a large biochemical automatic analyzer may be used or a small point-of-care testing (POCT) device may be used. Large automatic biochemical analyzers are installed in general hospitals and testing centers, have high sample processing capacity per unit time, relatively high measurement accuracy, and relatively low running costs. Suitable for inspection. On the other hand, with POCT equipment, the measurement accuracy is not as high as that of a large-scale automatic biochemical analyzer, but it is quick enough to obtain test results immediately after sampling. It is suitable for emergency tests such as outpatients, tests for outpatients, tests in ambulances, tests at clinics, self-tests at home such as self-blood glucose measurement.

  Devices for POCT often use electrical measurement methods. This is because the electrical measurement method can be used to reduce the size of the apparatus compared to the case of using the optical measurement method. The current measurement method, one of the electrical measurement methods, is an electrochemical method in which the product of reaction between a reagent and a sample (usually a redox substance) is reacted with an electrode and measured as a current value to determine the concentration of the measurement object. It is a measurement method. Usually, in the current measurement method, the current i flowing through the electrode is measured in accordance with the following Cottrell equation or an equation based on this equation.

As a device using the current measurement method, there is a glucose sensor for measuring a blood glucose level (for example, Patent Document 1), and the chip is made on the premise of being disposable.

  Unlike the current measurement method, the potential difference measurement method, which is another electrical measurement method, is an electrochemical measurement method in which the signal does not depend on the electrode area. The potentiometric method is composed of a measurement electrode (working electrode) made of gold or platinum and a reference electrode, and an enzyme and a redox substance are present in the measurement solution (Patent Documents 2 and 3). Further, the measurement electrode and the reference electrode are connected to a device for measuring a voltage such as a voltmeter. When the measurement target substance is added to the measurement solution, the measurement target substance is oxidized by the enzyme reaction, and at the same time, the oxidized redox substance is reduced. The surface potential of the measurement electrode generated at that time follows the following Nernst equation.

The above equation does not include the electrode area, and the surface potential does not depend on the electrode area.

JP-A-2-245650 Special table flat 9-500727 JP2008-128803

  When a measuring device was constructed using the potentiometric method with the above characteristics, it was found that a certain amount of time (response time) was required to generate a potential according to the Nernst equation. The response time tended to become longer with repeated use of the device.

  As a typical example of means for solving the above problems, there are a sample solution holding unit, a molecule having a hydrophilic group and a molecule having an electrochemically active substance in contact with the sample solution introduced into the sample solution holding unit. A measurement electrode coupled side by side on the surface, a reference electrode in contact with the sample solution introduced into the sample solution holding unit, and a potentiometer for measuring a potential difference between the measurement electrode and the reference electrode It is characterized by.

  With the above configuration, the response time can be shortened and the throughput of the apparatus can be improved. Also, when performing repeated measurements, the response time is kept shorter than the degree that affects measurement accuracy, so that high measurement reproducibility is maintained. Furthermore, although the response time tends to be inversely proportional to the concentration of the sample, the above configuration shortens the response time even when measuring a sample having a lower concentration, and as a result, the sensitivity of the apparatus is improved.

It is an example of a measurement electrode. It is an example of a block diagram of a potential difference measuring apparatus. It is an example of the flowchart explaining operation | movement of the apparatus of FIG. This is an example of the effect of using a measurement electrode in which a molecule having a hydrophilic group and a molecule having an electrochemically active substance are bonded side by side on the surface. It is an example of the equivalent circuit of a measurement electrode. This is an example of the effect of using a measurement electrode in which a molecule having a hydrophilic group and a molecule having an electrochemically active substance are bonded side by side on the surface. This is an example of the effect of using a measurement electrode in which a molecule having a hydrophilic group and a molecule having an electrochemically active substance are bonded side by side on the surface. This is an example of the effect of using a measurement electrode in which a molecule having a hydrophilic group and a molecule having an electrochemically active substance are bonded side by side on the surface. It is an example of a measurement electrode. It is an example of the result of having measured glucose concentration. It is an example of the result of having measured cholesterol concentration. It is an example of the result of having measured potential difference change.

  Hereinafter, embodiments will be described with reference to the drawings.

