JP6096608B2 - In vivo component concentration estimation method and in vivo component concentration estimation device - Google Patents

Description

The present invention relates to an in vivo component concentration estimation method and an in vivo component concentration estimation apparatus.

  Conventionally, various in-vivo component measuring methods or in-vivo component measuring apparatuses have been proposed that obtain analytical values relating to the amount of biological components contained in tissue fluid extracted through the skin of a subject. In addition, since the amount of extracted tissue fluid varies depending on the skin condition of the subject, the amount of biological component is corrected using ion information regarding the amount of inorganic ions contained in the tissue fluid in order to improve measurement accuracy. Has been proposed (see, for example, Patent Document 1).

  In the measuring method and measuring apparatus described in Patent Document 1, when measuring the amount of glucose in tissue fluid, the amount of glucose is analyzed using inorganic ions, specifically, the sodium ion concentration in the extracted tissue fluid as a correction parameter. ing.

JP 2010-169662 A

  However, in the measuring method and measuring apparatus described in Patent Document 1, depending on the component to be measured, the amount may not be accurately analyzed.

The present invention has been made in view of such circumstances, and provides an in vivo component concentration estimation method and an in vivo component concentration estimation apparatus capable of analyzing the amount of a measurement target component in tissue fluid with high accuracy. The purpose is that.

Physiological substance density estimating method according to the first aspect of the present invention (hereinafter, simply referred to as "density estimation method") is the biological extracted from the skin, the albumin in the set Oeki accumulated in the extraction medium a step of acquiring the concentration measurement value of the density measurements or protein TadashiSo amount,
Obtaining a concentration measurement value of a component to be measured in the accumulated tissue fluid; and
Based on the albumin concentration measurement value or the protein concentration measurement value, calculating the albumin skin permeability or the protein total skin permeability,
Based on the regression equation indicating the relationship between the skin permeability of the albumin skin permeability or the total amount of protein and the skin permeability of the component to be measured, the estimated skin permeability of the component to be measured is the skin of the albumin skin permeability or the total amount of protein. A step of calculating from the transmittance;
Based on said measured density values and the estimated skin permeation rate of the measurement target component, seen including a step of calculating a concentration estimated value of the measurement target component in the living body,
The component to be measured is a protein or a steroid hormone .

In the concentration estimation method of the present invention, the total amount of albumin or protein (total protein (TP)) is used as a correction parameter instead of conventional inorganic ions. The skin permeability of the total amount of albumin and protein shows a tendency to decrease with time. When a high-molecular substance such as protein having the same tendency is used as a measurement target component, the total amount of albumin or protein is corrected. By using it as a parameter, the estimated concentration value of the measurement target component can be obtained with high accuracy.

An in-vivo component concentration estimation device according to the second aspect of the present invention (hereinafter also simply referred to as “ concentration estimation device”) includes an introduction unit for introducing a sample containing tissue fluid extracted from the skin of a living body,
Contained in the sample introduced into the introduction part, the density measurement value of the measurement target component, and a detection unit for acquiring the density measurement value of the density measurements or protein TadashiSo amount of albumin,
Based on the albumin concentration measurement value or the protein concentration measurement value detected by the detection unit , albumin skin permeability or protein total skin permeability is calculated, and albumin skin permeability or protein total skin permeability and Based on the regression equation showing the relationship with the skin permeability of the measurement target component, the estimated skin permeability of the measurement target component is calculated from the albumin skin permeability or the skin permeability of the total amount of protein, and the concentration of the measurement target component Based on the measured value and the estimated skin permeability, an analysis unit that calculates a concentration estimated value of the measurement target component in vivo ,
The component to be measured is a protein or a steroid hormone .

According to the concentration estimation method and the concentration estimation apparatus of the present invention, it is possible to analyze the concentration estimation value of the measurement target component in the tissue fluid with high accuracy.

