WO2010055942A1 - Device for measurement of fertile egg respiratory activity and method for measurement of fertile egg respiratory activity - Google Patents

Device for measurement of fertile egg respiratory activity and method for measurement of fertile egg respiratory activity Download PDF

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Publication number
WO2010055942A1
WO2010055942A1 PCT/JP2009/069457 JP2009069457W WO2010055942A1 WO 2010055942 A1 WO2010055942 A1 WO 2010055942A1 JP 2009069457 W JP2009069457 W JP 2009069457W WO 2010055942 A1 WO2010055942 A1 WO 2010055942A1
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WIPO (PCT)
Prior art keywords
embryo
electrode
fertilized egg
current value
oxygen
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PCT/JP2009/069457
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French (fr)
Japanese (ja)
Inventor
仁 珠玖
智一 末永
真一朗 高野
安基 伊達
剛史 斉藤
正樹 横尾
隆広 伊藤
宏之 阿部
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クリノ株式会社
国立大学法人東北大学
国立大学法人山形大学
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Publication of WO2010055942A1 publication Critical patent/WO2010055942A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/404—Cells with anode, cathode and cell electrolyte on the same side of a permeable membrane which separates them from the sample fluid, e.g. Clark-type oxygen sensors

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  • the present invention mainly relates to an apparatus for measuring the respiratory activity of a fertilized egg and a respiratory activity of the fertilized egg, which can non-invasively determine the oxygen consumption (respiratory volume) of a fertilized mammal one by one based on an electrochemical measurement method. It relates to a measurement method.
  • the importance of developmental biology and reproductive engineering extends from basic science to the production of recombinant animals, breeding livestock, and medical fields.
  • Mammal in vitro fertilization and in vitro culture technologies are a major area of biotechnology, and have great ripple effects such as human infertility treatment, livestock production, and the production of cloned animals and transgenic animals.
  • the present inventors have developed a microsystem for quantifying oxygen consumption (respiration) in the early embryo of mammals (bovine, mouse, etc.). Initially, a fertilized egg was fixed with a micromanipulator and a micropipette, and an oxygen concentration profile in the vicinity thereof was observed with a microelectrode (for example, see Patent Document 1). Furthermore, the oxygen concentration change in the vicinity of a fertilized egg was increased by using an inverted conical well, and the measurement method was improved so that the sample fixing operation of the manipulator was unnecessary (see, for example, Patent Documents 2 and 3).
  • the method for obtaining the respiration rate from the oxygen concentration profile near the fertilized egg based on the diffusion equation is a general method. That is, the present invention is not limited to scanning a microelectrode, and if the sample-electrode distance is known, it is possible to obtain the respiration rate from the concentration profile using a fixed electrode device.
  • the amperometric oxygen sensor oxygen consumption occurs at the electrode of the detection unit, and the oxygen concentration gradient in the vicinity is not uniform. If the detected current value is sufficiently small, the disturbance of the concentration profile on the sample side can be ignored. However, when the detected current value increases, the disturbance of the concentration profile near the sample increases, and the true sample respiration rate, that is, the sample respiration rate when the oxygen consumption on the sensor side is zero cannot be obtained.
  • the fertilized egg respiration measuring device is a scanning electrochemical microscope. Because it is based on microscopy (SECM), it is necessary to manipulate the microelectrode as a probe. Although minute operations at the time of measurement are automated by a stepping motor, the movement of the electrode to the vicinity of the previous sample embryo and the determination of the measurement start position are performed manually, and skill is required to acquire the technique. In other words, the measurement principle of scanning a microelectrode has limitations in operability and throughput. Accordingly, the present inventors have produced a microfluidic device for respiratory measurement that combines a poly (dimethylsiloxane) (PDMS) channel for manipulating a fertilized egg and an independent microarray electrode (for example, non-patent literature). 1).
  • PDMS poly (dimethylsiloxane)
  • the fertilized egg sample was manipulated by the flow path, and the respiration measurement of a single fertilized egg based on the oxygen reduction current was successful.
  • the respiration measurement value apparatus sample respiration rate
  • Non-Patent Document 1 a flow channel device in which a flow channel and a stand-alone microelectrode array are combined has already been reported, but the oxygen reduction current value flowing through each electrode of the device is completely considered. There was a problem that there was no guarantee that accurate respiratory volume could be measured. Moreover, the comparison with the conventional method is also insufficient, and there is a problem that it is impossible to verify the validity of the measurement result. That is, the result of comparing the same sample between the device and the conventional method is not shown. Furthermore, since the polymer resist agent SU-8 is used as the insulating film, there is a problem that it is out of the definition of “non-invasive”.
  • the present invention has been made paying attention to such a problem, it is possible to measure the accurate respiration rate of the fertilized egg in consideration of the oxygen reduction current of the electrode, it is also possible to verify the validity of the measurement results, It is an object of the present invention to provide a respiratory activity measuring apparatus for fertilized eggs and a respiratory activity measuring method for fertilized eggs that can improve non-invasiveness.
  • the present inventors have studied in detail the oxidation-reduction current value flowing through the electrode of the device, and eliminate the influence of oxygen consumption at the electrode on the oxygen consumption of a fertilized egg (embryo) such as a mammal as a sample
  • a device design method showing an appropriate oxygen reduction current value and a device based on the methodology were successfully completed, and the present invention was completed.
  • silicon oxide (SiO 2 ) as the amount of electrode insulation material, we succeeded in improving the non-invasiveness to fertilized eggs.
  • the present inventors conducted a respiration measurement on the same fertilized egg using a conventional electrode scanning measurement system (fertilized egg respiration measuring device) and an electrode-fixed device, and detected the result.
  • a conventional electrode scanning measurement system ferrtilized egg respiration measuring device
  • an electrode-fixed device an electrode-fixed device
  • oxygen consumption at the electrode can affect the apparent sample respiration rate.
  • a method was proposed, and a device was actually fabricated based on this method.
  • the apparatus for measuring the respiratory activity of a fertilized egg comprises a chip comprising an electrode disposed on a substrate, and an animal embryo introduced in the vicinity of the electrode in a state where a constant potential is applied to the electrode.
  • a current measurement unit that measures a current value before introduction of the embryo and a current value after introduction, and a current value before introduction of the embryo and a current value after introduction measured by the current measurement unit, And an analysis unit for determining the oxygen consumption of the embryo.
  • the method for measuring the respiratory activity of a fertilized egg according to the present invention is the method before introducing an embryo when an animal embryo is introduced in the vicinity of the electrode in a state where a constant potential is applied to the electrode arranged on the substrate.
  • the oxygen consumption of the embryo is determined based on the current value and the current value after introduction.
  • the method for measuring the respiratory activity of a fertilized egg according to the present invention can be easily carried out by the apparatus for measuring the respiratory activity of a fertilized egg according to the present invention.
  • the oxygen consumption (respiratory volume) of the embryo (fertilized egg) can be determined easily and accurately, and the respiratory activity of the embryo Can be quantitatively evaluated.
  • the embryo to be introduced is preferably composed of a fertilized egg of a mammal. Moreover, it is preferable that the number of embryos to be introduced is one.
  • the electrodes are preferably composed of microelectrodes having a length and width of 10 ⁇ m or less, and may be one or a plurality of arrays.
  • the electrode is used in the oxygen reduction reaction of the electrode so that the influence of the oxygen consumption accompanying the oxygen reduction reaction of the electrode on the oxygen consumption of the embryo can be ignored.
  • the upper limit value of the oxygen reduction current value is determined based on the accompanying oxygen consumption and the oxygen consumption of the embryo.
  • the upper limit of the oxygen reduction current value of the electrode is preferably 1 nA. In this case, it is possible to almost eliminate the influence of the oxygen consumption accompanying the oxygen reduction reaction of the electrode on the oxygen consumption of the embryo, and the accurate oxygen consumption of the embryo, that is, the accurate respiration rate of the fertilized egg is measured. be able to.
  • the substrate is made of a quartz glass substrate, and the surface of the substrate is covered with a SiO 2 vapor-deposited insulating film so that at least the electrode is exposed.
  • a SiO 2 vapor-deposited insulating film so that at least the electrode is exposed.
  • the chip preferably has a well or a channel structure for introducing the embryo in the vicinity of the electrode. In this case, an embryo fixing operation is not necessary. Further, since the position of the introduced embryo is determined in advance, the distance between the embryo and the electrode is known, and the oxygen consumption of the embryo can be determined more accurately.
  • the analysis unit uses a spherical diffusion theory based on a current value before the introduction of the embryo and a current value after the introduction, and the oxygen concentration on the surface of the embryo It is preferable to obtain the oxygen consumption of the embryo from the oxygen concentration.
  • the method for measuring the respiratory activity of a fertilized egg according to the present invention uses the spherical diffusion theory to determine the oxygen concentration on the surface of the embryo based on the current value before the introduction of the embryo and the current value after the introduction. It is preferable to determine the oxygen consumption of the embryo from the concentration. In this case, the oxygen consumption of the embryo, that is, the respiration rate of the fertilized egg can be accurately determined.
  • the analysis unit obtains the oxygen consumption using a sample embryo whose oxygen consumption is already known as the embryo in advance, and the obtained oxygen consumption and the sample embryo A correction value may be obtained based on the known oxygen consumption, and the oxygen consumption obtained for an embryo different from the sample embryo may be corrected by the correction value.
  • the oxygen consumption is determined in advance using a sample embryo whose oxygen consumption is known as the embryo, and the determined oxygen consumption and the known oxygen consumption of the sample embryo Based on the above, a correction value may be obtained, and the oxygen consumption obtained for an embryo different from the sample embryo may be corrected by the correction value.
  • an accurate oxygen consumption can be obtained from the correction value.
  • the oxygen consumption obtained by a conventional method such as an electrode scanning measurement system (fertilized egg respiration measuring device) is used as the known oxygen consumption of the sample embryo, thereby measuring the respiratory activity of the fertilized egg according to the present invention.
  • the oxygen consumption of the embryo by the apparatus and the method for measuring the respiratory activity of the fertilized egg can be compared with the oxygen consumption of the embryo by the conventional method. Thereby, the validity of the measurement result can be verified.
  • an accurate respiration rate of a fertilized egg can be measured in consideration of the oxygen reduction current of the electrode, the validity of the measurement result can be verified, and non-invasiveness can be improved.
  • the respiratory activity measuring apparatus and the respiratory activity measuring method of a fertilized egg can be provided.
  • FIG. 1 It is a graph which shows the relationship between the normalized electric current value (i / i *) of a fertilized egg respiration measurement chip
  • FIG. 2 is a graph showing an average respiration rate F chip, sample [mol / s] for each stage of embryo development measured by the apparatus for measuring the respiratory activity of a fertilized egg shown in FIG. 1.
  • FIG. 9 is a graph obtained by excluding data having an oxygen reduction current value of 1 nA or more observed from an electrode of a fertilized egg respiration measuring chip of the fertilized egg respiration activity measuring apparatus shown in FIG. 1 from the graph of FIG. 8.
  • FIG. 9 is a graph obtained by excluding data having an oxygen reduction current value of 1 nA or more observed from an electrode of a fertilized egg respiration measuring chip of the fertilized egg respiration activity measuring apparatus shown in FIG. 1 from the graph of FIG. 8.
  • FIG. 1 shows a simulation result based on the finite element method of the oxygen concentration profile in the vicinity of the electrode-sample of the fertilized egg respiration measuring chip of the fertilized egg respiratory activity measuring apparatus shown in FIG. 1
  • the oxygen reduction current value observed at the electrode is 0 nA
  • B is a cross-sectional view when the oxygen reduction current value observed at the electrode is 1 nA.
  • the respiratory activity measuring device of a fertilized egg includes (a) an enlarged plan view of an electrode part at the time of measurement, and (b) a microscope in which the electrode part at the time of measurement is enlarged. It is a photograph.
  • FIG. 11 shows the relationship between the normalized current value (Normalized Current; ⁇ i / i *) and the dissolved oxygen concentration (Dissolved Oxygen) [mg / l] of the fertilized egg respiration measuring chip of the fertilized egg respiratory activity measuring apparatus shown in FIG. It is a graph. It is a graph which shows a time-dependent change of the normalized electric current (i / i *) when it measures with the respiratory activity measuring apparatus of a fertilized egg shown in FIG. It is the (a) top view at the time of a measurement in the case of having a PDMS microchannel of the respiration activity measuring device of a fertilized egg of an embodiment of the invention, and (b) the microscope picture which expanded the electrode part at the time of measurement.
  • Normalized Current ⁇ i / i *
  • Dissolved Oxygen Dissolved Oxygen
  • a fertilized egg respiration activity measuring apparatus includes a fertilized egg respiration measuring chip (chip) 11, a current measuring unit (not shown), and an analyzing unit (FIG. 1). Not shown).
  • the method for measuring the respiratory activity of a fertilized egg according to the embodiment of the present invention is implemented by the apparatus for measuring the respiratory activity of a fertilized egg according to the embodiment of the present invention.
  • the fertilized egg respiration measuring chip 11 is formed by disposing an electrode 13 on a substrate 12.
  • the substrate 12 is made of a quartz glass substrate.
  • the electrode 13 is composed of a microelectrode having a length and width of 10 ⁇ m or less, and an oxygen reduction current value is set to 1 nA or less.
  • the fertilized egg respiration measuring chip 11 cleans the quartz glass of the substrate 12 and deposits Ti and Pt in this order on the quartz glass substrate by metal sputtering deposition to produce a Pt electrode pattern. On top of that, it is manufactured by producing a SiO 2 layer for an insulating film by SiO 2 vapor deposition.
  • the entire substrate 12 is covered and insulated by SiO 2 . Only the cross-shaped portion at the center of the micrograph in FIG. 1B has SiO 2 removed by lift-off, and the Pt portion is exposed.
  • the surface of the substrate 12 is covered with the SiO 2 vapor-deposited insulating film so that the electrode 13 is exposed.
  • the fertilized egg respiration measuring chip 11 has a PDMS well 14 having three inverted cones for introduction of an embryo sample and position definition assistance. The PDMS well 14 is bonded to the electrode 13.