  FIG. 2 is an example of a configuration diagram of an apparatus for measuring a substance to be measured in a sample by a potentiometric method. 2A includes a drive unit 201 and a control unit 202, and the drive unit 201 and the control unit 202 are connected to each other. The driving unit 201 includes a sample transport unit 203, a sample placement unit 204, a sample introduction unit 205 that introduces a sample placed in the sample placement unit 204, and a sample solution holding unit 206 that holds a sample from the sample introduction unit in the flow path. , A measurement electrode 207 and a reference electrode 208 provided so as to be in contact with a sample in the flow path, a potentiometer 209 for measuring a voltage between the measurement electrode and the reference electrode, and a liquid feeding section for feeding the sample in the flow path 210, a waste liquid container 211 for draining a sample after measurement. The control unit 202 includes a data processing device 212 and a data display device 216. The data processing device 212 includes, for example, an arithmetic device 213, a temporary storage device 214, and a nonvolatile storage device 215. The sample placement unit 204 can be configured such that a plurality of sample containers can be placed and replaced.

  FIG. 2B shows an enlarged view of the flow channels in the measurement electrode 207 and the sample solution holding unit 206. The sample advances in the direction of the arrow by the liquid feeding unit 210 and contacts the measurement electrode. Instead of using the liquid feeding unit 210, the sample solution may be injected from the sample introduction unit 205 with a syringe or the like.

  FIG. 3 is an example of a diagram illustrating a measurement procedure. The sample solution is arranged in the sample arrangement unit 204 by the number of samples to be measured (302). The sample placement unit 204 is moved to the sample introduction unit 205 by the driving unit 201 (305), the sample solution in the sample placement unit 204 is sucked from the sample introduction unit 205 by the liquid feeding unit 210, and the sample solution holding unit 206 is sampled in the sample solution holding unit 206. Fill with solution (306). After waiting for a certain time (307), the potential difference between the measurement electrode and the reference electrode is measured and recorded by a potentiometer while the sample solution is in contact with the measurement electrode and the reference electrode (308). The sample solution in the sample solution holding unit is drained into the waste container by the liquid feeding unit (309). After performing the operations 304 to 309 on the sample to be measured (303), the concentration of the substance to be measured in the sample is calculated from the potential difference according to the Nernst equation or a modified equation (310), and the result is displayed ( 311).

  By using a mixture of the specimen and the reagent as the sample solution, the concentration of the substance (measurement item) to be measured in the specimen can be obtained. Examples of specimens include blood, urine, and saliva. The composition of the reagent and the measurement items are shown in the table.

  By using an antibody against the substance to be measured, various substances can be measured by enzyme immunoassay. An example of a combination of a labeling enzyme and a substrate is shown in the table.

  FIG. 1 is a diagram showing an example of surface modification of the measurement electrode 207. The measurement electrode 207 is preferably a measurement electrode in which molecules having a hydrophilic group and molecules having an electrochemically active substance are bonded side by side on the surface.

  Examples of the electrochemically active substance to be bonded to the surface of the measurement electrode include 6-ferrocenyl-1-hexanethiol (CAS number 134029-92-8), 8-ferrocenyl-1-octanethiol (CAS number 146056-20-4). ), 11-ferrocenyl-1-undecanthiol (CAS No. 127087-36-9).

  Examples of molecules having a hydrophilic group to be bonded to the surface of the measurement electrode include 6-hydroxy-1-hexanethiol (CAS number 1633-78-9), 8-hydroxy-1-octanethiol (CAS number 33065-54). -2), 11-hydroxy-1-undecanethiol (CAS number 73768-94-2), 16-hydroxy-1-hexadecanethiol (CAS number 114896-32-1) 11-mercaptoundecanol triethylene glycol ether ( CAS number 130727-41-2), 11-mercaptoundecanol hexaethylene glycol ether (CAS number 130727-44-5), 16-mercaptohexadecanol triethylene glycol ether, 16-mercaptohexadecanol hexaethylene glycol ether There is.

  FIG. 1 (A) shows a case where only 11-ferrocenyl-1-undecanethiol (11-FUT) is arranged as a molecule having an electrochemically active substance as a comparative example, and FIG. 1 (B) shows 11-FUT and 11-hydroxy. When -1-undecanethiol (11-HUT) is arranged, FIG. 1 (C) shows the case where 11-FUT and 11-mercaptoundecanol triethylene glycol ether (11-MUEG3) are arranged, FIG. 1 (D). Shows the case where 11-FUT, 11-HUT, and 11-MUEG3 are arranged at 1: 8: 1.