It is a flowchart which shows the measurement procedure of the C-reactive protein (CRP) density | concentration estimation method which concerns on the 1st Embodiment of this invention. It is a perspective view of the fine needle chip | tip with which the puncture tool used for the density | concentration estimation method of this invention is mounted | worn. It is perspective explanatory drawing of an example of the puncture tool used for the density | concentration estimation method of this invention. It is sectional explanatory drawing of the skin in which the micropore was formed with the puncture device. It is sectional explanatory drawing of one Embodiment of the density | concentration estimation apparatus of this invention. It is sectional explanatory drawing of the collection member used with the density | concentration estimation method which concerns on the 1st Embodiment of this invention. FIG. 7 is an explanatory cross-sectional view showing a state where the collecting member shown in FIG. 6 is affixed to the skin. It is a figure which shows the relationship between the skin permeability of albumin and the skin permeability of CRP. It is a figure which shows the relationship between the skin permeability | transmittance of a sodium ion, and the skin permeability | transmittance of CRP. It is a figure which shows the time-dependent change of the skin permeability of CRP. It is a figure which shows the time-dependent change of the skin permeability of albumin. It is a figure which shows a time-dependent change of the skin permeability | transmittance of a sodium ion. It is a graph which shows the relationship between the estimated CRP density | concentration by albumin correction | amendment, and a reference value. It is a graph which shows the relationship between the estimated CRP density | concentration by sodium ion correction | amendment, and a reference value. It is a flowchart which shows the measurement procedure of the CRP density | concentration estimation method which concerns on the 2nd Embodiment of this invention. It is a cross-sectional explanatory view of a tissue fluid extraction chamber used in the concentration estimation method according to the second embodiment of the present invention. It is explanatory drawing of the measurement procedure of the density | concentration estimation method which concerns on the 2nd Embodiment of this invention. It is a figure which shows the relationship between the skin permeability of albumin and the skin permeability of cortisol. It is a figure which shows the relationship between the skin permeability | transmittance of a sodium ion, and the skin permeability | transmittance of cortisol. It is a figure which shows the time-dependent change of the skin permeability of cortisol. It is a figure which shows the time-dependent change of the skin permeability of C-peptide. It is a figure which shows the time-dependent change of the skin permeability of DHEA-s. It is a graph which shows a time-dependent change of the skin permeability of total protein (TP). It is a figure which shows the image of the electrophoresis of the protein in an extraction sample. It is a figure which shows the time-dependent change of the skin transmittance | permeability of total protein (TP).

Hereinafter, embodiments of a concentration estimation method and a concentration estimation apparatus according to the present invention will be described in detail with reference to the accompanying drawings. In the following embodiments, the present invention is applied to the case of estimating the concentration of C-reactive protein (CRP) (first or second embodiment) or the case of estimating the concentration of cortisol (third embodiment). An example will be described.

[First embodiment]
A CRP concentration estimation method according to the first embodiment of the present invention will be described. FIG. 1 is a flowchart showing a measurement procedure of the CRP concentration estimation method according to the first embodiment of the present invention.

First, the outline of the concentration estimation procedure of the CRP concentration estimation method according to the first embodiment of the present invention will be described with reference to FIG. Of the steps shown in FIG. 1, steps S1 to S6 are steps performed by a person who performs concentration estimation , and step S7 is performed by the concentration estimation apparatus according to the present embodiment. It is a process.

  First, washing of the measurement site of the subject and formation of micropores at the measurement site using a puncture device described later are performed (step S1). The formation of the micropores is a process performed to promote extraction of tissue fluid from the skin of the subject. Next, the extraction time of the tissue fluid is set using a timer unit provided in the measuring device (step S2). Next, a collection member to be described later is attached to the measurement site, and extraction of tissue fluid and accumulation of CRP and albumin in the tissue fluid are started (step S3). Next, it is determined whether or not the end of the extraction time set in step S2 is notified by the alarm device of the timer unit (step S4). If notified, the collection member is removed and the extraction of the tissue fluid is ended. (Step S5). Next, the analyte accumulated in the collecting member is collected in pure water in a dedicated container (step S6). Finally, the measurement target component is measured using pure water in the dedicated container (step S7).

Hereinafter, each step will be described in detail.
(Step S1: Pretreatment process)
First, the subject cleans the application site of the skin S using cotton or the like impregnated with ethanol, and removes substances (sweat, dust, etc.) that cause disturbance in the measurement results. Then, micropores are formed in the cleaned skin S.

  The micropores can be formed using, for example, a puncture tool 2 (see FIG. 3) equipped with the microneedle chip 1 shown in FIG. The puncture device 2 shown in FIG. 3 is equipped with a microneedle chip 1 that has been sterilized, and the microneedle 1a of the microneedle chip 1 is brought into contact with the epidermis of the living body (subject's skin S). It is an apparatus for forming a tissue fluid extraction hole (micropore 3 in FIG. 4) in the skin S of a subject. The microneedle 1a of the microneedle chip 1 has a size and shape so that when the micropore 3 is formed by the puncture device 2, the micropore 3 stays in the epidermis of the skin S and does not reach the dermis. The puncture device 2 includes a housing 4, a release button 5 provided on the surface of the housing 4, and an array chuck 6 and a spring member 7 provided inside the housing 4. An opening (not shown) is formed in the lower portion 4 a of the housing 4. The spring member 7 has a function of urging the array chuck 6 in the puncturing direction. The array chuck 6 can be equipped with the fine needle chip 1 at the lower end. A plurality of fine needles 1 a are formed on the lower surface of the fine needle chip 1. The puncture device 2 has a fixing mechanism (not shown) that fixes the array chuck 6 in a state where the array chuck 6 is pushed upward (anti-puncture direction) against the urging force of the spring member 7. ) Is released, the array chuck 6 is released from being fixed by the fixing mechanism, and the array chuck 6 is moved in the puncture direction by the urging force of the spring member 7.