  • the current measuring unit is configured to measure a current value before the introduction of the embryo and a current value after the introduction when the animal embryo is introduced in the vicinity of the electrode 13 with a constant potential applied to the electrode 13. ing.
  • the analysis unit is configured to determine the oxygen consumption of the embryo based on the current value before the introduction of the embryo measured by the current measurement unit and the current value after the introduction.
  • the method for measuring the respiratory activity of a fertilized egg first, when an animal embryo is introduced in the vicinity of the electrode 13 with a constant potential applied to the electrode 13 arranged on the substrate 12.
  • the current value before the introduction of the embryo and the current value after the introduction are measured by the current measuring unit.
  • the embryo to be introduced is one fertilized egg of a mammal. Based on the current value before the introduction of the embryo measured by the current measurement unit and the current value after the introduction, the oxygen consumption of the embryo is obtained by the analysis unit.
  • the oxygen reduction current value of the electrode 13 is 1 nA or less, so The effect of consumption on the oxygen consumption of the embryo can be ignored, and the accurate oxygen consumption of the embryo, ie the exact respiration rate of the fertilized egg can be measured.
  • the oxygen consumption (respiration rate) of the embryo (fertilized egg) can be determined easily and accurately, and the respiratory activity of the embryo can be quantitatively evaluated.
  • the substrate 12 is made of a quartz glass substrate and is covered and insulated with SiO 2 , noninvasiveness to the embryo to be measured can be improved. By using the PDMS well 14, an embryo fixing operation is not required. Moreover, since the position of the introduced embryo is determined in advance, the distance between the embryo and the electrode 13 is known, and the oxygen consumption of the embryo can be determined more accurately.
  • the F electrode / F sample can be regarded as a parameter such as a capture rate in the generation-capture experiment of an electrochemical measurement method, but can be a value of 1 or more unlike the capture rate.
  • the effect of F electrode / F sample on the concentration gradient near the sample depends on many factors. Specific factors include electrode 13-sample distance, device design around electrode 13 and its surroundings, uniform and non-uniform chemical reaction rates in or on the sample, mass transfer rate on the sample surface, chemical reaction rate and substance.
  • the oxygen concentration dependence of the moving speed can be mentioned.
  • a microelectrode with an oxygen reduction current value of 1 nA or less is used as a probe.
  • the effect of F electrode on the oxygen consumption of one mammalian embryo was found to be negligible.
  • a microelectrode having an oxygen reduction current value of 3 to 5 nA is selected as a probe, the influence on the oxygen consumption of one mammalian embryo cannot be ignored.
  • the respiration rate of biological samples of various respiration rates is calculated according to the spherical diffusion formula.
  • the respiration rate estimated from the current value is larger than the true respiration rate only for samples with relatively low respiration rate (F sample ⁇ 1 ⁇ 10 -14 mol / s) (overestimate).
  • the allowable I * electrode value can be determined from the following equation and reflected in the device design.
  • I * electrode Oxygen reduction current value observed on the electrode 13.
  • F electrode ⁇ (I * electrode / nF).
  • n Number of reaction electrons.
  • N 4 for oxygen reduction reaction on platinum electrode 13.
  • F Faraday constant [C / mol]
  • A Correction term.
  • A 0.26 obtained empirically by the electrode scanning type measurement system which is a conventional method is a reference value. The value of A is determined by the shape of the device and the environmental conditions when the mammalian embryo sample is introduced and arranged near the electrode 13. In addition, even when F sample is affected by F electrode , the true sample-derived oxygen consumption can be converted by the correction term.
  • F sample Oxygen consumption of one true mammalian embryo on the device [mol / s]
  • FIG. 3A shows an optical micrograph in the vicinity of the electrode 13 at the time of measurement.
  • the bulk current was measured without inserting an embryo for about 60 s from the start of measurement (0 s), and the average value was defined as the bulk current value i *.
  • the embryo was moved into the PDMS well 14 using a glass capillary, and after the operation was completed, the current value was measured for 600 s until the current value was stabilized. The average of the current values during the last 60 s until the end of measurement was taken as the sample current value i. The same operation was performed without using an embryo, and the negative control was measured.
  • FIG. 3B shows a schematic diagram of xyz coordinates with the origin O as the center of the spherical sample 1.
  • Entire substrate 12 is insulated by being covered with SiO 2, the center of the cross-shaped portion only SiO 2 is removed Pt portions of the photomicrographs of FIGS. 3 (a) is exposed.
  • a Pt electrode 13 indicated by “A” in FIG. 3 was used as a detection electrode, and an Ag / AgCl electrode (external) was used as a counter electrode and a reference electrode.
  • the Pt electrode 13 indicated by “B” in FIG. 3 was not used.
  • the mouse embryo that has moved to the cross-shaped portion is designated as a spherical sample 1.
  • the distance L [ ⁇ m] from the center of the spherical sample 1 to the detection electrode A is the distance d [ ⁇ m] from the surface of the spherical sample 1 to the detection electrode A and the radius r s [ ⁇ m] is determined as shown in equation (2).
  • the oxygen reduction current i [nA] obtained by the measurement was normalized by the bulk (offshore) oxygen reduction current i * [nA].
  • the original definition of i * is the oxygen reduction current value of the detection electrode at a location sufficiently away from the oxygen concentration profile formed in the vicinity of the spherical sample 1.
  • C s is the oxygen concentration [mol cm ⁇ 3 ] in the vicinity of the spherical sample 1, and C it is the oxygen concentration [mol cm ⁇ 3 ] on the surface of the detection electrode A.
  • the oxygen flux f s [mol cm ⁇ 2 s ⁇ 1 ] on the surface of the spherical sample 1 in the x-axis direction is expressed by equation (5).
  • the diffusion coefficient D of oxygen is 2.10 ⁇ 10 ⁇ 5 cm ⁇ 2 s ⁇ 1 (25 ° C.).
  • the total F chip, sample [mol / s] of the oxygen flux obtained from the product of the surface area S x [cm 2 ] of the spherical sample 1 and the oxygen flux f s [mol cm ⁇ 2 s ⁇ 1 ] is It is represented by the following expression (6).
  • the sum of the oxygen fluxes on the surface of the spherical sample 1 F chip, sample [mol / s] is due to respiratory activity, so the oxygen consumption rate (respiration rate) [mol / s] of the spherical sample 1 is Expressed with F chip, sample .
  • the spherical surface area S L [cm 2 ] above the flat plate is expressed by equation (7).
  • the oxygen concentration profile shows a steady state due to the oxygen consumption of the spherical sample 1
  • the area S L of this sphere at an arbitrary L as shown in the equation (8).
  • the flux f L is approximately equal to F chip, sample .
  • FIG. 4 shows a cyclic voltamgram (CV) at the detection electrode A of the fertilized egg respiration measuring chip 11.
  • CV cyclic voltamgram
  • n is the number of reaction electrons
  • F is the Faraday constant [C / mol]
  • ⁇ is the width of the detection electrode A [cm]
  • v is the sweep speed [V s -1 ]
  • R is the gas constant [JK -1 mol -1 ]
  • T is the temperature [K]
  • D is the diffusion coefficient [cm 2 s -1 ] of the reaction species of the detection electrode A
  • C is the reaction species of the detection electrode A
  • I p is the peak current value [A]
  • b is the length [cm] of the detection electrode A.
  • the diffusion coefficient of Fe (CN) 6 is 6.5 ⁇ 10 ⁇ 6 cm 2 s ⁇ 1 , the width of the detection electrode A is 10 ⁇ m, the length of the detection electrode A is 6.3 ⁇ m from the micrograph, and the sweep rate is 20 mV s ⁇ It was set to 1 . From the equations (9) and (10), this peak current value was calculated to be 1.32 nA. The calculated peak current value agreed very well with the result of the peak current value in the vicinity of 0.7 V vs. Ag / AgCl in FIG. This indicates that the insulating coating by SiO 2 is effectively performed and the region of the detection electrode A is well defined. From the above, it was supported that the detection electrode A integrated on the substrate 12 functions well.
  • dissolved oxygen meter DO-5509, Fuso
  • mercury thermometer Ag / AgCl
  • counter electrode and reference electrode Ag / AgCl
  • fertilized egg respiration measuring chip for measuring dissolved oxygen concentration [mg / l] for control. 11 was installed.
  • the distance [cm] between Ag / AgCl and the fertilized egg respiration measuring chip 11 was set to about 1 cm.
  • the liquid temperature was confirmed with a mercury thermometer and stirred with a magnetic stirrer (HS-50E, iuchi).
  • the measurement was performed at room temperature (21 ⁇ 1 ° C.), and the potential of the electrode 13 was set to ⁇ 0.5 V vs. Ag / AgCl 3.
  • the oxygen reduction current (i) obtained by the measurement was normalized by the bulk (offshore) oxygen reduction current (i *).
  • FIG. 5 shows the relationship between the normalized current value (i / i *) of the fertilized egg respiration measuring chip 11 and the oxygen concentration value [mg / l].
  • the value of the normalized current (i / i *) was determined using the value of the oxygen reduction current (i) after about 15 minutes had elapsed after dropping 1 M Na 2 SO 3 .
  • the dissolved oxygen concentration [mg / l] value was recorded at the same time.
  • the normalized current value (i / i *) obtained by the fertilized egg respiration measuring chip 11 was confirmed to have a residual current of about 7% at a dissolved oxygen concentration of 0 mg / l by a dissolved oxygen meter.
  • the oxygen concentration value there is a correlation between the oxygen concentration value and the detected value of the fertilized egg respiration measuring chip 11, suggesting that the oxygen concentration change can be regarded as the current value change.
  • the respiratory activity of the mouse embryo was examined.
  • a two-cell embryo (2cell) collected by in vitro fertilization (IVF) was cultured for 2 days and developed to a blastocyst.
  • IVF in vitro fertilization
  • a chronoamperometry method was used in which the potential of the electrode 13 was set to -0.5 V vs. Ag / AgCl, and the change in the oxygen reduction current with time was monitored while a constant potential was applied.
  • the measurement was performed at room temperature (24 ⁇ 1 ° C.), and the oxygen reduction current (i) obtained by the measurement was normalized by the bulk (offshore) oxygen reduction current (i *).
  • Fig. 6 (a) shows the relationship between the time [s] of mouse embryos not immobilized (Alive embryo) and the normalized current (i / i *), and Fig. 6 (b) shows the immobilized mouse embryos. The relationship between the time [s] of (Dead embryo) and the normalized current (i / i *) is shown. Immobilization of Dead embryos was performed using glutaraldehyde. The sample was immersed in glutaraldehyde for 30 min, washed with a PBS ( ⁇ ) solution, and used for measurement. Bulk current i * was measured for about 60 s from the start of measurement (0 s).
  • the Alive / Dead embryo is moved into the PDMS well 14 at the position of the downward arrow ( ⁇ ) in FIG. 6, and after the operation of about 120 s, the 600 s redox current is measured, and the last 60 s ⁇ ⁇ ⁇ ⁇ is sampled
  • the oxidation-reduction current i was used.
  • the sample measurement start point is indicated by an upward arrow ( ⁇ ) in FIG.
  • i * is normalized using i *.
  • the size of the Alive / Dead embryo was determined from an image taken with a computer-controlled inverted microscope (DM ⁇ ⁇ IRE 2, Leica).
  • the Alive embryo showed a decrease of about 1% i / i *.
  • FIG. 6B no change in i / i * i was observed in the Dead embryo. From this result, it was suggested that using the fertilized egg respiration measurement chip 11, it is possible to detect a change in i / i * due to the respiratory activity of the mouse embryo. In addition, it was suggested that the viability of mouse embryos can be determined using i / i * ⁇ as an index.
  • FIG. 7 shows the average respiratory volume F chip, sample [mol / s] for each stage of embryo development.
  • LSD least significant difference
  • the average respiratory volume at each developmental stage is 2 cell stage (0.29 ⁇ 0.08) ⁇ 10 ⁇ 14 mol s ⁇ 1 , morula stage (0.38 ⁇ 0.05) ⁇ 10 ⁇ 14 mol s ⁇ 1 , blastocyst stage It was (0.62 ⁇ 0.11) ⁇ 10 ⁇ 14 mol s ⁇ 1 .
  • FIG. 8 shows the relationship between F Well [mol / s] and F chip, sample [mol / s].
  • R 0.32
  • FIG. 10 shows a simulation result of the oxygen concentration profile in the vicinity of the electrode 13-sample based on the finite element method.
  • the cases where the oxygen reduction current values observed at the electrode 13 of the fertilized egg respiration measuring chip 11 are 0 nA and 1 nA are calculated, respectively, and are shown in FIGS. 10 (a) and 10 (b), respectively.
  • Both oxygen consumption rates on the spherical sample side were 1 ⁇ 10 ⁇ 14 mol / s.
  • FIGS. 10A and 10B it can be seen that the oxygen concentration profiles of the two do not necessarily match. If simulation of an oxygen concentration profile based on the finite element method is used, in principle, the fertilized egg respiration measuring chip 11 of any design can be verified.
  • the oxygen reduction current values of the electrodes 13 used in the following examples are all designed to be 1 nA or less.
  • the fertilized egg respiration measuring chip 11 has three electrodes 13 (W1 to W3), and a well for assisting in defining the embryo position is formed on the substrate 12 by wet etching.
  • the PDMS well 14 was bonded to the substrate 12 for measurement.
  • the potential of the electrode 13 is ⁇ 0.5-V vs. Ag / AgCl. The change in oxygen reduction current with respect to time was monitored while a constant potential was applied.
  • the bulk current was measured without the embryo 2 for about 60 s from the start of measurement (0 s), and the average value was defined as the bulk current value i *. Thereafter, the embryo 2 is moved into the PDMS well 14 using a glass capillary (RE). After the operation is completed, the current value is measured for 600 s until the current value is stabilized, and the current value of the last 60 s until the measurement is completed. The average was taken as the sample current value i. The same operation was performed without using embryo 2, and the negative control was measured.