  In FIG. 1, the gold electrode is modified with alkanethiol to form a measurement electrode in which molecules having hydrophilic groups and molecules having electrochemically active substances are bonded side by side on the surface. Chemical modification by ionic bond or covalent bond may be applied to noble metals such as. In addition to the above modification using alkanethiol, for example, a thiol compound having thiol benzene or the like at its end can be used, and a molecule modified with an electrochemically active substance and a molecule modified with a hydrophilic group are arranged side by side. What is necessary is just to be able to arrange. It is sufficient that the heights of the respective molecules are aligned when they are arranged in order to make the atmosphere in which the electrochemically active substance exists hydrophilic, and to replace the sample at the time of sample replacement.

  FIG. 4 is a measurement result showing an example of the effect of using a measurement electrode in which a molecule having a hydrophilic group and a molecule having an electrochemically active substance are bonded side by side on the surface. 11-ferrocenyl-1-undecanethiol (11-FUT) as an electrochemically active substance and 11-hydroxy-1-undecanethiol (11-HUT) as a molecule having a hydrophilic group were bonded side by side on the surface. A gold electrode with a diameter of 1.6 mm was used as a measurement electrode (hereinafter referred to as 11-HUT mixed electrode). Specifically, the gold electrode was immersed in an ethanol solution containing 500 μmol / L of 11-HUT and 11-FUT for 1 hour.

  As a comparative control, 11-ferrocenyl-1-undecanethiol (11-FUT), which is an electrochemically active substance, and 11-undecanethiol (11-UT) that does not contain a hydrophilic group are bound side by side on the surface. A 1.6 mm gold electrode was used as the measurement electrode (hereinafter referred to as 11-UT mixed electrode). Specifically, the gold electrode was immersed in an ethanol solution containing 500 μmol / L of 11-HUT and 11-FUT for 1 hour.

A measuring electrode, a reference electrode (silver silver chloride reference electrode), and a counter electrode (platinum electrode) are placed in a phosphate buffer containing 5 mmol / L potassium ferricyanide and 5 mmol / L potassium ferrocyanide. AC impedance was measured. The obtained AC impedance was approximated by an equivalent circuit shown in FIG. In FIG. 5, the resistance R _s represents the solution resistance, the resistance R _dl represents the resistance on the electrode surface, and the capacitance C _dl represents the capacitance on the electrode surface.

The electrical relaxation time τ is defined as τ = R _dl C _dl, and the ratio of 11-FUT to the total concentration of 11-FUT and 11-HUT or 11-UT to the total concentration of 11-FUT and 11-UT 4 is plotted in FIG. 4 with the ratio of 11-FUT, the horizontal axis of 11-FUT, and the vertical axis of electrical relaxation time. Black circles represent 11-HUT mixed electrodes and white circles represent 11-UT mixed electrodes.

  It has been shown that the 11-HUT mixed electrode having a hydrophilic group has a shorter electrical relaxation time than the 11-UT mixed electrode. And when 11-FUT and 11-HUT are coupled side by side (11-FUT ratio is greater than 0 and 1) than when 11-FUT alone is coupled to the surface (11-FUT ratio is 1). The smaller) the shorter the electrical relaxation time. In the 11-HUT mixed electrode, the relaxation time was minimized when the ratio of 11-FUT was about 0.2, and the electrical relaxation time increased as the ratio of 11-FUT decreased. In addition, the change in capacitance was almost constant compared to the change in resistance, and the change in electrical relaxation time was dominated by the change in resistance.

  Examining the details of FIG. 4 here, it can be seen that the electrical relaxation time decreases if the ratio of 11-FUT is in the range of 0.01 to 0.9. Furthermore, if the 11-FUT ratio is 0.5 or less, the electrical relaxation time can be 0.01 seconds or less.

  From the above results, the following can be considered. Compared to a gold electrode in which only 11-FUT is bonded to the surface, a gold electrode in which 11-HUT is also bonded to the surface makes the gold electrode surface hydrophilic by the action of hydroxyl groups. Therefore, hydrophilic ferricyanide and potassium ferrocyanide easily approach the gold electrode surface, and the redox reaction amount per unit time between potassium ferricyanide and potassium ferrocyanide and ferrocene group, that is, the redox reaction rate is improved. However, if the ratio of 11-FUT is further reduced, the surface density of the ferrocene group that reacts with potassium ferricyanide and potassium ferrocyanide decreases, so the redox reaction rate decreases.