  As a puncturing procedure using such a puncture device 2, first, the puncture device 2 is prepared in a state where the array chuck 6 is pushed upward (anti-puncture direction) against the urging force of the spring member 7. With the opening (not shown) of the lower part 4a of the puncture device 2 disposed at a predetermined site of the skin S to be formed, the release button 5 is pressed. Thereby, the fixation of the array chuck 6 by the fixing mechanism described above is released, and the array chuck 6 moves to the skin S side by the biasing force of the spring member 7. Then, the fine needle 1a of the fine needle chip 1 attached to the lower end of the array chuck 6 comes into contact with the skin S of the subject at a predetermined speed. Thereby, as shown in FIG. 4, the micropore 3 is formed in the epidermis portion of the skin S of the subject.

  After the release button 5 is pressed, the state in which the fine needle 1a of the fine needle chip 1 is punctured into the skin S of the subject is held for a predetermined time (for example, 3 minutes). Thereby, the quantity of the tissue fluid extracted from a micropore can be increased.

(Step S2: Timer setting process)
Next, a test subject sets the time (extraction time) of the timer part 50 of the said measuring apparatus 40 by operating the operation button (not shown) provided in the measuring apparatus 40 shown by FIG. The set time can be, for example, 5 to 120 minutes.

(Steps S3 to S5: Extraction-accumulation step)
Next, tissue fluid is extracted and stored using the collection member 30 shown in FIG. The collection member 30 shown in FIG. 6 has a structure in which a support member 32 supports a gel 31 having a water retention property capable of holding tissue fluid extracted from the skin S of a subject. This gel 31 contains pure water as an extraction medium.

  The support member 32 includes a support portion main body 32a having a recess and a flange portion 32b formed on the outer peripheral side of the support portion main body 32a, and the gel 31 is held in the recess of the support portion main body 32a. An adhesive layer 33 is provided on the surface of the collar portion 32b, and a release paper 34 that seals the gel 31 held in the recess is stuck to the adhesive layer 33 before the measurement. When performing the measurement, the release paper 34 is peeled from the adhesive layer 33 to expose the gel 31 and the adhesive layer 33, and the collecting member 30 is attached to the subject's skin S by the adhesive layer 33 in a state where the gel 31 is in contact with the subject's skin S. It can be stuck and fixed on the skin S of the subject.

  In this extraction-accumulation step, the subject removes the release paper 34 (see FIG. 6) of the collection member 30, and then affixes the collection member 30 to the site where the micropores 3 are formed as shown in FIG. Step S3). Thereby, the site | part in which the micropore 3 was formed, and the gel 31 contact, and the tissue fluid containing CRP and albumin begins to move to the gel 31 via the micropore 3, and extraction is started. The subject turns on the timer unit 50 of the measuring device 40 simultaneously with the start of extraction. Thereafter, the collecting member 30 is left in a state of being stuck on the skin S until a predetermined time (alarm setting time) elapses (step S4). Then, the subject removes the collecting member 30 from the skin S when a predetermined time elapses and an alarm sounds (step S5). Here, since the alarm of the timer unit is set to 5 to 120 minutes, the tissue fluid is continuously extracted from the skin over a period of 5 to 120 minutes. This completes the extraction-accumulation process.

(Step S6: Extract recovery process)
Subsequently, the extract accumulated in the gel 31 is recovered. In this collection, the collection member 30 removed from the skin S of the subject is kept in the collection liquid L for a predetermined time (for example, 3 to 3) in the collection tube 34 (see FIG. 5) containing the collection liquid L made of pure water. 48 hours).

(Step S7: Measurement process)
After the recovery of the analyte is completed, the recovery liquid L in the recovery tube 34 is moved to the measurement unit 42 via the introduction unit 41 of the measurement device 40 using the syringe 35 as shown in FIG. The measurement unit 42 serving as a detection unit is provided with a CRP concentration measurement sensor 43 and an albumin concentration measurement sensor 44. The electrical control unit 45 and the analysis unit 46 measure the CRP concentration and the albumin concentration. The obtained result is output on the display unit 47. Each of the CRP concentration measurement sensor 43 and the albumin concentration measurement sensor 44 is an immunosensor using an antigen-antibody reaction, and optically changes a refractive index due to binding of a target molecule to an antibody formed on a substrate. (SPR method).

  The analysis unit 46 calculates the estimated biological concentration of CRP (the concentration of CRP in tissue fluid or the concentration of CRP in blood) using the obtained measured values of albumin concentration and CRP concentration. Hereinafter, this calculation method will be described in detail.

<Acquisition of correction formula using albumin>
It is known that the following equation (1) holds for the relationship between the extraction rate (J) of a substance and the membrane permeability (P) when the substance permeates the semipermeable membrane. This relationship is obtained when the measurement target component or albumin in the tissue fluid extracted from the living body is a “certain substance” and the skin of the subject from which the measurement target component is extracted is a “semipermeable membrane”. It can be applied to measurement or analysis.
J = A × P × C (1)
Here, A is the area of the permeable part, and C is the concentration of the biological component in the tissue fluid.