  • RE glass capillary
  • FIG. 13 shows the change with time of the normalized current value i / i *. As shown in FIG. 13, no decrease in the current value was observed in the negative control. In contrast, the electrode 13 (W1 to W3) using the sample embryo 2 showed a steady value after noise due to the embryo 2 introduction operation, but the value decreased from the bulk current value.
  • the respiration rate F chip sample [mol / s] of the sample embryo 2 can be obtained.
  • the distance L [ ⁇ m] from the center of the spherical sample (embryo 2) to the electrode 13 the following equation is used instead of the equation (2).
  • h well is an etching depth ( ⁇ m).
  • measurement was performed using a chip having a PDMS (poly-dimethylsiloxane) microchannel 15 for sample manipulation instead of the PDMS well 14 as the fertilized egg respiration measuring chip 11.
  • the electrode 13 is a 10 ⁇ m ⁇ 10 ⁇ m Pt micro band electrode.
  • the PDMS microchannel 15 is made using a photoresist SU-8 as a mold, and includes a sample introduction channel and a measurement chamber (width: 200 ⁇ m, height: 150 ⁇ m).
  • the PDMS microchannel 15 is permanently bonded to the substrate 12 by oxygen plasma processing.
  • the measurement embryo 3 As the measurement embryo 3, a two-cell embryo, a blastocyst and a fixed embryo were used. After filling the measurement solution ERAM-2 (Functional Peptide Laboratory Co., Ltd.) in the PDMS microchannel, the fertilized egg respiration measuring chip 11 is connected to a multipotentiostat, and the electrode 13 is an oxygen reduction potential. 0.5 V vs. Ag / AgCl was applied. Waiting for the current value to stabilize, chronoamperometry measurement was started to measure the change in current value for a constant voltage. After placing the measurement embryo 3 in the sample inlet, as shown in FIG. 14, the embryo 3 was introduced into the measurement chamber by a syringe pump, and the change in the current value was measured.
  • ERAM-2 Federal Peptide Laboratory Co., Ltd.
  • a fertilized egg respiration measuring chip 11 As shown in FIG. 16 (a), as a fertilized egg respiration measuring chip 11, a substrate 12 having a measurement site in which an electrode 13 for electrochemical measurement is integrated on a quartz glass substrate, and at the time of embryo culture and respiration measurement Measurements were made using a PDMS microchannel 15 having a chamber for sample manipulation. As shown in FIG. 16 (b), the fertilized egg respiration measuring chip 11 is kept horizontal at the time of culture, and the embryo 4 is arranged at the culture site. On the other hand, at the time of measurement, the angle of the fertilized egg respiration measuring chip 11 is changed from horizontal to 60 °, whereby the embryo 4 is guided to the measurement site.
  • the fertilized egg respiration measuring chip 11 was connected to a multipotentiostat, and an oxygen reduction potential ( ⁇ 0.5 V Vs. V Ag / AgCl) was applied to the working electrode 13.
  • the embryo 4 was introduced into the measurement site by changing the fertilized egg respiration measuring chip 11 placed horizontally and waiting 60 [deg.] From the horizontal while waiting for the potential to stabilize. Changes in oxygen concentration in the vicinity of the electrode 13 due to respiration were monitored by chronoamperometry.
  • FIG. 17 shows changes in the oxygen reduction current.
  • the respiration rate of embryo 4 was calculated to be 4.2 ⁇ 10 ⁇ 15 mol ⁇ s ⁇ 1 . From the above results, the possibility of embryo respiration measurement using the fertilized egg respiration measuring chip 11 was shown. In this fertilized egg respiration measurement chip 11, both the embryo culture in a microenvironment and the respiration evaluation by electrochemical measurement are performed on a mouse fertilized egg only by an operation on a single substrate 12 with low stress on the cells. Is possible.
  • the apparatus for measuring the respiratory activity of a fertilized egg and the method for measuring the respiratory activity of a fertilized egg by using the fertilized egg respiration measuring chip 11, the operation step of the electrode 13 is omitted, and the embryo is placed. You can make measurements with just " In Examples 1 to 5, the function evaluation of the fertilized egg respiration measuring chip 11 and the respiratory activity of the mouse embryo are evaluated.
  • the apparatus for measuring the respiratory activity of a fertilized egg and the method for measuring the respiratory activity of a fertilized egg according to an embodiment of the present invention, the evaluation of the activity of an embryo based on the respiratory activity is performed in a non-invasive manner using an electrochemical technique.
  • a quantitative method was used.
  • the electrode 13 was integrated on the board

Abstract

Disclosed are a device for measurement of the respiratory activity of a fertile egg and a method for measurement of the respiratory activity of a fertile egg, which are able to accurately measure the amount of respiration of a fertile egg while taking into account the oxygen reduction current of an electrode, for which the appropriateness of the measurement results can be verified, and which provides an improvement with respect to non-invasiveness. A chip (11) for measurement of the respiration of fertile eggs is formed with electrodes (13) disposed on a substrate (12) and has in the vicinity of the electrodes (13) a PDMS well (14) or a PDMS micro flow channel (15) for the purpose of introducing embryos. The substrate (12) is comprised of a quartz glass substrate the surface of which is covered by means of an SiO2 deposition insulation film such that the electrodes (13) are exposed. The upper limit value for the oxygen reduction current value of the electrodes (13) is set, based on the amount of oxygen consumed in response to the oxygen reduction reaction thereof and the amount of oxygen consumed by the embryos, such that the influence of the amount of oxygen consumed in response to the oxygen reduction reaction on the amount of oxygen consumed by the embryos can be ignored.

Description

受精卵の呼吸活性測定装置および受精卵の呼吸活性測定方法Apparatus for measuring respiratory activity of fertilized egg and method for measuring respiratory activity of fertilized egg
　本発明は、主に哺乳動物受精卵の酸素消費量（呼吸量）を電気化学測定法に基づき無侵襲的に１個ずつ定量することができる受精卵の呼吸活性測定装置および受精卵の呼吸活性測定方法に関する。 The present invention mainly relates to an apparatus for measuring the respiratory activity of a fertilized egg and a respiratory activity of the fertilized egg, which can non-invasively determine the oxygen consumption (respiratory volume) of a fertilized mammal one by one based on an electrochemical measurement method. It relates to a measurement method.
　発生生物学・生殖工学の重要性は、基礎科学から組換え動物作出、家畜繁殖、医療の現場など多方面に及んでいる。哺乳動物の体外受精・体外培養技術は、バイオテクノロジーのなかでも大きな柱となる領域であり、ヒト不妊治療や家畜の生産、クローン動物やトランスジェニック動物の作出など波及効果も大きい。本発明者等は、哺乳動物（ウシ、マウスなど）初期胚の酸素消費（呼吸）を定量するマイクロシステムを開発してきた。当初、マイクロマニピュレータおよびマイクロピペットで受精卵を固定し、その近傍の酸素濃度プロファイルを微小電極により観測した（例えば、特許文献１参照）。さらに、逆円錐形ウェルを用いて受精卵近傍の酸素濃度変化を増大させ、マニピュレータの試料固定操作が不要な測定法に改良した（例えば、特許文献２および３参照）。 The importance of developmental biology and reproductive engineering extends from basic science to the production of recombinant animals, breeding livestock, and medical fields. Mammal in vitro fertilization and in vitro culture technologies are a major area of biotechnology, and have great ripple effects such as human infertility treatment, livestock production, and the production of cloned animals and transgenic animals. The present inventors have developed a microsystem for quantifying oxygen consumption (respiration) in the early embryo of mammals (bovine, mouse, etc.). Initially, a fertilized egg was fixed with a micromanipulator and a micropipette, and an oxygen concentration profile in the vicinity thereof was observed with a microelectrode (for example, see Patent Document 1). Furthermore, the oxygen concentration change in the vicinity of a fertilized egg was increased by using an inverted conical well, and the measurement method was improved so that the sample fixing operation of the manipulator was unnecessary (see, for example, Patent Documents 2 and 3).
　受精卵近傍の酸素濃度プロファイルから拡散方程式に基づき呼吸量を求める方法は、一般的な方法である。つまり、微小電極を走査する場合に限らず、サンプル－電極間距離が既知であれば、電極固定型デバイスを用いても、濃度プロファイルから呼吸量を求めることは可能である。アンペロメトリック酸素センサでは、検出部の電極にて酸素消費が起こり、近傍の酸素濃度勾配は不均一となっている。検出電流値が十分小さければ、サンプル側の濃度プロファイルの乱れは無視することができる。しかしながら、検出電流値が大きくなると、サンプル近傍の濃度プロファイルの乱れが大きくなり、真のサンプル呼吸量、即ちセンサ側の酸素消費がゼロの場合のサンプル呼吸量を求めることができなくなる。検出電極とサンプルの拡散層とのクロスオーバー、およびサンプル由来の酸素消費量への影響を詳細かつ正確に議論するには、拡散方程式のデジタルシミュレーションが有効であると考えられるが、実際に数値あるいは指標を提示する方法論はこれまでほとんど議論されてこなかった。したがって、センサの検出電流値を指標にしたデバイス設計、デバイスデザインの検証、デバイスの作製を試みた例はこれまで報告されていない。 The method for obtaining the respiration rate from the oxygen concentration profile near the fertilized egg based on the diffusion equation is a general method. That is, the present invention is not limited to scanning a microelectrode, and if the sample-electrode distance is known, it is possible to obtain the respiration rate from the concentration profile using a fixed electrode device. In the amperometric oxygen sensor, oxygen consumption occurs at the electrode of the detection unit, and the oxygen concentration gradient in the vicinity is not uniform. If the detected current value is sufficiently small, the disturbance of the concentration profile on the sample side can be ignored. However, when the detected current value increases, the disturbance of the concentration profile near the sample increases, and the true sample respiration rate, that is, the sample respiration rate when the oxygen consumption on the sensor side is zero cannot be obtained. In order to discuss in detail and accurately the crossover between the detection electrode and the sample diffusion layer and the effect on the oxygen consumption from the sample, digital simulation of the diffusion equation is considered effective. There has been little discussion of methodologies for presenting indicators. Therefore, there have been no reports of examples of device design, device design verification, and device fabrication using the detected current value of the sensor as an index.
　受精卵呼吸測定装置は、走査型電気化学顕微鏡(scanning electrochemical
microscopy, SECM)を基にしているために、プローブである微小電極の操作を行う必要がある。測定時の微小操作こそステッピングモーターにより自動化されているものの、それ以前のサンプル胚の近傍までの電極の移動および測定開始位置の決定は手動で行われており、技術の習得に熟練を要する。つまり、微小電極を走査する測定原理では、操作性、スループットに限界があった。そこで、本発明者等は、受精卵を操作するためのポリ（ジメチルシロキサン）（PDMS）製流路と独立型マイクロアレイ電極とを組合せた呼吸測定用マイクロ流体デバイスを作製した（例えば、非特許文献１参照）。これにより、流路により受精卵試料を操作すると共に、酸素還元電流に基づく単一受精卵の呼吸測定に成功した。 しかし、既に述べたとおり、検出電極の電流値が受精卵サンプルの呼吸測定値（みかけのサンプル呼吸量）に与える影響に関しては全く情報が得られていない。 The fertilized egg respiration measuring device is a scanning electrochemical microscope.
Because it is based on microscopy (SECM), it is necessary to manipulate the microelectrode as a probe. Although minute operations at the time of measurement are automated by a stepping motor, the movement of the electrode to the vicinity of the previous sample embryo and the determination of the measurement start position are performed manually, and skill is required to acquire the technique. In other words, the measurement principle of scanning a microelectrode has limitations in operability and throughput. Accordingly, the present inventors have produced a microfluidic device for respiratory measurement that combines a poly (dimethylsiloxane) (PDMS) channel for manipulating a fertilized egg and an independent microarray electrode (for example, non-patent literature). 1). As a result, the fertilized egg sample was manipulated by the flow path, and the respiration measurement of a single fertilized egg based on the oxygen reduction current was successful.   However, as already mentioned, no information is obtained regarding the influence of the current value of the detection electrode on the respiration measurement value (apparent sample respiration rate) of the fertilized egg sample.
特許第３６９３９０７号公報Japanese Patent No. 3693907 特許第３６８８６７１号公報Japanese Patent No. 3688671 特許第４０９７４９２号公報Japanese Patent No. 4097492
　非特許文献１に記載のように、流路と独立型微小電極アレイとを組合せた流路デバイスがすでに報告されているが、デバイスの各電極に流れる酸素還元電流値には全く配慮がされておらず、正確な呼吸量が測定できている保証がないという課題があった。また、従来法との比較も不十分であり、測定結果の妥当性を検証することが不可能であるという課題もあった。即ち、同一サンプルをデバイスと従来法とで比較した結果は示されていない。さらに、絶縁膜として高分子レジスト剤SU-8を用いているため、「無侵襲的」の定義から外れるという課題もあった。 As described in Non-Patent Document 1, a flow channel device in which a flow channel and a stand-alone microelectrode array are combined has already been reported, but the oxygen reduction current value flowing through each electrode of the device is completely considered. There was a problem that there was no guarantee that accurate respiratory volume could be measured. Moreover, the comparison with the conventional method is also insufficient, and there is a problem that it is impossible to verify the validity of the measurement result. That is, the result of comparing the same sample between the device and the conventional method is not shown. Furthermore, since the polymer resist agent SU-8 is used as the insulating film, there is a problem that it is out of the definition of “non-invasive”.
　本発明は、このような課題に着目してなされたもので、電極の酸素還元電流を考慮して受精卵の正確な呼吸量を測定可能であり、測定結果の妥当性の検証も可能で、無侵襲性を向上させることができる受精卵の呼吸活性測定装置および受精卵の呼吸活性測定方法を提供することを目的としている。 The present invention has been made paying attention to such a problem, it is possible to measure the accurate respiration rate of the fertilized egg in consideration of the oxygen reduction current of the electrode, it is also possible to verify the validity of the measurement results, It is an object of the present invention to provide a respiratory activity measuring apparatus for fertilized eggs and a respiratory activity measuring method for fertilized eggs that can improve non-invasiveness.