  FIG. 9 schematically shows the arrangement of molecules at an electrode in which 11-ferrocenyl-1-undecanethiol (11-FUT) and 11-hydroxy-1-undecanethiol (11-HUT) are bonded side by side on the surface. It is shown in Black circles represent ferrocene and white circles represent hydroxyl groups. When these molecules are arranged in a honeycomb shape, the ratio of 11-FUT is about 0.33 as shown in (A), and the ferrocenes are not adjacent to each other and surrounded by hydroxyl groups. However, even in this case, if the density is uneven, ferrocenes may be adjacent to each other. As shown in (B), the ratio of 11-FUT is about 0.125, and two or more ferrocenes can be separated. Probability of not matching increases. In addition, when the molecules are arranged in a grid, the ratio of 11-FUT is about 0.5 as shown in (C), and ferrocenes are not adjacent to each other, and as shown in (D), two or more ferrocenes are separated from each other. it can. Note that the arrangement of alkanethiols such as 11-FUT on the electrode surface depends on the crystal plane of the electrode, so in what arrangement (whether honeycomb or grid) the electrode is produced without controlling the crystal plane. It will not be determined.

  FIG. 6 is a graph showing the relationship between the electrical relaxation time and the relaxation time actually measured by the measurement apparatus of FIG. Using the measurement device of FIG. 2, a phosphate buffer (1: 1 sample) containing 500 μmol / L potassium ferricyanide and 500 μmol / L potassium ferrocyanide is placed in the first sample placement section, and 900 μmol / L in the second sample placement section. A phosphate buffer solution (9: 1 sample) containing potassium L ferricyanide and 100 μmol / L potassium ferrocyanide was placed, and measurements were performed in order. Here, 11-HUT mixed electrodes or 11-UT mixed electrodes prepared at various ratios were used as measurement electrodes. First, the 1: 1 sample is held in the sample solution holding unit, and then the 9: 1 sample is sucked into the sample solution holding unit by driving by the driving unit such as rotating the sample transport unit 203. The potential difference of the reference electrode changes from the potential difference for the 1: 1 sample to the potential difference for the 9: 1 sample. The response time of the potential difference is defined as the time at which the change of 1 / e (the base of natural logarithm) can be obtained with respect to the total change. In FIG. 6, the vertical axis is the measured value of the response time of the potential difference, and the horizontal axis is the electrode Was plotted as the electrical relaxation time. The electrical relaxation time and the response time of the potential difference coincided in the time range longer than that with the electrical relaxation time of around 0.2 seconds, and the response time of the potential difference became almost constant in the shorter time range. this is,

Can be explained. That is, the response time of the potential difference is governed by the electrical relaxation time of the measurement electrode and the time required for the sample solution to be replaced in the sample solution holding portion, and the larger factor is dominant. In this measurement, the solution exchange time was about 0.2 seconds, and at this time, the electrical relaxation time became the dominant factor in the longer time zone, and the solution exchange time became the dominant factor in the shorter time zone. . The solution exchange time referred to here is the time from when the sample solution starts to be replaced from the 1: 1 sample to the 9: 1 sample at the measurement electrode until the replacement is completed.

  FIG. 7 is a measurement result showing an example of the effect of using a measurement electrode in which a molecule having a hydrophilic group and a molecule having an electrochemically active substance are bonded side by side on the surface. Using the measurement device of FIG. 2, a phosphate buffer (1: 1 sample) containing 500 μmol / L potassium ferricyanide and 500 μmol / L potassium ferrocyanide is placed in the first sample placement section, and 900 μmol / L in the second sample placement section. A phosphate buffer solution (9: 1 sample) containing potassium L ferricyanide and 100 μmol / L potassium ferrocyanide was placed, and measurements were performed in order.

  In FIG. 7, circles indicate the case where an electrode having only 11-FUT bonded to the surface is used as a measurement electrode, and triangles indicate that a molecule having a hydrophilic group and a molecule having an electrochemically active substance are present on the measurement electrode. , Shows the case of using electrodes connected side by side on the surface. In addition, each white is a clean case, and black is a case where the electrode is contaminated as described below.