When the equation (1) is transformed, the following equation (2) is obtained, and it can be seen that the skin permeability of the biological component can be obtained from the extraction speed J, the permeation area A, and the concentration of the biological component in the tissue fluid.
P = J / (A × C) (2)
The extraction speed J is obtained by dividing the extracted biological component amount, which is the product of the concentration of the extracted biological component and the amount of the extraction medium, by the extraction time.

  Now, the CRP, albumin and sodium ion concentrations corresponding to the above-mentioned concentration C obtained by serum concentration analysis for a plurality of subjects, and the plurality of subjects were collected in steps S1 to S6 described above. CRP concentration measured using tissue fluid (measured using High-Sensitivity CRP ELISA Kit manufactured by Cyclex Co., Ltd.) and albumin concentration (measured using Human Album ELISA Kit manufactured by Takara Bio Inc.) and ion chromatography Based on the sodium ion concentration obtained from the graph, each skin permeability of CRP, albumin and sodium ion was calculated according to the formula (2).

Regarding the calculated skin permeability, FIG. 8 shows the relationship between albumin skin permeability and CRP skin permeability, and FIG. 9 shows the relation between sodium ion skin permeability and CRP skin permeability. 8 and 9, it can be seen that the skin permeability of albumin has a higher correlation with the skin permeability of CRP than the skin permeability of sodium ions.
When a regression line between the skin permeability of albumin and the skin permeability of CRP is obtained, y = 0.70x + 0.0006, and the correlation coefficient R is 0.78.
On the other hand, when a regression line between the skin permeability of sodium ions and the skin permeability of CRP is obtained, y = 0.032x + 0.0011, and the correlation coefficient R is 0.02.

  As a result of intensive studies on the cause of the large difference in the correlation coefficient between albumin and sodium ion, the present inventors have found that albumin and sodium ion have different skin permeability changes over time, and albumin skin. It has been found that the change in transmittance over time tends to be closer to the change in skin permeability of CRP over time than that of sodium ions.

  FIGS. 10-12 is a figure which shows the time-dependent change of the skin permeability | transmittance of CRP, albumin, and sodium ion, respectively. 10 to 12, the loop number on the x-axis indicates the elapsed time in 15-minute units after puncturing. That is, “1” on the x-axis indicates 15 minutes after puncturing, “2” indicates 30 minutes after puncturing, and “3” indicates 45 minutes after puncturing.

10-12, the skin permeability of sodium ions is almost constant regardless of the elapsed time after the puncture, whereas the skin permeability of CRP gradually decreases with the passage of time after the puncture. It can be seen that the change over time is greatly different. On the other hand, the change with time of the skin permeability of albumin shows the same tendency as that of CRP, and it can be seen that it gradually attenuates over time after puncture. Thus, since the time-dependent change of skin permeability shows the same tendency, it is understood that albumin has a higher correlation of skin permeability with CRP than sodium ion. Therefore, it can be seen that the skin permeability P (CRP) of CRP can be estimated from the skin permeability P (Albumin) of albumin using the following equation (3).
P (CRP) = 0.70 × P (Albumin) +0.0006 (3)

<Estimation of P (CRP)>
The estimated skin permeability pdcP (CRP) of CRP is calculated from the skin permeability P (Albumin) of albumin using the above formula (3).
P (Albumin) can be calculated according to the above formula (2), and the concentration C (Albumin) of albumin in the tissue fluid required for the calculation is not greatly different depending on the subject, and is a common concentration. Is given as a constant. Specifically, C (Albumin) can be set to, for example, 1.125 g / dL (assuming that the tissue fluid concentration is 1/4 of 4.5 g / dL, which is the reference value for blood concentration).

<Calculation of C (CRP)>
The CRP concentration C (CRP) in the tissue fluid is calculated from the estimated skin permeability pdcP (CRP) of CRP calculated by the expression (3) and the expression (4) obtained by modifying the expression (1). be able to. The CRP concentration in the blood is calculated by multiplying the CRP concentration C (CRP) in the tissue fluid by 4 times, assuming that the CRP concentration in the tissue fluid is 1/4 of the CRP concentration in the blood. Can do.
C (CRP) = J / (A × pdcP (CRP)) (4)

  FIG. 13 is a diagram showing a correlation between the CRP concentration in blood estimated from the CRP concentration in tissue fluid calculated by the above-described procedure and the CRP concentration in serum (serum) obtained as a reference value. FIG. 14 is a diagram showing the correlation between the CRP concentration in tissue fluid calculated using sodium ions as a correction parameter instead of albumin and the CRP concentration in serum obtained as a reference value. In the experiment shown in FIG. 14, the skin permeability of CRP was estimated from the skin permeability of sodium ions using the above-described regression line (y = 0.032x + 0.0011).

  From FIG. 13 and FIG. 14, the correlation coefficient R of the CRP concentration obtained by the albumin correction is larger than the correlation coefficient R of the CRP concentration obtained by the sodium ion correction, and the albumin correction is more measured than the sodium ion correction. It can be seen that the accuracy increases.