　本発明者等は、デバイスの電極に流れる酸化還元電流値について詳細に検討し、電極における酸素消費が、サンプルである哺乳動物等の受精卵（胚）の酸素消費量に与える影響を排除する方法について鋭意研究を重ねた結果、妥当な酸素還元電流値を示すデバイスデザインの方法、その方法論に立脚したデバイスの作製に成功し、本発明を完成させた。さらに、電極絶縁材量に酸化ケイ素（SiO2）を用いることで、受精卵に対する無侵襲性を向上させることにも成功した。 The present inventors have studied in detail the oxidation-reduction current value flowing through the electrode of the device, and eliminate the influence of oxygen consumption at the electrode on the oxygen consumption of a fertilized egg (embryo) such as a mammal as a sample As a result of extensive research on the device, a device design method showing an appropriate oxygen reduction current value and a device based on the methodology were successfully completed, and the present invention was completed. Furthermore, by using silicon oxide (SiO 2 ) as the amount of electrode insulation material, we succeeded in improving the non-invasiveness to fertilized eggs.
　また、本発明者等は、従来法である電極走査型測定システム（受精卵呼吸測定装置）と電極固定型デバイスとを用いて、同一受精卵について呼吸量測定を実施し比較検討した結果、検出電極における酸素消費量が見かけのサンプル呼吸量に影響を与える可能性があることを見出した。さらにこの結果から、真のサンプル呼吸量を得るために許容される検出電極電流値を決定可能であることを示し、その方法を提案すると共に、実際にこの方法に基づきデバイスを作製した。 In addition, the present inventors conducted a respiration measurement on the same fertilized egg using a conventional electrode scanning measurement system (fertilized egg respiration measuring device) and an electrode-fixed device, and detected the result. We have found that oxygen consumption at the electrode can affect the apparent sample respiration rate. Furthermore, from this result, it was shown that it is possible to determine the allowable detection electrode current value for obtaining a true sample respiration rate, a method was proposed, and a device was actually fabricated based on this method.
　本発明に係る受精卵の呼吸活性測定装置は、基板上に電極を配置して成るチップと、前記電極に一定の電位を印加した状態で、前記電極の近傍に動物の胚を導入したときの、前記胚の導入前の電流値と導入後の電流値を測定する電流測定部と、前記電流測定部で測定された前記胚の導入前の電流値と導入後の電流値に基づいて、前記胚の酸素消費量を求める解析部とを、有することを特徴とする。 The apparatus for measuring the respiratory activity of a fertilized egg according to the present invention comprises a chip comprising an electrode disposed on a substrate, and an animal embryo introduced in the vicinity of the electrode in a state where a constant potential is applied to the electrode. A current measurement unit that measures a current value before introduction of the embryo and a current value after introduction, and a current value before introduction of the embryo and a current value after introduction measured by the current measurement unit, And an analysis unit for determining the oxygen consumption of the embryo.
　本発明に係る受精卵の呼吸活性測定方法は、基板上に配置された電極に一定の電位を印加した状態で、前記電極の近傍に動物の胚を導入したときの、前記胚の導入前の電流値と導入後の電流値とに基づいて、前記胚の酸素消費量を求めることを、特徴とする。 The method for measuring the respiratory activity of a fertilized egg according to the present invention is the method before introducing an embryo when an animal embryo is introduced in the vicinity of the electrode in a state where a constant potential is applied to the electrode arranged on the substrate. The oxygen consumption of the embryo is determined based on the current value and the current value after introduction.
　本発明に係る受精卵の呼吸活性測定方法は、本発明に係る受精卵の呼吸活性測定装置により容易に実施することができる。本発明に係る受精卵の呼吸活性測定装置および受精卵の呼吸活性測定方法によれば、胚（受精卵）の酸素消費量（呼吸量）を容易かつ正確に求めることができ、胚の呼吸活性を定量的に評価することができる。導入する胚は、哺乳動物の受精卵から成ることが好ましい。また、導入する胚は、１個であることが好ましい。電極は、長さおよび幅が１０μｍ以下の微小電極から成ることが好ましく、１つであっても、複数からなるアレイであってもよい。 The method for measuring the respiratory activity of a fertilized egg according to the present invention can be easily carried out by the apparatus for measuring the respiratory activity of a fertilized egg according to the present invention. According to the apparatus for measuring the respiratory activity of a fertilized egg and the method for measuring the respiratory activity of a fertilized egg according to the present invention, the oxygen consumption (respiratory volume) of the embryo (fertilized egg) can be determined easily and accurately, and the respiratory activity of the embryo Can be quantitatively evaluated. The embryo to be introduced is preferably composed of a fertilized egg of a mammal. Moreover, it is preferable that the number of embryos to be introduced is one. The electrodes are preferably composed of microelectrodes having a length and width of 10 μm or less, and may be one or a plurality of arrays.
　本発明に係る受精卵の呼吸活性測定装置で、前記電極は、前記電極の酸素還元反応に伴う酸素消費量が前記胚の酸素消費量に与える影響を無視できるよう、前記電極の酸素還元反応に伴う酸素消費量と前記胚の酸素消費量とに基づいて、酸素還元電流値の上限値が定められることが好ましい。特に、前記電極は酸素還元電流値の上限値が１ｎＡであることが好ましい。この場合、電極の酸素還元反応に伴う酸素消費量が、胚の酸素消費量に与える影響をほぼ排除することができ、胚の正確な酸素消費量、すなわち受精卵の正確な呼吸量を測定することができる。 In the apparatus for measuring the respiratory activity of a fertilized egg according to the present invention, the electrode is used in the oxygen reduction reaction of the electrode so that the influence of the oxygen consumption accompanying the oxygen reduction reaction of the electrode on the oxygen consumption of the embryo can be ignored. It is preferable that the upper limit value of the oxygen reduction current value is determined based on the accompanying oxygen consumption and the oxygen consumption of the embryo. In particular, the upper limit of the oxygen reduction current value of the electrode is preferably 1 nA. In this case, it is possible to almost eliminate the influence of the oxygen consumption accompanying the oxygen reduction reaction of the electrode on the oxygen consumption of the embryo, and the accurate oxygen consumption of the embryo, that is, the accurate respiration rate of the fertilized egg is measured. be able to.
　本発明に係る受精卵の呼吸活性測定装置で、前記基板は石英ガラス基板から成り、前記チップは少なくとも前記電極が露出するよう、前記基板の表面がＳｉＯ２蒸着絶縁膜により覆われていることが好ましい。この場合、受精卵に対する無侵襲性を向上させることができる。 In the apparatus for measuring the respiratory activity of a fertilized egg according to the present invention, the substrate is made of a quartz glass substrate, and the surface of the substrate is covered with a SiO 2 vapor-deposited insulating film so that at least the electrode is exposed. preferable. In this case, the noninvasiveness with respect to a fertilized egg can be improved.
　本発明に係る受精卵の呼吸活性測定装置で、前記チップは前記電極の近傍に前記胚を導入するためのウェルまたは流路構造を有することが好ましい。この場合、胚の固定操作が不要である。また、導入された胚の位置があらかじめ決められているため、胚と電極間の距離が既知となり、胚の酸素消費量をより正確に求めることができる。 In the apparatus for measuring the respiratory activity of a fertilized egg according to the present invention, the chip preferably has a well or a channel structure for introducing the embryo in the vicinity of the electrode. In this case, an embryo fixing operation is not necessary. Further, since the position of the introduced embryo is determined in advance, the distance between the embryo and the electrode is known, and the oxygen consumption of the embryo can be determined more accurately.
　本発明に係る受精卵の呼吸活性測定装置で、前記解析部は、前記胚の導入前の電流値と導入後の電流値に基づいて、球面拡散理論を用いて、前記胚の表面の酸素濃度を求め、その酸素濃度から前記胚の酸素消費量を求めることが好ましい。本発明に係る受精卵の呼吸活性測定方法は、前記胚の導入前の電流値と導入後の電流値に基づいて、球面拡散理論を用いて、前記胚の表面の酸素濃度を求め、その酸素濃度から前記胚の酸素消費量を求めることが好ましい。この場合、胚の酸素消費量、すなわち受精卵の呼吸量を正確に求めることができる。 In the apparatus for measuring the respiratory activity of a fertilized egg according to the present invention, the analysis unit uses a spherical diffusion theory based on a current value before the introduction of the embryo and a current value after the introduction, and the oxygen concentration on the surface of the embryo It is preferable to obtain the oxygen consumption of the embryo from the oxygen concentration. The method for measuring the respiratory activity of a fertilized egg according to the present invention uses the spherical diffusion theory to determine the oxygen concentration on the surface of the embryo based on the current value before the introduction of the embryo and the current value after the introduction. It is preferable to determine the oxygen consumption of the embryo from the concentration. In this case, the oxygen consumption of the embryo, that is, the respiration rate of the fertilized egg can be accurately determined.
　本発明に係る受精卵の呼吸活性測定装置で、前記解析部は、あらかじめ前記胚として酸素消費量が既知の試料胚を用いて酸素消費量を求め、その求めた酸素消費量と前記試料胚の既知の酸素消費量とに基づいて補正値を求めておき、前記試料胚とは異なる胚について求めた酸素消費量を前記補正値により補正してもよい。本発明に係る受精卵の呼吸活性測定方法は、あらかじめ前記胚として酸素消費量が既知の試料胚を用いて酸素消費量を求め、その求めた酸素消費量と前記試料胚の既知の酸素消費量とに基づいて補正値を求めておき、前記試料胚とは異なる胚について求めた酸素消費量を前記補正値により補正してもよい。この場合、補正値により、正確な酸素消費量を求めることができる。また、電極走査型測定システム（受精卵呼吸測定装置）等の従来法で得られた酸素消費量を、試料胚の既知の酸素消費量とすることにより、本発明に係る受精卵の呼吸活性測定装置および受精卵の呼吸活性測定方法による胚の酸素消費量と、従来法による胚の酸素消費量とを比較することができる。これにより、測定結果の妥当性の検証も可能である。 In the apparatus for measuring the respiratory activity of a fertilized egg according to the present invention, the analysis unit obtains the oxygen consumption using a sample embryo whose oxygen consumption is already known as the embryo in advance, and the obtained oxygen consumption and the sample embryo A correction value may be obtained based on the known oxygen consumption, and the oxygen consumption obtained for an embryo different from the sample embryo may be corrected by the correction value. In the method for measuring the respiratory activity of a fertilized egg according to the present invention, the oxygen consumption is determined in advance using a sample embryo whose oxygen consumption is known as the embryo, and the determined oxygen consumption and the known oxygen consumption of the sample embryo Based on the above, a correction value may be obtained, and the oxygen consumption obtained for an embryo different from the sample embryo may be corrected by the correction value. In this case, an accurate oxygen consumption can be obtained from the correction value. Further, the oxygen consumption obtained by a conventional method such as an electrode scanning measurement system (fertilized egg respiration measuring device) is used as the known oxygen consumption of the sample embryo, thereby measuring the respiratory activity of the fertilized egg according to the present invention. The oxygen consumption of the embryo by the apparatus and the method for measuring the respiratory activity of the fertilized egg can be compared with the oxygen consumption of the embryo by the conventional method. Thereby, the validity of the measurement result can be verified.
　本発明によれば、電極の酸素還元電流を考慮して受精卵の正確な呼吸量を測定可能であり、測定結果の妥当性の検証も可能で、無侵襲性を向上させることができる受精卵の呼吸活性測定装置および受精卵の呼吸活性測定方法を提供することができる。 According to the present invention, an accurate respiration rate of a fertilized egg can be measured in consideration of the oxygen reduction current of the electrode, the validity of the measurement result can be verified, and non-invasiveness can be improved. The respiratory activity measuring apparatus and the respiratory activity measuring method of a fertilized egg can be provided.