  First, the potassium ferrocyanide concentration in the 9: 1 sample was measured repeatedly. When a gold electrode with only 11-FUT bonded to the surface was used as the measurement electrode, the measurement reproducibility (the standard deviation of the measurement value The value divided by the average value was 0.3%. Similarly, when a gold electrode in which 11-FUT and 11-HUT are bonded to the surface using an ethanol solution containing 50 μmol / L 11-FUT and 5 μmol / L 11-HUT is used as the measurement electrode, the measurement reproducibility is It was 0.3%.

  On the other hand, when each electrode was immersed in serum for 1 hour and the same measurement was performed, the reproducibility of the measurement with a gold electrode in which only 11-FUT was bound to the surface was 9.7%. The measurement reproducibility with a gold electrode combined with -FUT and 11-HUT was 0.3%, unchanged from that before immersion in serum.

  As a result of examining the cause, as shown in FIG. 7, a difference in response was observed when the sample solution was switched from the 1: 1 sample to the 9: 1 sample. On the horizontal axis, the solution replacement instant was taken as 0 seconds. The response time of each potential difference was 0.6 seconds before serum immersion and 1.8 seconds after serum immersion for the gold electrode with only 11-FUT bonded to the surface, compared with 11-FUT and 11-HUT. The bonded gold electrode was constant at 0.6 seconds before and after serum immersion.

  The electrical relaxation time was measured using the above three-electrode method. The gold electrode with only 11-FUT bonded to the surface showed 0.2 seconds before serum immersion and 1.8 seconds after serum immersion. On the other hand, the gold electrode combined with 11-FUT and 11-HUT was 0.2 milliseconds before serum immersion and 3 milliseconds after serum immersion.

  If the solution exchange time in this measurement is about 0.6 seconds, the response time of the potential difference can be explained by the electrical relaxation time. In addition, even when whole blood was used instead of serum, the response time of the potential difference was constant at 0.6 seconds before and after the whole blood immersion even with the gold electrode combined with 11-FUT and 11-HUT.

  FIG. 8 also shows another effect of using a measurement electrode in which a molecule having a hydrophilic group and a molecule having an electrochemically active substance are bonded side by side on the surface. FIG. 8 shows the results of evaluating the electrical relaxation time by impedance measurement immediately after the measurement electrode was prepared and after immersion in serum for 1 hour using the measurement electrodes (A) to (D) shown in FIG. First, as shown in Fig. 1 (A), a gold electrode in which only 11-FUTs are aligned and bonded has a large electrical relaxation time of 0.16 seconds before serum immersion and 1.6 seconds after serum immersion, and also has an effect on contamination. Was seen (FIG. 8 (a)).

  In addition, as shown in Fig. 1 (B), the gold electrode combined with 11-FUT and 11-HUT is 0.2 milliseconds before serum immersion and 3 milliseconds after serum immersion. As a result, the electrical relaxation time increased by about 15 times (FIG. 8B).

  Then, as shown in FIG. 1C, 11-FUT and 11-FUT are formed on the surface using an ethanol solution containing 50 μmol / L 11-FUT and 5 μmol / L 11-mercaptoundecanol triethylene glycol ether (11-MUEG3). When a gold electrode combined with MUEG3 is used as a measurement electrode, the electrical relaxation time is about 1.25 times due to the effect of electrode contamination due to immersion in serum, 4 milliseconds before serum immersion and 5 milliseconds after serum immersion. (FIG. 8 (c)).

  It is thought that the binding of serum contaminants to the electrode surface was suppressed by the ethylene glycol group binding to the electrode surface. That is, molecules having a hydrophilic group and molecules having an electrochemically active substance are bonded side by side on the surface, so that (1) the atmosphere surrounding the electrochemically active substance is made hydrophilic, thereby making the hydrophilicity in the sample solution Two effects are obtained: improving the reaction rate of the redox material with (2) suppressing the adsorption of contaminants by making the electrode surface hydrophilic.

  Based on this result, 11-FUT, 11-HUT, and 11-MUEG3 are 1: 8: 1 as shown in Fig. 1 (D) as an electrode having both features (1) and (2). When the surface modification of the gold electrode was performed with the mixed solution, the obtained electrode had a small initial electrical relaxation time (2.5 milliseconds) and a small increase in the electrical relaxation time after serum immersion (2.5 millisecond). Second to 3 milliseconds) (FIG. 8 (d)).