[Second Embodiment]
Next, a CRP concentration estimation method according to the second embodiment of the present invention will be described. FIG. 15 is a flowchart showing a concentration estimation procedure of the CRP concentration estimation method according to the second embodiment of the present invention.

First, an outline of the concentration estimation procedure of the CRP concentration estimation method according to the second embodiment of the present invention will be described with reference to FIG. In addition, each process of step S10 to S19 shown in FIG. 15 is a process performed by the person who performs concentration estimation , and the process (measurement process) of step S19 is a process performed using a concentration measurement kit. is there. In this embodiment, unlike the first embodiment concentration estimation process is performed by the density estimating apparatus having a measuring unit for density measurement sensor is disposed, using a commercial density measurement kit described later concentration An estimation process is performed.

  First, washing of the measurement site of the subject, formation of micropores in the measurement site using a puncture tool described later, and fixation of the tissue fluid extraction chamber to the measurement site are performed (step S10). Next, a tissue fluid extraction time is set using an appropriate timer (not shown) (step S11). Next, the tissue fluid extraction medium is supplied into the chamber fixed to the measurement site (step S12). Next, extraction of tissue fluid and accumulation of CRP, albumin, etc. in the tissue fluid are started (step S13). Next, it is determined whether or not the end of the extraction time set in step S12 has been notified by the timer alarm device (step S14). If notified, the tissue fluid extraction medium is collected in a predetermined container. (Step S15). Next, the tissue fluid extraction chamber is washed (step S16). Next, it is determined whether or not the extraction of the tissue fluid has been completed a predetermined number of times (step S17). If it is determined that the extraction of the tissue fluid has been completed a predetermined number of times, the tissue fluid extraction and accumulation process is completed (step S18). When it is determined that the extraction of the tissue fluid has not been completed a predetermined number of times, the process returns to step S11, the tissue fluid extraction medium is supplied into the chamber, and the steps S12 to S17 are repeated. Finally, the tissue fluid is measured (step S19).

Hereinafter, each step will be described in detail. Note that the description common to the first embodiment is omitted for the sake of simplicity.
(Step S10: Pretreatment process)
First, the subject cleans the application site of the skin S using cotton or the like impregnated with ethanol, and removes substances (sweat, dust, etc.) that cause disturbance in the measurement results. Then, micropores are formed in the cleaned skin S. The formation of the micropores can be performed using the puncture device 2 in the first embodiment.

  Next, the tissue fluid extraction chamber 10 shown in FIG. 16 is fixed to the micropore formation site of the skin S of the subject. A tissue fluid extraction chamber 10 capable of holding tissue fluid is composed of a short cylindrical support member 11 having openings in the upper and lower sides, and an adhesive layer 12 is formed on one end surface of the support member 11. Before use, a release paper 13 is affixed to the adhesive layer 12. At the time of use, the release paper 13 is peeled off, and the adhesive layer 12 of the tissue fluid extraction chamber 10 is pressed against the skin S of the subject so that the micropore forming portion is disposed in the opening of the support member 11. The chamber 10 is fixed to the skin S of the subject.

(Step S11: Timer setting process)
Next, the subject sets the extraction time using a timer. The set time can be set to 15 minutes, for example.

(Step S12: Supply process of tissue fluid extraction medium)
Next, the tissue fluid extraction chamber 10 is cleaned using 200 μL of tissue fluid extraction medium (purified water manufactured by Wako Pure Chemical Industries, Ltd.). After washing, as shown in FIG. 17, a predetermined amount (100 μL) of the tissue fluid extraction medium 14 is supplied into the tissue fluid extraction chamber 10 by a pipette (not shown), and the tissue fluid is extracted to prevent evaporation of the supplied tissue fluid extraction medium 14. The upper opening of the extraction chamber 10 is sealed with a mending tape 15. Thereby, the site | part in which the micropore 3 was formed, and the tissue fluid extraction medium 14 contact, and the tissue fluid containing CRP and albumin begins to move into the tissue fluid extraction medium 14 via the micropore 3, and extraction is started. Also, the subject turns on the alarm device of the timer simultaneously with the start of extraction.

(Steps S13 to S14: Extraction-accumulation step)
Thereafter, the tissue fluid extraction chamber 10 is left on the skin S until a predetermined time (alarm setting time) elapses (steps S13 and S14).

(Step S15: Collection of tissue fluid extraction medium)
When the alarm sounds after a predetermined time has elapsed, the subject removes the mending tape 15 and pipettes the liquid in the tissue fluid extraction chamber 10 (the tissue fluid extraction medium 14 from which the tissue fluid has been extracted) into a predetermined container. to recover.