本発明の実施の形態の受精卵の呼吸活性測定装置の（ａ）受精卵呼吸測定用チップの顕微鏡写真、（ｂ）電極部分を拡大した顕微鏡写真である。It is (a) the microscope picture of the chip | tip for a fertilized egg respiration measurement of the fertilized egg respiratory activity measuring apparatus of embodiment of this invention, (b) The microscope picture which expanded the electrode part. 図１に示す受精卵の呼吸活性測定装置の受精卵呼吸測定用チップを示す（ａ）斜視図、（ｂ）電極部分の断面図である。It is the (a) perspective view which shows the chip | tip for fertilized egg respiration measurement of the respiratory activity measuring apparatus of a fertilized egg shown in FIG. 1, (b) It is sectional drawing of an electrode part. 図１に示す受精卵の呼吸活性測定装置の、測定時の電極の近傍を示す（ａ）光学顕微鏡写真、（ｂ）球状サンプルの中心を原点とするxyz座標の断面図である。2A is an optical micrograph showing the vicinity of an electrode at the time of measurement of the respiratory activity measuring apparatus for a fertilized egg shown in FIG. 1, and FIG. 図１に示す受精卵の呼吸活性測定装置の、受精卵呼吸測定チップの電極におけるサイクリックボルタムグラム（CV）を示すグラフである。It is a graph which shows the cyclic voltamgram (CV) in the electrode of the fertilized egg respiration measurement chip | tip of the respiratory activity measuring apparatus of a fertilized egg shown in FIG. 図１に示す受精卵の呼吸活性測定装置の、受精卵呼吸測定チップの規格化電流値(i/i*)と溶存酸素濃度[mg/l]との関係を示すグラフである。It is a graph which shows the relationship between the normalized electric current value (i / i *) of a fertilized egg respiration measurement chip | tip, and dissolved oxygen concentration [mg / l] of the respiration activity measuring apparatus of a fertilized egg shown in FIG. 図１に示す受精卵の呼吸活性測定装置により（ａ）固定化していないマウス胚(Alive胚)について測定したときの規格化電流(i/i*)の経時変化を示すグラフ、（ｂ）固定化処理したマウス胚(Dead胚)について測定したときの規格化電流(i/i*)の経時変化を示すグラフである。(A) a graph showing the time course of the normalized current (i / i *) when measured on a non-immobilized mouse embryo (Alive embryo) using the fertilized egg respiratory activity measuring apparatus shown in FIG. 1, (b) fixed It is a graph which shows the time-dependent change of the normalized electric current (i / i *) when measuring about the mouse embryo (Dead embryo) which carried out the chemical treatment. 図１に示す受精卵の呼吸活性測定装置により測定された胚の発生ステージごとの呼吸量平均Fchip, sample[mol/s]を示すグラフである。2 is a graph showing an average respiration rate F chip, sample [mol / s] for each stage of embryo development measured by the apparatus for measuring the respiratory activity of a fertilized egg shown in FIG. 1. 図１に示す受精卵の呼吸活性測定装置により測定された胚の呼吸量（FWell[mol/ s]）と、従来の受精卵呼吸測定装置により測定された同じ胚の呼吸量（Fchip, sample[mol/s]）との関係を示すグラフである。The embryo respiration rate (F Well [mol / s]) measured with the fertilized egg respiratory activity measuring device shown in FIG. 1 and the same embryo respiration rate (F chip, It is a graph which shows the relationship with sample [mol / s]). 図８のグラフから、図１に示す受精卵の呼吸活性測定装置の受精卵呼吸測定チップの電極で観測される酸素還元電流値が1nA以上のデータを排除したグラフである。FIG. 9 is a graph obtained by excluding data having an oxygen reduction current value of 1 nA or more observed from an electrode of a fertilized egg respiration measuring chip of the fertilized egg respiration activity measuring apparatus shown in FIG. 1 from the graph of FIG. 8. 図１に示す受精卵の呼吸活性測定装置の受精卵呼吸測定チップの電極－サンプル近傍における酸素濃度プロファイルの有限要素法に基づくシミュレーション結果を示す（ａ）電極で観測される酸素還元電流値が0nAの場合の断面図、（ｂ）電極で観測される酸素還元電流値が1nAの場合の断面図である。FIG. 1 shows a simulation result based on the finite element method of the oxygen concentration profile in the vicinity of the electrode-sample of the fertilized egg respiration measuring chip of the fertilized egg respiratory activity measuring apparatus shown in FIG. 1 (a) The oxygen reduction current value observed at the electrode is 0 nA (B) is a cross-sectional view when the oxygen reduction current value observed at the electrode is 1 nA. 本発明の実施の形態の受精卵の呼吸活性測定装置の、３本の電極を有する場合の（ａ）測定時の電極部分を拡大した平面図、（ｂ）測定時の電極部分を拡大した顕微鏡写真である。The respiratory activity measuring device of a fertilized egg according to an embodiment of the present invention includes (a) an enlarged plan view of an electrode part at the time of measurement, and (b) a microscope in which the electrode part at the time of measurement is enlarged. It is a photograph. 図１１に示す受精卵の呼吸活性測定装置の、受精卵呼吸測定チップの規格化電流値(Normalized Current; i/i*)と溶存酸素濃度（Dissolved Oxygen）[mg/l]との関係を示すグラフである。FIG. 11 shows the relationship between the normalized current value (Normalized Current; 測定 i / i *) and the dissolved oxygen concentration (Dissolved Oxygen) [mg / l] of the fertilized egg respiration measuring chip of the fertilized egg respiratory activity measuring apparatus shown in FIG. It is a graph. 図１１に示す受精卵の呼吸活性測定装置により測定したときの規格化電流(i/i*)の経時変化を示すグラフである。It is a graph which shows a time-dependent change of the normalized electric current (i / i *) when it measures with the respiratory activity measuring apparatus of a fertilized egg shown in FIG. 本発明の実施の形態の受精卵の呼吸活性測定装置の、PDMSマイクロ流路を有する場合の（ａ）測定時の平面図、（ｂ）測定時の電極部分を拡大した顕微鏡写真である。It is the (a) top view at the time of a measurement in the case of having a PDMS microchannel of the respiration activity measuring device of a fertilized egg of an embodiment of the invention, and (b) the microscope picture which expanded the electrode part at the time of measurement. 図１４に示す受精卵の呼吸活性測定装置により測定したときの、受精卵呼吸測定チップの酸素還元電流変化率の経時変化を示すグラフである。It is a graph which shows the time-dependent change of the oxygen reduction current change rate of a fertilized egg respiration measuring chip when it measures with the respiratory activity measuring apparatus of a fertilized egg shown in FIG. 本発明の実施の形態の受精卵の呼吸活性測定装置の、PDMSマイクロ流路を有する場合の（ａ）サンプル胚を誘導する方法を示す側面図、（ｂ）サンプル胚の培養時（t=0sec）の状態を示す培養部位を拡大した顕微鏡写真、（ｃ）サンプル胚の測定時（t=90sec）の状態を示す測定部位を拡大した顕微鏡写真である。(A) Side view showing a method for inducing a sample embryo in the case of having a PDMS microchannel in the apparatus for measuring the respiratory activity of a fertilized egg according to an embodiment of the present invention, (b) at the time of culturing the sample embryo (t = 0 sec) And (c) a photomicrograph of an enlarged measurement site showing a state at the time of measurement of a sample embryo (t = 90 sec). 図１６に示す受精卵の呼吸活性測定装置により測定したときの、受精卵呼吸測定チップの酸素還元電流変化率の経時変化を示すグラフである。It is a graph which shows the time-dependent change of the oxygen reduction current change rate of a fertilized egg respiration measuring chip when it measures with the respiratory activity measuring apparatus of a fertilized egg shown in FIG.
　以下、図面に基づき本発明の実施の形態について説明する。
　図１乃至図１７は、本発明の実施の形態の受精卵の呼吸活性測定装置および受精卵の呼吸活性測定方法を示している。
　図１および図２に示すように、本発明の実施の形態の受精卵の呼吸活性測定装置は、受精卵呼吸測定用チップ（チップ）１１と電流測定部（図示せず）と解析部（図示せず）とを有している。なお、本発明の実施の形態の受精卵の呼吸活性測定方法は、本発明の実施の形態の受精卵の呼吸活性測定装置により実施される。 Hereinafter, embodiments of the present invention will be described with reference to the drawings.
1 to 17 show a device for measuring the respiratory activity of a fertilized egg and a method for measuring the respiratory activity of a fertilized egg according to an embodiment of the present invention.
As shown in FIGS. 1 and 2, a fertilized egg respiration activity measuring apparatus according to an embodiment of the present invention includes a fertilized egg respiration measuring chip (chip) 11, a current measuring unit (not shown), and an analyzing unit (FIG. 1). Not shown). The method for measuring the respiratory activity of a fertilized egg according to the embodiment of the present invention is implemented by the apparatus for measuring the respiratory activity of a fertilized egg according to the embodiment of the present invention.
　図１および図２に示すように、受精卵呼吸測定用チップ１１は、基板１２上に電極１３を配置して形成されている。基板１２は、石英ガラス基板から成っている。電極１３は、長さおよび幅が１０μｍ以下の微小電極から成り、酸素還元電流値が１ｎＡ以下に設定されている。受精卵呼吸測定用チップ１１は、まず、基板１２の石英ガラスを洗浄し、その石英ガラス基板上に金属スパッタ蒸着法によりTi、Ptの順に蒸着し、Pt電極パターンを作製する。その上に、SiO2蒸着により絶縁膜用SiO2層を作製することにより製造される。 As shown in FIGS. 1 and 2, the fertilized egg respiration measuring chip 11 is formed by disposing an electrode 13 on a substrate 12. The substrate 12 is made of a quartz glass substrate. The electrode 13 is composed of a microelectrode having a length and width of 10 μm or less, and an oxygen reduction current value is set to 1 nA or less. First, the fertilized egg respiration measuring chip 11 cleans the quartz glass of the substrate 12 and deposits Ti and Pt in this order on the quartz glass substrate by metal sputtering deposition to produce a Pt electrode pattern. On top of that, it is manufactured by producing a SiO 2 layer for an insulating film by SiO 2 vapor deposition.
　図１に示すように、基板１２の全体はSiO2で覆われ絶縁されている。図１（ｂ）の顕微鏡写真の中央の十字型の部分のみ、リフトオフによりSiO2が除去され、Pt部分が露出している。このように、受精卵呼吸測定用チップ１１は、電極１３が露出するよう、基板１２の表面がＳｉＯ２蒸着絶縁膜により覆われている。さらに、図２に示すように、受精卵呼吸測定用チップ１１は、胚試料の導入、位置規定補助用の３つの逆円錐を有するPDMSウェル１４を有している。PDMSウェル１４は、電極１３と接着されている。 As shown in FIG. 1, the entire substrate 12 is covered and insulated by SiO 2 . Only the cross-shaped portion at the center of the micrograph in FIG. 1B has SiO 2 removed by lift-off, and the Pt portion is exposed. As described above, in the fertilized egg respiration measuring chip 11, the surface of the substrate 12 is covered with the SiO 2 vapor-deposited insulating film so that the electrode 13 is exposed. Further, as shown in FIG. 2, the fertilized egg respiration measuring chip 11 has a PDMS well 14 having three inverted cones for introduction of an embryo sample and position definition assistance. The PDMS well 14 is bonded to the electrode 13.
　電流測定部は、電極１３に一定の電位を印加した状態で、電極１３の近傍に動物の胚を導入したときの、胚の導入前の電流値と導入後の電流値を測定するよう構成されている。
　解析部は、電流測定部で測定された胚の導入前の電流値と導入後の電流値に基づいて、胚の酸素消費量を求めるよう構成されている。 The current measuring unit is configured to measure a current value before the introduction of the embryo and a current value after the introduction when the animal embryo is introduced in the vicinity of the electrode 13 with a constant potential applied to the electrode 13. ing.
The analysis unit is configured to determine the oxygen consumption of the embryo based on the current value before the introduction of the embryo measured by the current measurement unit and the current value after the introduction.
　本発明の実施の形態の受精卵の呼吸活性測定方法では、まず、基板１２上に配置された電極１３に一定の電位を印加した状態で、電極１３の近傍に動物の胚を導入したときの、胚の導入前の電流値と導入後の電流値とを電流測定部で測定する。このとき、導入する胚は、哺乳動物の１個の受精卵である。電流測定部で測定された胚の導入前の電流値と導入後の電流値とに基づいて、解析部で胚の酸素消費量を求める。 In the method for measuring the respiratory activity of a fertilized egg according to an embodiment of the present invention, first, when an animal embryo is introduced in the vicinity of the electrode 13 with a constant potential applied to the electrode 13 arranged on the substrate 12. The current value before the introduction of the embryo and the current value after the introduction are measured by the current measuring unit. At this time, the embryo to be introduced is one fertilized egg of a mammal. Based on the current value before the introduction of the embryo measured by the current measurement unit and the current value after the introduction, the oxygen consumption of the embryo is obtained by the analysis unit.
　本発明の実施の形態の受精卵の呼吸活性測定装置および受精卵の呼吸活性測定方法によれば、電極１３の酸素還元電流値が１ｎＡ以下とすることにより、電極１３の酸素還元反応に伴う酸素消費量が、胚の酸素消費量に与える影響を無視することができ、胚の正確な酸素消費量、すなわち受精卵の正確な呼吸量を測定することができる。このように、胚（受精卵）の酸素消費量（呼吸量）を容易かつ正確に求めることができ、胚の呼吸活性を定量的に評価することができる。 According to the apparatus for measuring the respiratory activity of a fertilized egg and the method for measuring the respiratory activity of a fertilized egg according to an embodiment of the present invention, the oxygen reduction current value of the electrode 13 is 1 nA or less, so The effect of consumption on the oxygen consumption of the embryo can be ignored, and the accurate oxygen consumption of the embryo, ie the exact respiration rate of the fertilized egg can be measured. Thus, the oxygen consumption (respiration rate) of the embryo (fertilized egg) can be determined easily and accurately, and the respiratory activity of the embryo can be quantitatively evaluated.
　また、基板１２が石英ガラス基板から成り、SiO2で覆われて絶縁されているため、測定対象の胚に対する無侵襲性を向上させることができる。PDMSウェル１４を利用することにより、胚の固定操作が不要である。また、導入された胚の位置があらかじめ決められているため、胚と電極１３との間の距離が既知となり、胚の酸素消費量をより正確に求めることができる。 In addition, since the substrate 12 is made of a quartz glass substrate and is covered and insulated with SiO 2 , noninvasiveness to the embryo to be measured can be improved. By using the PDMS well 14, an embryo fixing operation is not required. Moreover, since the position of the introduced embryo is determined in advance, the distance between the embryo and the electrode 13 is known, and the oxygen consumption of the embryo can be determined more accurately.
＜電極に流れる電流値の許容範囲について＞
　アンペロメトリック酸素センサで検知される酸素還元電流値がI*electrodeの際、電極１３由来の酸素消費速度Felectrodeは、Felectrode= I*electrode/nF で表される(n は反応電子数、F はファラデー定数[C/mol])。FelectrodeとFsampleとの比較は、センサ上での電極１３の反応がサンプル（胚）由来の代謝活性に影響し得るかどうかの指標となる。定性的に、センサ上で検知される電流値が小さいほど、サンプル由来の代謝活性に与える影響が小さくなる。即ち、FelectrodeがFsampleと較べて小さいことが望ましいことは明らかである。しかしながら、FelectrodeがFsampleに及ぼす影響を定量的に議論することはそれほど簡単ではない。有限要素法に基づく拡散方程式のシミュレーションなどにより、これを解決可能である。 <About the allowable range of the current value flowing through the electrode>
When the oxygen reduction current value detected by the amperometric oxygen sensor is I * electrode , the oxygen consumption rate F electrode derived from the electrode 13 is expressed by F electrode = I * electrode / nF (n is the number of reaction electrons, F is the Faraday constant [C / mol]). The comparison between F electrode and F sample is an indicator of whether the reaction of electrode 13 on the sensor can affect the metabolic activity derived from the sample (embryo). Qualitatively, the smaller the current value detected on the sensor, the smaller the effect on sample-derived metabolic activity. That is, it is clear that the F electrode is desirably smaller than the F sample . However, it is not so easy to quantitatively discuss the effect of F electrode on F sample . This can be solved by simulation of diffusion equations based on the finite element method.