The effect of electrode contamination is essentially different between current measurement and potentiometry. In the current measurement method, when the electrode is contaminated, the electrode area A in the Cottrell equation is effectively reduced, or the diffusion coefficient D _{0 of the} redox material is effectively reduced. As a result, if the electrode is contaminated by repeated use, the current value decreases even if the same concentration of the measurement target substance is measured. On the other hand, in the potentiometric method, the Nernst equation does not include the electrode area and the diffusion coefficient of the redox material, so that the potential difference does not change theoretically even if the electrode is contaminated by repeated use. However, as can be seen from the experimental results, when the electrode is contaminated by repeated use, the electrical relaxation time increases, possibly due to the influence of the diffusion coefficient, etc. It can no longer be obtained. On the other hand, this also means that if the potential difference is measured after waiting for a time longer than the electrical relaxation time, it is less susceptible to electrode contamination. Therefore, shortening the initial electrical relaxation time or suppressing an increase in the electrical relaxation time due to repeated use is effective in the potentiometric measurement method. This is because, in the current measurement method, when the state of the electrode changes, the measurement is affected regardless of the initial state and its degree. In addition, as can be seen from the relationship between the response time of the potential difference, the electrical relaxation time, and the solution exchange time, the electrical relaxation time << solution exchange time is set to satisfy the electrical contamination caused by electrode contamination and individual differences. Variations and variations in the relaxation time can be prevented from affecting the waiting time.

  Another effect can be obtained by using a measurement electrode in which a molecule having a hydrophilic group and a molecule having an electrochemically active substance are bonded side by side on the surface.

  A gold electrode having a diameter of 1.6 mm in which 11-ferrocenyl-1-undecanthiol (11-FUT) and 11-hydroxy-1-undecanthiol (11-HUT) were bonded side by side on the surface was used as a measurement electrode. Phosphoric acid with a concentration ratio of potassium ferricyanide and potassium ferrocyanide of 1: 1 (1: 1 solution) or 9: 1 (9: 1 solution) as the sample solution, and total concentrations of 10 mmol / L, 1 mmol / L, and 100 μmol / L A buffer solution was used.

  When the 1: 1 and 9: 1 solutions with the same total concentration were measured alternately, 11-FUT and 11-HUT were bonded side by side as compared to the gold electrode with only 11-FUT bonded to the surface. With the gold electrode, the potential change at 100 μmol / L increased and approached the theoretical value (56.5 mV). This is because the gold electrode with 11-FUT and 11-HUT bonded side by side has improved the reaction rate between the ferrocene group on the electrode surface and potassium ferricyanide and potassium ferrocyanide in the solution, so that the sample solution with a lower concentration can be used. It is because it became possible to measure.

  Furthermore, when each electrode was immersed in serum for 1 hour to simulate electrode contamination by the specimen, the potential change at a concentration of 1 mmol / L or less was greatly reduced in the gold electrode in which only 11-FUT was bound to the surface. On the other hand (FIG. 12 (B)), the influence of the contamination on the potential change was hardly observed in the gold electrode in which 11-FUT and 11-HUT were combined side by side (FIG. 12 (A)).

  FIG. 10 shows an example of the result of measuring serum glucose using the apparatus of FIG. A sample mixed with a reagent containing potassium ferricyanide, potassium ferrocyanide, glucose kinase, glucose 6-phosphate dehydrogenase, diaphorase, adenosine triphosphate, and nicotinamide adenine dinucleotide, and reacted for 5 minutes was used as a sample solution. As a measurement electrode here, a 1.6 mm diameter gold electrode in which 11-ferrocenyl-1-undecanethiol (11-FUT) and 11-hydroxy-1-undecanethiol (11-HUT) are bonded side by side on the surface. Was used. Similar results were obtained when other measurement electrodes were used.

  FIG. 10 (A) shows the results of measurement using 25 to 200 mg / dL glucose aqueous solution on the specimen. When the glucose concentration of the sample was plotted on the horizontal axis as the set glucose concentration and the glucose concentration obtained from the obtained potential difference was plotted on the vertical axis as the measured glucose concentration, good linearity was obtained. Subsequently, when the measurement was repeated 25 times using serum as a specimen, the transition of the measured glucose concentration shown in FIG. 10 (B) was observed. The standard deviation was 1.0 mg / dL against the average value of 92.6 mg / dL.