(Step S16: Washing of tissue fluid extraction chamber)
Next, in step S16, using a predetermined amount (for example, 100 μL) of the tissue fluid extraction medium 14, the discharge / suction by the pipette is repeated 2 to 3 times in the tissue fluid extraction chamber 10 to thereby store the tissue fluid extraction chamber 10 in the tissue fluid extraction chamber 10. Wash.
Next, in step S17, it is determined whether or not the extraction of the predetermined number of tissue fluids has been completed. If it is determined that the extraction of the predetermined number of tissue fluids has been completed, the tissue fluid extraction and accumulation process ends (step S18). When it is determined that the extraction of the tissue fluid has not been completed a predetermined number of times, the process returns to step S11, the alarm is set again, and then the tissue fluid extraction medium is supplied into the chamber (step S12). S13 to step S16 are repeated.

(Step S19: measurement process)
Subsequently, the CRP concentration and albumin concentration of the recovered extraction medium are measured. The concentration of CRP can be measured using a commercially available measurement kit, for example, High-Sensitivity CRP ELISA Kit manufactured by Cyclex Corporation. The concentration of albumin can also be measured using a commercially available measurement kit, for example, Human Albumin ELISA Kit manufactured by Takara Bio Inc.

  Using the obtained measured values of the albumin concentration and the CRP concentration, the estimated biological concentration of CRP is calculated by the same method as in the first embodiment.

[Third Embodiment]
In the present embodiment, unlike the first or second embodiment in which CRP, which is a type of protein, is a measurement target component, cortisol, which is a type of steroid hormone, is used as a measurement target component. As an index (correction index) used for estimating cortisol skin permeability, albumin is adopted as in the first or second embodiment.

  Each measurement value of albumin concentration and cortisol is obtained by a measurement procedure similar to the measurement procedure flow (step S1 to step S7) shown in FIG. However, in the measurement apparatus used in the present embodiment, instead of the CRP concentration measurement sensor 43 (see FIG. 5), a cortisol concentration measurement sensor capable of measuring the concentration of cortisol is disposed in the measurement unit 42. ing. Then, the estimated biological concentration of cortisol is calculated using the obtained measured values of albumin concentration and cortisol concentration.

<Acquisition of correction formula using albumin>
With respect to a plurality of subjects, each concentration of cortisol, albumin, and sodium ion corresponding to the concentration C in the above formulas (1) to (2) obtained by serum concentration analysis, and the subject with respect to the first embodiment Cortisol concentration (ELISA Kit manufactured by Salimetrics) and albumin concentration (measured using Human Album ELISA Kit manufactured by Takara Bio Inc.) and ions measured using the tissue fluid collected in the same steps S1 to S6 Based on the concentration of sodium ions determined by chromatography, the skin permeability of cortisol, albumin and sodium ions was calculated according to the above formula (2).

FIG. 18 shows the relationship between the skin permeability of albumin and the skin permeability of cortisol, and FIG. 19 shows the relation between the skin permeability of sodium ion and the skin permeability of cortisol. FIG. 18 and FIG. 19 show that the skin permeability of albumin has a higher correlation with the skin permeability of cortisol than the skin permeability of sodium ions.
When a regression line between the skin permeability of albumin and the skin permeability of cortisol is obtained, y = 0.2493x + 0.0023, and the correlation coefficient R is 0.5.
On the other hand, when a regression line between the skin permeability of sodium ions and the skin permeability of cortisol is obtained, y = 0.0344x + 0.0022, and the correlation coefficient R is 0.1.

  The reason for the large difference in the correlation coefficient between albumin and sodium ion is that the change in skin permeability of albumin with time is more time-dependent with cortisol than with sodium ions. This is because it tends to be close to.

  FIG. 20 is a diagram showing the change with time of the skin permeability of cortisol. In FIG. 20, the loop number on the x-axis indicates the elapsed time in units of 15 minutes after puncturing, as in FIGS.

From FIG. 12, the skin permeability of sodium ions is almost constant regardless of the elapsed time after the puncture, whereas the skin permeability of cortisol shown in FIG. 20 is gradually increased with the passage of time after the puncture. It is attenuated, and it can be seen that the change over time of both is greatly different. On the other hand, as shown in FIG. 11, the time-dependent change in the skin permeability of albumin shows the same tendency as that of cortisol, and it can be seen that it gradually attenuates over time after puncture. Thus, since the time-dependent change of skin permeability shows the same tendency, it is understood that albumin has a higher correlation with cortisol than sodium ions. Therefore, it can be seen that the skin permeability P (cortisol) of cortisol can be estimated from the skin permeability P (Albumin) of albumin using the following equation (5).
P (cortisol) = 0.2493 × P (Albumin) +0.0023 (5)

<Estimation of P (cortisol)>
When albumin is used as a correction index, the estimated skin permeability pdcP (cortisol) of cortisol is calculated from the skin permeability P (Albumin) of albumin using the above equation (5).
P (Albumin) can be calculated according to the above equation (2) in the same manner as in the first embodiment, and the concentration C (Albumin) of albumin in the tissue fluid required for the calculation is large depending on the subject. There is no difference, and it is given as a constant (for example, 1.125 g / dL) on the assumption that the concentration is common.