　Felectrode/Fsampleは、電気化学測定法の生成-捕捉実験における捕捉率のようなパラメータと位置付けることができるが、捕捉率と異なり１以上の値に成り得る。Felectrode/Fsampleのサンプル近傍の濃度勾配への影響は多くの因子に依存している。この因子として具体的には、電極１３-サンプル間距離、電極１３およびその周辺のデバイスデザイン、サンプル内あるいは表面の、均一および不均一化学反応速度、サンプル表面の物質移動速度、化学反応速度および物質移動速度の酸素濃度依存性などを挙げる事ができる。 The F electrode / F sample can be regarded as a parameter such as a capture rate in the generation-capture experiment of an electrochemical measurement method, but can be a value of 1 or more unlike the capture rate. The effect of F electrode / F sample on the concentration gradient near the sample depends on many factors. Specific factors include electrode 13-sample distance, device design around electrode 13 and its surroundings, uniform and non-uniform chemical reaction rates in or on the sample, mass transfer rate on the sample surface, chemical reaction rate and substance. The oxygen concentration dependence of the moving speed can be mentioned.
　電極走査型センサによる呼吸量計測方法では、Fsample = 1×10-14 mol/sレベルのウシ胚およびマウス胚の呼吸量を測定する際、酸素還元電流値が1nA以下の微小電極をプローブとして選択すると、Felectrodeが哺乳動物胚１個の酸素消費量に与える影響を無視できることが分かった。これに対し、酸素還元電流値が3～５nAの微小電極をプローブとして選択した場合には、哺乳動物胚１個の酸素消費量に与える影響を無視できない。 In the respiration rate measurement method using an electrode scanning sensor, when measuring the respiration rate of bovine and mouse embryos at F sample = 1 × 10 -14 mol / s level, a microelectrode with an oxygen reduction current value of 1 nA or less is used as a probe. When selected, the effect of F electrode on the oxygen consumption of one mammalian embryo was found to be negligible. On the other hand, when a microelectrode having an oxygen reduction current value of 3 to 5 nA is selected as a probe, the influence on the oxygen consumption of one mammalian embryo cannot be ignored.
　「無視できない」とする根拠として、以下の実験事実を挙げる事ができる。すなわち、酸素還元電流値が3～５nAの微小電極を用いて様々な呼吸量の生体サンプル（哺乳動物受精卵に加え、藻類ハネモのプロトプラストや癌細胞スフェロイド）の呼吸量を球面拡散の式に従い算出した場合に、比較的呼吸量の低いサンプル（Fsample < 1×10-14 mol/s）に限って、電流値から見積もられる呼吸量が真の呼吸量よりも大きくなる（オーバーエスティメイト）。 The following experimental facts can be cited as the basis for “cannot be ignored”. That is, using a microelectrode with an oxygen reduction current value of 3 to 5 nA, the respiration rate of biological samples of various respiration rates (along with mammalian fertilized eggs, algal honey protoplasts and cancer cell spheroids) is calculated according to the spherical diffusion formula. In this case, the respiration rate estimated from the current value is larger than the true respiration rate only for samples with relatively low respiration rate (F sample <1 × 10 -14 mol / s) (overestimate).
　すなわち、
　I*electrode = 1nA、 Fsample = 1×10-14 mol/sの場合、Felectrode / Fsample = 0.26となる。
　I*electrode = 3～5nA、 Fsample = 1×10-14 mol/sの場合、Felectrode / Fsample = 0.78～1.3となる。 That is,
When I * electrode = 1nA and F sample = 1 × 10 -14 mol / s, F electrode / F sample = 0.26.
When I * electrode = 3 to 5nA and F sample = 1 × 10 -14 mol / s, F electrode / F sample = 0.78 to 1.3.
　以上の結果から、次式より、許容されるI*electrode値を判定し、デバイスデザインに反映させることができる。
Figure JPOXMLDOC01-appb-M000001
 From the above results, the allowable I * electrode value can be determined from the following equation and reflected in the device design.
Figure JPOXMLDOC01-appb-M000001
　ここにおいて、
　I*electrode：電極１３上で観測される酸素還元電流値。電極１３が複数本ある場合は、各々を加算しFelectrode＝Σ(I*electrode/nF)で判定する。
　ｎ：反応電子数。白金の電極１３上での酸素還元反応の場合n = 4。
　F：ファラデー定数[C/mol]
　A：補正項。従来法である電極走査型測定系により、経験的に求めたA = 0.26が基準値となる。Aの値は、デバイスの形状、哺乳動物胚サンプルを電極１３の近傍に導入配置した際の環境条件により決定される。さらに、FelectrodeによりFsampleが影響を受ける場合も、補正項により真のサンプル由来酸素消費量を換算できる。
　Fsample：デバイス上での真の哺乳動物胚１個の酸素消費量[mol/s] put it here,
I * electrode : Oxygen reduction current value observed on the electrode 13. When there are a plurality of electrodes 13, they are added together and determined by F electrode = Σ (I * electrode / nF).
n: Number of reaction electrons. N = 4 for oxygen reduction reaction on platinum electrode 13.
F: Faraday constant [C / mol]
A: Correction term. A = 0.26 obtained empirically by the electrode scanning type measurement system which is a conventional method is a reference value. The value of A is determined by the shape of the device and the environmental conditions when the mammalian embryo sample is introduced and arranged near the electrode 13. In addition, even when F sample is affected by F electrode , the true sample-derived oxygen consumption can be converted by the correction term.
F sample : Oxygen consumption of one true mammalian embryo on the device [mol / s]
＜受精卵呼吸測定用チップの検証＞
　6-9週齢のB6C3F1系統マウスから回収した２細胞期胚から胚盤胞期胚について、呼吸計測を実施した。電極１３の電位を-0.5 V vs. Ag/AgClに設定し、定電位のまま時間に対する酸素還元電流の変化をモニタリングした。図３（ａ）に、測定時の電極１３の近傍の光学顕微鏡写真を示す。測定開始(0 s)から60 s程度、胚を入れずにバルク電流を測定し、その平均値をバルク電流値i*とした。その後、ガラスキャピラリーを用いて胚をPDMSウェル１４内に移動し、操作終了後、電流値が安定するまで600s間電流値を測定した。測定終了までの最後の60s間の電流値の平均を、サンプル電流値i とした。同様の操作を、胚を用いずに行いネガティブコントロールを測定した。 <Verification of chip for measuring fertilized egg respiration>
Respiration measurements were performed on blastocyst stage embryos from 2-cell stage embryos collected from 6-9 week old B6C3F1 strain mice. The potential of the electrode 13 was set to -0.5 V vs. Ag / AgCl, and the change in the oxygen reduction current with respect to time was monitored while maintaining the constant potential. FIG. 3A shows an optical micrograph in the vicinity of the electrode 13 at the time of measurement. The bulk current was measured without inserting an embryo for about 60 s from the start of measurement (0 s), and the average value was defined as the bulk current value i *. Thereafter, the embryo was moved into the PDMS well 14 using a glass capillary, and after the operation was completed, the current value was measured for 600 s until the current value was stabilized. The average of the current values during the last 60 s until the end of measurement was taken as the sample current value i. The same operation was performed without using an embryo, and the negative control was measured.
［受精卵呼吸測定チップにおける酸素濃度プロファイルの解析］
　アンペロメトリー法により得られる球状サンプル近傍の酸素還元電流応答から、球面拡散理論を用いて、受精卵呼吸測定チップ１１における球状サンプルが消費する酸素濃度および酸素消費速度を算出した。以下に、受精卵呼吸測定チップ１１における酸素濃度プロファイルの解析方法を述べる。 [Analysis of oxygen concentration profile in fertilized egg respiration measuring chip]
From the oxygen reduction current response in the vicinity of the spherical sample obtained by the amperometry method, the oxygen concentration and the oxygen consumption rate consumed by the spherical sample in the fertilized egg respiration measuring chip 11 were calculated using spherical diffusion theory. Below, the analysis method of the oxygen concentration profile in the fertilized egg respiration measuring chip 11 will be described.
　図３（ｂ）に、球状サンプル１の中心を原点Oとするxyz座標の概略図を示す。基板１２全体はSiO2で覆われて絶縁されており、図３（ａ）の顕微鏡写真の中央の十字型の部分のみのSiO2が除去されてPt部分が露出している。測定には、検出電極として図３中の「Ａ」で示すPt電極１３を用い、対極兼参照極としてAg/AgCl電極(外付け)を用いた。図３中の「Ｂ」で示したPt電極１３は使用しなかった。十字型の部分に移動したマウス胚を、球状サンプル１とする。 FIG. 3B shows a schematic diagram of xyz coordinates with the origin O as the center of the spherical sample 1. Entire substrate 12 is insulated by being covered with SiO 2, the center of the cross-shaped portion only SiO 2 is removed Pt portions of the photomicrographs of FIGS. 3 (a) is exposed. In the measurement, a Pt electrode 13 indicated by “A” in FIG. 3 was used as a detection electrode, and an Ag / AgCl electrode (external) was used as a counter electrode and a reference electrode. The Pt electrode 13 indicated by “B” in FIG. 3 was not used. The mouse embryo that has moved to the cross-shaped portion is designated as a spherical sample 1.
　図３（ｂ）より、球状サンプル１の中心から検出電極Ａまでの距離L［μm］は、球状サンプル１の表面から検出電極Ａまでの距離d［μm］と球状サンプル１の半径rs［μm］により、（２）式のように決定される。
Figure JPOXMLDOC01-appb-M000002
 From FIG. 3B, the distance L [μm] from the center of the spherical sample 1 to the detection electrode A is the distance d [μm] from the surface of the spherical sample 1 to the detection electrode A and the radius r s [ μm] is determined as shown in equation (2).
Figure JPOXMLDOC01-appb-M000002
　ここで、球状サンプル１内の酸素濃度は一定であり、球状サンプル１の大きさは一定であると仮定する。測定によって得られた酸素還元電流i [nA]は、バルク（沖合い）の酸素還元電流i＊[nA]によって規格化した。i＊の本来の定義は、球状サンプル１の近傍に形成される酸素濃度プロファイルよりも十分離れた場所における検出電極の酸素還元電流値である。受精卵呼吸測定チップ１１上で検出電極Ａの近傍に受精卵の球状サンプル１を導入－導出する場合、導入直前の電流値がi＊に相当する。 Here, it is assumed that the oxygen concentration in the spherical sample 1 is constant and the size of the spherical sample 1 is constant. The oxygen reduction current i [nA] obtained by the measurement was normalized by the bulk (offshore) oxygen reduction current i * [nA]. The original definition of i * is the oxygen reduction current value of the detection electrode at a location sufficiently away from the oxygen concentration profile formed in the vicinity of the spherical sample 1. When the spherical sample 1 of a fertilized egg is introduced and derived near the detection electrode A on the fertilized egg respiration measuring chip 11, the current value immediately before the introduction corresponds to i * .
　検出電極Ａの表面の酸素濃度差(ΔCi-t=C*－Ci－t)は、酸素還元電流i [nA]と、バルクの酸素還元電流i＊[nA]およびバルクの酸素濃度C*(0.21 mM)により、(３)式のように決定される。
Figure JPOXMLDOC01-appb-M000003
 The oxygen concentration difference (ΔC it = C * −C i−t ) on the surface of the detection electrode A is determined by the oxygen reduction current i [nA], the bulk oxygen reduction current i * [nA], and the bulk oxygen concentration C * ( 0.21 mM) is determined as shown in equation (3).
Figure JPOXMLDOC01-appb-M000003
　球状サンプル１の表面の酸素濃度差(ΔCs=C*-Cs)は、(４)式のように、検出電極Ａの表面の酸素濃度差(ΔCi-t=C*－Ci－t)を外挿することで得られる。
Figure JPOXMLDOC01-appb-M000004
 The oxygen concentration difference (ΔC s = C * −C s ) on the surface of the spherical sample 1 is the difference in oxygen concentration (ΔC it = C * −C i−t ) on the surface of the detection electrode A as shown in the equation (4). Is obtained by extrapolating.
Figure JPOXMLDOC01-appb-M000004
　ここで、Csは球状サンプル１の近傍の酸素濃度［mol cm-3］、Ci-t は検出電極Ａの表面の酸素濃度［mol cm-3］である。Fickの第一法則より、x軸方向の球状サンプル１の表面の酸素流束fs［mol cm-2 s-1］は、(５)式で表される。
Figure JPOXMLDOC01-appb-M000005
 Here, C s is the oxygen concentration [mol cm −3 ] in the vicinity of the spherical sample 1, and C it is the oxygen concentration [mol cm −3 ] on the surface of the detection electrode A. From Fick's first law, the oxygen flux f s [mol cm −2 s −1 ] on the surface of the spherical sample 1 in the x-axis direction is expressed by equation (5).
Figure JPOXMLDOC01-appb-M000005
　ここで、酸素の拡散係数Dは2.10×10-5 cm-2 s-1 (25℃)である。球状サンプル１の表面積Sx ［cm2］と酸素流束fs ［mol cm-2 s-1］との積から得られる酸素流束の総和Fchip, sample [mol/s]は、次に示す（６）式で表される。
Figure JPOXMLDOC01-appb-M000006
 Here, the diffusion coefficient D of oxygen is 2.10 × 10 −5 cm −2 s −1 (25 ° C.). The total F chip, sample [mol / s] of the oxygen flux obtained from the product of the surface area S x [cm 2 ] of the spherical sample 1 and the oxygen flux f s [mol cm −2 s −1 ] is It is represented by the following expression (6).
Figure JPOXMLDOC01-appb-M000006
　ここで、球状サンプル１の表面の酸素流束の総和Fchip, sample[mol/s]は、呼吸活動に起因することから、球状サンプル１の酸素消費速度（呼吸量）[mol/s]をFchip, sample で表現した。 Here, the sum of the oxygen fluxes on the surface of the spherical sample 1 F chip, sample [mol / s] is due to respiratory activity, so the oxygen consumption rate (respiration rate) [mol / s] of the spherical sample 1 is Expressed with F chip, sample .