  FIG. 11 shows an example of the result of measuring serum cholesterol using the apparatus of FIG. As a measurement electrode here, a 1.6 mm diameter gold electrode in which 11-ferrocenyl-1-undecanethiol (11-FUT) and 11-hydroxy-1-undecanethiol (11-HUT) are bonded side by side on the surface. Was used. Similar results were obtained when other electrodes were used. A sample was mixed with a reagent containing potassium ferricyanide, potassium ferrocyanide, cholesterol esterase, cholesterol dehydrogenase, diaphorase, and nicotinamide adenine dinucleotide and allowed to react for 5 minutes. FIG. 11A shows the result of measurement using serum with a known cholesterol concentration as a specimen. When the cholesterol concentration of the sample was plotted on the horizontal axis as the set cholesterol concentration and the cholesterol concentration obtained from the obtained potential difference was plotted on the vertical axis as the measured cholesterol concentration, good linearity was obtained. Subsequently, measurement was repeated 11 times using serum as a specimen. As a result, the average value was 202.4 mg / dL and the standard deviation was 2.2 mg / dL as shown in FIG.

201 driving unit 202 control unit 203 sample transport unit 204 sample placement unit 205 sample introduction unit 206 sample solution holding unit 207 measurement electrode 208 reference electrode 209 potentiometer 210 liquid feeding unit 211 waste liquid container 212 data processing unit 213 arithmetic unit 214 temporary storage unit 215 Nonvolatile memory device 216 Data display device

Claims (15)

A sample solution holder,
A measurement electrode that is in contact with the sample solution introduced into the sample solution holding unit and in which a molecule having a hydrophilic group and a molecule having an electrochemically active substance are bonded side by side on the surface;
A reference electrode in contact with the sample solution introduced into the sample solution holding unit;
A potentiometer comprising a potentiometer that measures a potential difference between the measurement electrode and the reference electrode.   The potentiometer according to claim 1, wherein the sample solution holding part is a flow path having a sample introduction part.   The potentiometric apparatus according to claim 1, wherein the hydrophilic group is an OH group.   The potentiometer according to claim 3, wherein the molecule having a hydrophilic group further has an ethylene glycol group.   The potentiometric apparatus according to claim 1, wherein the electrochemically active substance is a ferrocene derivative.   The potential difference according to claim 1, wherein a ratio of the electrochemically active substance to a total amount of the molecule having the hydrophilic group and the molecule having the electrochemically active substance is 0.01 or more and 0.9 or less. measuring device.   The potential difference according to claim 1, wherein a ratio of the electrochemically active substance to a total amount of the molecule having the hydrophilic group and the molecule having the electrochemically active substance is 0.01 or more and 0.5 or less. measuring device.   The potential difference according to claim 1, wherein a ratio of the electrochemically active substance to a total amount of the molecule having the hydrophilic group and the molecule having the electrochemically active substance is 0.01 or more and 0.34 or less. measuring device.   The potential difference according to claim 1, wherein a ratio of the electrochemically active substance to a total amount of the molecule having the hydrophilic group and the molecule having the electrochemically active substance is 0.01 or more and 0.125 or less. measuring device.   The potential difference according to claim 1, wherein a ratio of the electrochemically active substance to a total amount of the molecule having the hydrophilic group and the molecule having the electrochemically active substance is 0.01 or more and 0.1 or less. measuring device.   The potentiometer according to claim 1, wherein the measurement electrode is made of a noble metal.   The potentiometer according to claim 11, wherein the noble metal is made of gold, platinum, or silver.   The potentiometer according to claim 1, wherein the sample solution includes a whole blood sample, a serum sample, or a urine sample.   3. The potentiometric apparatus according to claim 2, further comprising a sample solution installing unit for installing a plurality of sample solutions, wherein one of the plurality of installed sample solutions is provided so as to touch the sample introducing unit. A potential difference measuring device.   The potential difference measuring apparatus according to claim 14, further comprising a drive unit that is driven so as to exchange a sample solution that touches the sample introduction unit.