<Calculation of C (cortisol)>
The cortisol concentration C (cortisol) can be calculated from the estimated skin permeability pdcP (cortisol) of cortisol calculated by the expression (5) and the expression (6) obtained by modifying the above expression (1). .
C (cortisol) = J / (A × pdcP (cortisol)) (6)

In the above-described embodiment, CRP (first and second embodiments) or cortisol (third embodiment) in tissue fluid is used as a concentration estimation target component. However, the concentration estimation method of the present invention uses these components. However, other biological components can also be used as the concentration estimation target. The biological components in tissue fluid that can be subjected to concentration estimation by the concentration estimation method of the present invention can be broadly classified into two types: high molecular substances and low molecular substances bonded to high molecular substances. it can.

  Examples of the former polymer substance include, for example, proteins such as CRP, insulin, and C-peptide; nucleic acids that are DNA or RNA; polysaccharides such as glycogen.

  For example, as shown in FIG. 21, the time-dependent change in the skin permeability of C-peptide is gradually attenuated with the passage of time after puncture as in the above-mentioned CRP and cortisol, and has the same tendency as albumin. Show. Therefore, it can be seen that the skin permeability of C-peptide can also be estimated using the skin permeability of albumin.

  Examples of the low molecular weight substance bonded to the latter high molecular weight substance include, for example, cortisol, steroid hormones such as DHEA-s and testosterone; lipoproteins in which apoproteins such as ApoB48 and ApoAI are bound to low molecular weight lipids. Can be mentioned. Further, antibiotics such as vancomycin and low molecular weight drugs such as warfarin can be exemplified.

  For example, the change with time in the skin permeability of DHEA-s, which is a kind of steroid hormone, is gradually attenuated with the passage of time after puncture, as in the case of CRP and cortisol described above, as shown in FIG. It shows the same tendency as albumin. Therefore, it can be seen that the skin permeability of DHEA-s can also be estimated using the skin permeability of albumin.

As described above, in the concentration estimation method of the present invention, a polymer substance or a substance obtained by binding a low molecular substance to the polymer substance (hereinafter collectively referred to as “polymer substance”) is used as a suitable concentration estimation target substance. However, the reason will be described below.
It was confirmed by experiments that the extraction rate of the protein as an example of the polymer substance was decreased with time. The protein extraction rate in the following three time zones (a to c) (15 minutes each) was calculated. In FIG. 23, “d” is data of a non-puncture site. Further, as described above, the extraction rate (J) is obtained by dividing the amount of protein, which is the product of the extracted protein concentration and the amount of extraction medium, by the extraction time.
a: Immediately after puncture to 15 minutes b: 15 minutes to 30 minutes c: 30 minutes to 45 minutes The number of measurement sites is 3 for both puncture and non-puncture, and after measuring the total amount of protein extracted for each site, Samples extracted from the three sites were mixed and subjected to electrophoresis. FIG. 23 shows the relationship between the lapse of time after puncture and the protein extraction rate, and FIG. 24 shows an image obtained by electrophoresis.

  FIG. 23 shows that the amount of protein extracted into the tissue fluid decreases with time. On the other hand, in glucose, for example, there is almost no decrease with time. Therefore, as a correction parameter, a component to be measured using albumin whose extraction amount decreases with time, such as a high molecular weight substance such as protein, the amount that is extracted into the tissue fluid decreases with time. It turns out that it is suitable.

  Since the molecular weight in the range of 5.6 to 102 kDa can be confirmed from the electrophoresis image of FIG. 24, the molecular weight in the range of 5.6 to 102 kDa is used as the protein as the measurement target component of the present invention. From the viewpoint that the degree of reduction is relatively large, it can preferably have a molecular weight in the range of 8.9 to 94 kDa.

[Other variations]
The embodiments and experimental examples disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is shown not by the above description of the embodiments and experimental examples, but by the scope of claims, and further includes all modifications within the meaning and scope equivalent to the scope of claims.

  For example, in the above-described embodiment, albumin is used as a correction index used when estimating the skin permeability of a measurement target component such as CRP, but the total amount of protein (total protein (TP)) may be used. it can. The skin permeability of this total protein (TP) also shows the same tendency as albumin, as shown in FIG. That is, the skin permeability of total protein (TP) is gradually attenuated with the passage of time after puncture, as is the skin permeability of albumin. Total protein (TP) is the total amount of many kinds of proteins measured by the biuret method, and albumin and γ globulin account for the majority. Since the total protein (TP) has a small change in the concentration in the living body compared to other in vivo proteins (reference value: 6.7 to 8.3 g / dL), like albumin, as a correction index in the present invention It can be used.

  In the embodiment described above, as a process for promoting the extraction of tissue fluid from the living body, fine needles are formed on the subject's skin using a puncture device. However, the extraction of tissue fluid is promoted by other methods. You can also. For example, the extraction of tissue fluid may be promoted by so-called peeling that removes keratin of the skin, and further extraction of tissue fluid by iontophoresis, electroporation, laser poration, thermal poration, sonophoresis, chemical enhancer, etc. May be promoted. Examples of chemical enhancers include alcohols, terpenes, surfactants, and the like.