　球状サンプル１と共通の中心をもつ半径Lの球のうち、平板より上側の球表面積SL［cm2］は（７）式で表される。平板上に球状サンプル１が静置されており、球状サンプル１の酸素消費により酸素濃度プロファイルが定常状態を示す場合、（８）式に示すように、任意のLにおいて、この球の面積SLと流束fLの積は、近似的にFchip, sampleと等しくなる。
Figure JPOXMLDOC01-appb-M000007
 Of the spheres of radius L having the same center as the spherical sample 1, the spherical surface area S L [cm 2 ] above the flat plate is expressed by equation (7). When the spherical sample 1 is placed on a flat plate, and the oxygen concentration profile shows a steady state due to the oxygen consumption of the spherical sample 1, the area S L of this sphere at an arbitrary L as shown in the equation (8). And the flux f L is approximately equal to F chip, sample .
Figure JPOXMLDOC01-appb-M000007
　図４に、受精卵呼吸測定チップ１１の検出電極Ａにおけるサイクリックボルタムグラム（CV）を示す。本実験は、検出電極Ａの形状およびサイズの妥当性を確認するため、測定溶液に4 mM フェロシアン化カリウムを用いており、酸素還元電流I*chipとは直接関係ない。検出電極Ａの形状からバンド電極と仮定すると、CVはディスク電極の場合と類似したシグモイダルな形状をしているが、定常電流は存在しない事が既に知られている。そこで、次に示すピーク電流の（９）および（１０）式から、ピーク電流値を決定した。
Figure JPOXMLDOC01-appb-M000008
 FIG. 4 shows a cyclic voltamgram (CV) at the detection electrode A of the fertilized egg respiration measuring chip 11. In this experiment, 4 mM potassium ferrocyanide was used for the measurement solution in order to confirm the validity of the shape and size of the detection electrode A, and it was not directly related to the oxygen reduction current I * chip . Assuming that the detection electrode A is a band electrode, it is already known that the CV has a sigmoidal shape similar to that of the disk electrode, but there is no steady current. Therefore, the peak current value was determined from the following peak current expressions (9) and (10).
Figure JPOXMLDOC01-appb-M000008
　ここで、p は無次元化された掃引速度パラメーター、n は反応電子数、F はファラデー定数[C/mol]、ω は検出電極Ａの幅[cm]、v は掃引速度[V s-1]、R は気体定数[J K-1 mol-1]、T は温度[K]、D は検出電極Ａの反応種の拡散係数[cm2 s-1]、C は検出電極Ａの反応種の濃度[mol cm-3]、Ip はピーク電流値[A]、b は検出電極Ａの長さ[cm]である。Fe(CN)6 の拡散係数は、6.5 × 10-6cm2 s-1、検出電極Ａの幅は10μm、検出電極Ａの長さは顕微鏡写真より6.3μmとし、掃引速度は20 mV s-1 とした。（９）および（１０）式より、このピーク電流値は1.32nA と算出された。算出されたピーク電流値は、図４の0.7 V vs. Ag/AgCl 付近のピーク電流値の結果と極めて良く一致した。これは、SiO2 による絶縁被覆が効果的になされており、検出電極Ａの領域が良好に規定されている事を示す。以上より、基板１２上に集積化した検出電極Ａが、良好に機能している事が支持された。 Where p is the dimensionless sweep speed parameter, n is the number of reaction electrons, F is the Faraday constant [C / mol], ω is the width of the detection electrode A [cm], and v is the sweep speed [V s -1 ], R is the gas constant [JK -1 mol -1 ], T is the temperature [K], D is the diffusion coefficient [cm 2 s -1 ] of the reaction species of the detection electrode A, and C is the reaction species of the detection electrode A The concentration [mol cm −3 ], I p is the peak current value [A], and b is the length [cm] of the detection electrode A. The diffusion coefficient of Fe (CN) 6 is 6.5 × 10 −6 cm 2 s −1 , the width of the detection electrode A is 10 μm, the length of the detection electrode A is 6.3 μm from the micrograph, and the sweep rate is 20 mV s − It was set to 1 . From the equations (9) and (10), this peak current value was calculated to be 1.32 nA. The calculated peak current value agreed very well with the result of the peak current value in the vicinity of 0.7 V vs. Ag / AgCl in FIG. This indicates that the insulating coating by SiO 2 is effectively performed and the region of the detection electrode A is well defined. From the above, it was supported that the detection electrode A integrated on the substrate 12 functions well.
［検量線：受精卵呼吸測定チップの酸素濃度検出能評価］
　受精卵呼吸測定チップ１１の酸素濃度検出能について検討を行った。ビーカーにPBS(-)(1.47 mM KH2PO4、4.30 mM Na2HPO4、2.68 mM KCl、136.9 mM NaCl)溶液を100 ml 満たし、1M亜硫酸ナトリウム(Na2SO3)水溶液を適当量滴下し、PBS(-)溶液中の酸素濃度を決定した。ビーカー中には、コントロールの溶存酸素濃度[mg/l]測定のために、溶存酸素計(DO-5509、Fuso)、水銀温度計、対極兼参照極(Ag/AgCl)および受精卵呼吸測定チップ１１を設置した。この際、Ag/AgCl と受精卵呼吸測定チップ１１との距離[cm]は、1cm程度に設定した。また、溶存酸素濃度[mg/l]を一定に保つために、水銀温度計による液温確認とマグネチックスターラー(HS-50E、iuchi)による攪拌とを行った。ここで、測定は室温(21±1 ℃)で行い、電極１３の電位は-0.5 V vs. Ag/AgCl に設定した。測定によって得られた酸素還元電流(i)は、バルク（沖合い）の酸素還元電流(i*)により規格化した。 [Calibration curve: Evaluation of oxygen concentration detection ability of fertilized egg respiration measuring chip]
The oxygen concentration detection ability of the fertilized egg respiration measuring chip 11 was examined. Fill a beaker with 100 ml of PBS (-) (1.47 mM KH 2 PO 4 , 4.30 mM Na 2 HPO 4 , 2.68 mM KCl, 136.9 mM NaCl) solution, and add 1M sodium sulfite (Na 2 SO 3 ) aqueous solution dropwise. The oxygen concentration in the PBS (−) solution was determined. In the beaker, dissolved oxygen meter (DO-5509, Fuso), mercury thermometer, counter electrode and reference electrode (Ag / AgCl), and fertilized egg respiration measuring chip for measuring dissolved oxygen concentration [mg / l] for control. 11 was installed. At this time, the distance [cm] between Ag / AgCl and the fertilized egg respiration measuring chip 11 was set to about 1 cm. In order to keep the dissolved oxygen concentration [mg / l] constant, the liquid temperature was confirmed with a mercury thermometer and stirred with a magnetic stirrer (HS-50E, iuchi). Here, the measurement was performed at room temperature (21 ± 1 ° C.), and the potential of the electrode 13 was set to −0.5 V vs. Ag / AgCl 3. The oxygen reduction current (i) obtained by the measurement was normalized by the bulk (offshore) oxygen reduction current (i *).
　図５に、受精卵呼吸測定チップ１１の規格化電流値(i/i*)と酸素濃度の値[mg/l]との関係を示す。1 M Na2SO3 の滴下を行う前に、バルクの酸素還元電流(i*)および、溶存酸素濃度[mg/l]を測定した。1 M Na2SO3 滴下後、15min程度経過した後の酸素還元電流(i)の値を用い、規格化電流(i/i*)の値を決定した。この際、同時に溶存酸素濃度[mg/l]の値を記録した。図５に示すように、溶存酸素濃度の現象は直線性を示した(R=0.984)。また、受精卵呼吸測定チップ１１により得られた規格化電流値(i/i*)は、溶存酸素計による溶存酸素濃度0mg/lにおいて、7％程度の残余電流が確認された。しかしながら、酸素濃度の値と受精卵呼吸測定チップ１１の検出値との間には相関性があり、酸素濃度変化を電流値の変化として捉えることが出来るということが示唆された。 FIG. 5 shows the relationship between the normalized current value (i / i *) of the fertilized egg respiration measuring chip 11 and the oxygen concentration value [mg / l]. Before dropping 1 M Na 2 SO 3 , bulk oxygen reduction current (i *) and dissolved oxygen concentration [mg / l] were measured. The value of the normalized current (i / i *) was determined using the value of the oxygen reduction current (i) after about 15 minutes had elapsed after dropping 1 M Na 2 SO 3 . At this time, the dissolved oxygen concentration [mg / l] value was recorded at the same time. As shown in FIG. 5, the phenomenon of dissolved oxygen concentration showed linearity (R = 0.984). Further, the normalized current value (i / i *) obtained by the fertilized egg respiration measuring chip 11 was confirmed to have a residual current of about 7% at a dissolved oxygen concentration of 0 mg / l by a dissolved oxygen meter. However, there is a correlation between the oxygen concentration value and the detected value of the fertilized egg respiration measuring chip 11, suggesting that the oxygen concentration change can be regarded as the current value change.
　受精卵呼吸測定チップ１１を用いて、マウス胚の呼吸活性について検討した。サンプルとして、体外受精(IVF)により回収した２細胞期胚(2cell)を２日間培養し、胚盤胞(Blastocyst)まで発生したものを用いた。電極１３の電位を-0.5V vs. Ag/AgCl に設定し、一定電位を印加しながら、時間に対する酸素還元電流変化をモニタリングする、クロノアンペロメトリー法を用いた。ここで、測定は室温(24±1℃)で行い、測定により得られた酸素還元電流(i)はバルク（沖合い）の酸素還元電流(i*)により規格化した。 Using the fertilized egg respiration measuring chip 11, the respiratory activity of the mouse embryo was examined. As a sample, a two-cell embryo (2cell) collected by in vitro fertilization (IVF) was cultured for 2 days and developed to a blastocyst. A chronoamperometry method was used in which the potential of the electrode 13 was set to -0.5 V vs. Ag / AgCl, and the change in the oxygen reduction current with time was monitored while a constant potential was applied. Here, the measurement was performed at room temperature (24 ± 1 ° C.), and the oxygen reduction current (i) obtained by the measurement was normalized by the bulk (offshore) oxygen reduction current (i *).
　図６（ａ）に、固定化していないマウス胚(Alive胚)の時間[s]と規格化電流(i/i*)との関係を、図６（ｂ）に、固定化処理したマウス胚(Dead胚)の時間[s]と規格化電流(i/i*)との関係を示す。Dead胚の固定化処理は、グルタルアルデヒドを用いて行った。グルタルアルデヒドに30min 浸漬後、PBS(-)溶液で洗浄してから測定に用いた。測定開始(0s)から60s 程度バルク電流i* を測定した。続いて、図６中の下向きの矢印(↓)の箇所でPDMSウェル１４内にAlive/Dead胚を移動し、120s 程度の操作終了後、600s 間酸化還元電流を測定し、最後の60s をサンプル酸化還元電流i とした。この際、サンプル測定開始点を、図６中に上向きの矢印(↑)で示す。ここで、i* を用いてi を規格化した。また、Alive/Dead胚のサイズは、コンピューター制御型倒立顕微鏡(DM IRE 2、Leica)により撮影した画像から決定した。 Fig. 6 (a) shows the relationship between the time [s] of mouse embryos not immobilized (Alive embryo) and the normalized current (i / i *), and Fig. 6 (b) shows the immobilized mouse embryos. The relationship between the time [s] of (Dead embryo) and the normalized current (i / i *) is shown. Immobilization of Dead embryos was performed using glutaraldehyde. The sample was immersed in glutaraldehyde for 30 min, washed with a PBS (−) solution, and used for measurement. Bulk current i * was measured for about 60 s from the start of measurement (0 s). Subsequently, the Alive / Dead embryo is moved into the PDMS well 14 at the position of the downward arrow (↓) in FIG. 6, and after the operation of about 120 s, the 600 s redox current is measured, and the last 60 s サ ン プ ル is sampled The oxidation-reduction current i was used. At this time, the sample measurement start point is indicated by an upward arrow (↑) in FIG. Here, i * is normalized using i *. The size of the Alive / Dead embryo was determined from an image taken with a computer-controlled inverted microscope (DM 顕 微鏡 IRE 2, Leica).
　図６（ａ）に示すように、Alive胚では1%程度のi/i* の減少が見られた。一方、図６（ｂ）に示すように、Dead胚では、i/i* の変化は見られなかった。この結果から、受精卵呼吸測定チップ１１を用いて、マウス胚の呼吸活性に起因したi/i* の変化を検出可能であることが示唆された。また、i/i* を指標としたマウス胚の生死判断が可能であることが示唆された。 As shown in FIG. 6 (a), the Alive embryo showed a decrease of about 1% i / i *. On the other hand, as shown in FIG. 6B, no change in i / i * i was observed in the Dead embryo. From this result, it was suggested that using the fertilized egg respiration measurement chip 11, it is possible to detect a change in i / i * due to the respiratory activity of the mouse embryo. In addition, it was suggested that the viability of mouse embryos can be determined using i / i * 指標 as an index.
　図７に、胚の発生ステージごとの呼吸量平均Fchip, sample[mol/s]を示す。なお、有意差の検定には、一元配置分散解析およびFisherの最小有意差法(LSD : Least significant difference)を用い、全ての値は平均値±標準誤差(SEM : standard error of the mean)で表した。それぞれの発生ステージでの呼吸量平均は、２細胞期(0.29±0.08)×10-14 mol s-1、桑実胚期(0.38±0.05)×10-14 mol s-1、胚盤胞期(0.62±0.11)×10-14 mol s-1 であった。２細胞期と桑実胚期および桑実胚期と胚盤胞期との間には有意な差は見られなかったが、２胞期と胚盤胞期の間には有意な差が見られた。 FIG. 7 shows the average respiratory volume F chip, sample [mol / s] for each stage of embryo development. For the significance test, one-way analysis of variance and Fisher's least significant difference (LSD) method were used, and all values were expressed as mean ± standard error (SEM). did. The average respiratory volume at each developmental stage is 2 cell stage (0.29 ± 0.08) × 10 −14 mol s −1 , morula stage (0.38 ± 0.05) × 10 −14 mol s −1 , blastocyst stage It was (0.62 ± 0.11) × 10 −14 mol s −1 . There was no significant difference between the 2-cell stage and the morula stage and between the morula stage and the blastocyst stage, but there was a significant difference between the 2-cell stage and the blastocyst stage. It was.