  In the first embodiment described above, the CRP concentration and the albumin concentration are measured using a sensor that optically detects the refractive index change caused by the antigen-antibody reaction. By irradiating, the photoelectric conversion dye labeled on the antibody can be photoexcited and measured using another sensor such as a photoelectrochemical sensor that measures the generated current.

DESCRIPTION OF SYMBOLS 1 Fine needle tip 1a Fine needle 2 Puncture tool 3 Micro hole 4 Case 4a Lower part 5 Release button 6 Array chuck 7 Spring member 10 Tissue fluid extraction chamber 30 Collection member 31 Gel 40 Measuring device 41 Introduction part 42 Measurement part 43 CRP concentration measurement Sensor 44 Albumin concentration measurement sensor 45 Electric control unit 46 Analysis unit

Claims (17)

Obtaining a concentration measurement value of albumin concentration or a protein concentration measurement in tissue fluid extracted from the skin of a living body and accumulated in an extraction medium;
Obtaining a concentration measurement value of a component to be measured in the accumulated tissue fluid; and
Based on the albumin concentration measurement value or the protein concentration measurement value, calculating the albumin skin permeability or the protein total skin permeability,
Based on the regression equation indicating the relationship between the skin permeability of the albumin skin permeability or the total amount of protein and the skin permeability of the component to be measured, the estimated skin permeability of the component to be measured is the skin of the albumin skin permeability or the total amount of protein. A step of calculating from the transmittance;
Calculating a concentration estimation value of the measurement target component in vivo based on the concentration measurement value of the measurement target component and the estimated skin permeability, and
The in vivo component density | concentration estimation method whose said measurement object component is protein or a steroid hormone.   The in vivo component concentration estimation method according to claim 1, wherein the protein has a molecular weight of 5.6 to 102 kDa.   The in vivo component concentration estimation method according to claim 1, wherein the protein has a molecular weight of 8.9 to 94 kDa.   The in vivo component concentration estimation method according to any one of claims 1 to 3, wherein the protein is C-reactive protein, insulin, or C-peptide.   The in vivo component concentration estimation method according to any one of claims 1 to 4, wherein the steroid hormone is cortisol or DHEA-s. The in vivo component concentration estimation method according to any one of claims 1 to 5, wherein the estimated concentration value of the measurement target component in the living body is an estimated value of the blood concentration of the measurement target component.   The in vivo component concentration estimation method according to any one of claims 1 to 6, wherein the extraction time of the tissue fluid is 5 to 120 minutes.   The in vivo component concentration estimation method according to any one of claims 1 to 7, wherein tissue fluid from the skin of a living body that has been subjected to an extraction promoting process that promotes extraction of tissue fluid is accumulated in the extraction medium.   The in vivo component concentration estimation method according to claim 8, wherein tissue fluid from the skin of a living body that has undergone micropore formation as an extraction promoting process is accumulated in the extraction medium.   The in vivo component concentration estimation method according to any one of claims 1 to 9, wherein the albumin skin permeability is calculated from an albumin concentration measurement value, an extraction medium amount, an extraction time, and a permeation portion area.   The in vivo component concentration estimation according to any one of claims 1 to 10, wherein the skin permeability of the total protein amount is calculated from a concentration measurement value of the total protein amount, an extraction medium amount, an extraction time, and a permeation part area. Method. The concentration estimation value of the measurement target component in the living body is calculated from the concentration measurement value of the measurement target component, the extraction medium amount, the extraction time, the permeation area, and the estimated skin permeability of the measurement target component. The in-vivo component density | concentration estimation method as described in any one of -11. An introduction part for introducing a sample containing tissue fluid extracted from the skin of a living body;
A detection unit for obtaining a concentration measurement value of a measurement target component and a concentration measurement value of albumin or a total protein concentration contained in a sample introduced into the introduction unit;
Based on the albumin concentration measurement value or the protein concentration measurement value detected by the detection unit, albumin skin permeability or protein total skin permeability is calculated, and albumin skin permeability or protein total skin permeability and Based on the regression equation showing the relationship with the skin permeability of the measurement target component, the estimated skin permeability of the measurement target component is calculated from the albumin skin permeability or the skin permeability of the total amount of protein, and the concentration of the measurement target component Based on the measured value and the estimated skin permeability, an analysis unit that calculates a concentration estimated value of the measurement target component in vivo,
The in-vivo component density | concentration estimation apparatus whose said measurement object component is protein or a steroid hormone.   The in vivo component concentration estimation apparatus according to claim 13, wherein the protein has a molecular weight of 5.6 to 102 kDa.   The in vivo component concentration estimation apparatus according to claim 13 or 14, wherein the protein has a molecular weight of 8.9 to 94 kDa.   The in vivo component concentration estimation apparatus according to any one of claims 13 to 15, wherein the protein is C-reactive protein, insulin, or C-peptide. The in vivo component concentration estimation device according to any one of claims 13 to 16, wherein the steroid hormone is cortisol or DHEA-s.