［受精卵呼吸測定装置と受精卵呼吸測定チップとの相関性］
　従来の受精卵呼吸測定装置(HSV-403、ペプチド機能性研究所(株))と受精卵呼吸測定チップ１１との相関性について、検討を行った。受精卵呼吸測定装置において、プローブとして、白金マイクロ電極を用いた。また、XYZステージ(K701-20RMS、(株)駿河精器)上の電極ホルダーに、電極を固定した。６個の逆円錐型マイクロウェルを有する受精卵呼吸測定用Well((株)北斗電工)に、測定溶液(ERAM2、(株)機能性ペプチド研究所)でウェルを満たし、ウェルの中心にサンプルを配し、測定を行った。測定専用プレートを37℃に維持するため、Warm plate (MATS-502NLR、(株)東海ヒット))をXYZステージ上に設置した。 [Correlation between fertilized egg respiration measuring device and fertilized egg respiration measuring chip]
The correlation between the conventional fertilized egg respiration measuring device (HSV-403, Peptide Functional Laboratory Co., Ltd.) and the fertilized egg respiration measuring chip 11 was examined. In the fertilized egg respiration measuring apparatus, a platinum microelectrode was used as a probe. Moreover, the electrode was fixed to the electrode holder on the XYZ stage (K701-20RMS, Suruga Seiki Co., Ltd.). Fill well with measurement solution (ERAM2, Functional Peptide Laboratory Co., Ltd.) in Well (Hokuto Denko Co., Ltd.) for fertilized egg respiration measurement with 6 inverted conical microwells, and sample at the center of well And measured. A Warm plate (MATS-502NLR, Tokai Hit Co., Ltd.)) was placed on the XYZ stage in order to maintain the dedicated measurement plate at 37 ° C.
　サンプルとして、グルタルアルデヒドにより固定化処理を行った胚(Dead胚)および体外受精(IVF)によって回収したマウス胚を培養した２細胞期(2-cell)、桑実胚期(Morula)、胚盤胞期(Blastocyst)の異なる発生ステージの胚を用いた。まず、受精卵呼吸測定装置を用いて呼吸量(Fchip, sample [mol/s])を測定した。続いて、同サンプルを用いて受精卵呼吸測定チップ１１により呼吸量(Fwell [mol/s])を測定した。装置の設置場所の都合により、37℃で保温された細胞培養輸送器(MEA451、Fujihira industry co., Ltd)を用いてサンプルの輸送を行った。受精卵呼吸測定装置で測定後、10~15 min 程度輸送を行い、輸送後すぐに37℃、5% CO2、95% air の条件下の炭酸ガスインキュベーター(Model-9200、Wakenyaku Co., Ltd)中に移動した。なお、測定は室温(24℃±1)で行った。 As samples, embryos fixed with glutaraldehyde (Dead embryos) and mouse embryos recovered by in vitro fertilization (IVF) were cultured in the 2-cell stage (2-cell), morula stage (Morula), scutellum Embryos with different developmental stages at the blast stage (Blastocyst) were used. First, the respiration rate (F chip, sample [mol / s]) was measured using a fertilized egg respiration measuring device. Subsequently, the respiration rate (F well [mol / s]) was measured with the fertilized egg respiration measuring chip 11 using the sample. Samples were transported using a cell culture transporter (MEA451, Fujihira industry co., Ltd) kept at 37 ° C. depending on the location of the apparatus. After measurement with a fertilized egg respiration measuring device, transport for about 10 to 15 min. Immediately after transport, carbon dioxide incubator under conditions of 37 ° C, 5% CO2, 95% air (Model-9200, Wakenyaku Co., Ltd) Moved in. The measurement was performed at room temperature (24 ° C. ± 1).
　図８に、FWell[mol/ s]とFchip, sample[mol/ s]との関係を示す。プロットは比較的直線的であり、Fwell[mol/ s]とFchip, sample[mol/ s]との間にはある程度の相関性(R=0.32)が有ることが示された。また、Dead胚ついては、FWell[mol/ s]、Fchip, sample[mol/ s]共に0に近い値を示した。これらのことから、受精卵呼吸測定チップ１１は、受精卵呼吸測定装置と同様に胚の呼吸量を測定できることが示された。しかしながら、FWell[mol/s]の値とFchip, sample[mol/s]の値とを比較すると、Fchip, sample[mol/s]の方が大きくなる傾向が見られた。 FIG. 8 shows the relationship between F Well [mol / s] and F chip, sample [mol / s]. The plot is relatively linear, indicating that there is a certain degree of correlation (R = 0.32) between F well [mol / s] and F chip, sample [mol / s]. For Dead embryos, both F Well [mol / s], F chip and sample [mol / s] showed values close to 0. From these facts, it was shown that the fertilized egg respiration measuring chip 11 can measure the respiration rate of the embryo in the same manner as the fertilized egg respiration measuring device. However, when comparing the values of F Well [mol / s] and F chip, sample [mol / s], F chip, sample [mol / s] tended to be larger.
　受精卵呼吸測定チップ１１の電極１３で観測される酸素還元電流値が大きすぎると、電極１３の酸素消費が原因でサンプルの酸素消費量を正確に測定できていない可能性がある。そこで図８のうち、受精卵呼吸測定チップ１１の電極１３で観測される酸素還元電流値が1nA以上のデータを排除し、その結果を図９に示す。この結果、相関係数が大幅に向上した（R=0.78）。このことから、電極１３の電流値を指標に、受精卵呼吸測定チップ１１を設計・検証することが極めて重要であることが示唆された。 If the oxygen reduction current value observed at the electrode 13 of the fertilized egg respiration measuring chip 11 is too large, the oxygen consumption of the sample may not be accurately measured due to the oxygen consumption of the electrode 13. Therefore, in FIG. 8, data having an oxygen reduction current value of 1 nA or more observed at the electrode 13 of the fertilized egg respiration measuring chip 11 is excluded, and the result is shown in FIG. As a result, the correlation coefficient was greatly improved (R = 0.78). This suggests that it is extremely important to design and verify the fertilized egg respiration measurement chip 11 using the current value of the electrode 13 as an index.
　図１０は、有限要素法に基づく、電極１３－サンプル近傍における酸素濃度プロファイルのシミュレーション結果である。受精卵呼吸測定チップ１１の電極１３で観測される酸素還元電流値が0nAおよび1nAの場合を各々計算し、それぞれ図１０（ａ）および（ｂ）に示す。球状サンプル側の酸素消費速度は共に1x10-14 mol/s とした。図１０（ａ）および（ｂ）に示すように、両者の酸素濃度プロファイルが必ずしも一致していないことが分かる。有限要素法に基づく酸素濃度プロファイルのシミュレーションを利用すれば、原理的にあらゆるデザインの受精卵呼吸測定チップ１１の検証が可能となる。 FIG. 10 shows a simulation result of the oxygen concentration profile in the vicinity of the electrode 13-sample based on the finite element method. The cases where the oxygen reduction current values observed at the electrode 13 of the fertilized egg respiration measuring chip 11 are 0 nA and 1 nA are calculated, respectively, and are shown in FIGS. 10 (a) and 10 (b), respectively. Both oxygen consumption rates on the spherical sample side were 1 × 10 −14 mol / s. As shown in FIGS. 10A and 10B, it can be seen that the oxygen concentration profiles of the two do not necessarily match. If simulation of an oxygen concentration profile based on the finite element method is used, in principle, the fertilized egg respiration measuring chip 11 of any design can be verified.
　以上の結果から、以下の実施例で用いる電極１３の酸素還元電流値は、すべて1nA以下となるよう設計されている。 From the above results, the oxygen reduction current values of the electrodes 13 used in the following examples are all designed to be 1 nA or less.
　図１１に示すように、受精卵呼吸測定用チップ１１として、電極１３を３本（W1～Ｗ3）とし、胚位置規定補助のためのウェルを湿式エッチングにより基板１２上に作製し、逆円錐型のPDMSウェル１４を基板１２と接着したものを使用して測定を行った。測定手順として、まず、B6C3F1系統マウスから回収した２細胞期胚を培養して得た胚盤胞について、受精卵呼吸測定用チップ１１を用い、電極１３の電位を-0.5 V vs. Ag/AgClに設定し、一定電位を印加したまま、時間に対する酸素還元電流の変化をモニタリングした。測定開始(0s)から60s程度、胚２を入れずにバルク電流を測定し、その平均値をバルク電流値i*とした。その後、ガラスキャピラリー（ＲＥ）を用いて胚２をPDMSウェル１４内に移動し、操作終了後、電流値が安定するまで600s間電流値を測定し、測定終了までの最後の60sの電流値の平均をサンプル電流値i とした。同様の操作を、胚２を用いずに行い、ネガティブコントロールを測定した。 As shown in FIG. 11, the fertilized egg respiration measuring chip 11 has three electrodes 13 (W1 to W3), and a well for assisting in defining the embryo position is formed on the substrate 12 by wet etching. The PDMS well 14 was bonded to the substrate 12 for measurement. As a measurement procedure, first, for a blastocyst obtained by culturing a two-cell stage embryo collected from a B6C3F1 strain mouse, using a fertilized egg respiration measuring chip 11, the potential of the electrode 13 is −0.5-V vs. Ag / AgCl. The change in oxygen reduction current with respect to time was monitored while a constant potential was applied. The bulk current was measured without the embryo 2 for about 60 s from the start of measurement (0 s), and the average value was defined as the bulk current value i *. Thereafter, the embryo 2 is moved into the PDMS well 14 using a glass capillary (RE). After the operation is completed, the current value is measured for 600 s until the current value is stabilized, and the current value of the last 60 s until the measurement is completed. The average was taken as the sample current value i. The same operation was performed without using embryo 2, and the negative control was measured.
　受精卵呼吸測定チップ１１の酸素濃度検出能について検討を行った。図５と同様に、1M亜硫酸ナトリウム(Na2SO3)水溶液を適当量滴下し、PBS(-)溶液中の酸素濃度を決定した。図１２に示すように、酸素濃度と電流値とは直線性を示した(R = 0.982)。規格化電流値i/i* の経時変化を、図１３に示す。図１３に示すように、ネガティブコントロールでは、電流値の減少は認められなかった。それに対して、サンプルの胚２を用いた電極１３（W1～Ｗ3）においては、胚２の導入操作によるノイズの後に定常値を示したが、その値はバルク電流値から減少していた。また、3つの異なる位置にある電極１３（W1～Ｗ3）で同様の電流値変化が見られることから、同時に多方向から胚２の酸素消費量測定を行うことが出来ることが示された。胚２は、発生段階が進むにつれ極性を持つことが知られている。そのため、胚２の向きにより、同一の胚２でも呼吸量が変化することが考えられる。今回の結果から、受精卵呼吸測定チップ１１を用いることで胚２の極性に起因する同一胚内での呼吸量の差異を測定できることが予想される。 The oxygen concentration detection ability of the fertilized egg respiration measuring chip 11 was examined. As in FIG. 5, an appropriate amount of 1M sodium sulfite (Na 2 SO 3 ) aqueous solution was dropped, and the oxygen concentration in the PBS (−) solution was determined. As shown in FIG. 12, the oxygen concentration and the current value showed linearity (R   = 0.982). FIG. 13 shows the change with time of the normalized current value i / i *. As shown in FIG. 13, no decrease in the current value was observed in the negative control. In contrast, the electrode 13 (W1 to W3) using the sample embryo 2 showed a steady value after noise due to the embryo 2 introduction operation, but the value decreased from the bulk current value. In addition, since similar current value changes were observed at the electrodes 13 (W1 to W3) at three different positions, it was shown that the oxygen consumption of the embryo 2 can be measured simultaneously from multiple directions. Embryo 2 is known to have polarity as the developmental stage proceeds. Therefore, it is conceivable that the respiration rate of the same embryo 2 changes depending on the orientation of the embryo 2. From this result, it is expected that the difference in respiration rate in the same embryo due to the polarity of the embryo 2 can be measured by using the fertilized egg respiration measuring chip 11.
　実施例２と同様にして、サンプルの胚２の呼吸量Fchip, sample[mol/s]を求めることができる。ただし、球状サンプル（胚２）の中心から電極１３までの距離L［μm］は、式（２）の代わりに次式を用いる。ここで、hwellはエッチング深さ(μm)である。
Figure JPOXMLDOC01-appb-M000009
 Similarly to Example 2, the respiration rate F chip, sample [mol / s] of the sample embryo 2 can be obtained. However, for the distance L [μm] from the center of the spherical sample (embryo 2) to the electrode 13, the following equation is used instead of the equation (2). Here, h well is an etching depth (μm).
Figure JPOXMLDOC01-appb-M000009
　図１４に示すように、受精卵呼吸測定用チップ１１として、PDMSウェル１４の代わりに、サンプル操作用のPDMS（poly-dimethylsiloxane）マイクロ流路１５を有するものを使用して測定を行った。電極１３は、10μm×10μmのPtマイクロ帯電極である。また、PDMSマイクロ流路１５は、フォトレジストSU-8 を鋳型として作製され、試料導入流路および測定チャンバ（幅：200μm、高さ：150μm）で構成されている。PDMSマイクロ流路１５は、酸素プラズマ処理により基板１２と永久接合されている。 As shown in FIG. 14, measurement was performed using a chip having a PDMS (poly-dimethylsiloxane) microchannel 15 for sample manipulation instead of the PDMS well 14 as the fertilized egg respiration measuring chip 11. The electrode 13 is a 10 μm × 10 μm Pt micro band electrode. The PDMS microchannel 15 is made using a photoresist SU-8 as a mold, and includes a sample introduction channel and a measurement chamber (width: 200 μm, height: 150 μm). The PDMS microchannel 15 is permanently bonded to the substrate 12 by oxygen plasma processing.
　測定胚３として、２細胞胚、胚盤胞および固定胚を用いた。PDMSマイクロ流路内に測定溶液ERAM-2（（株）機能性ペプチド研究所）を満たした後、受精卵呼吸測定用チップ１１をマルチポテンショスタットと接続し、電極１３に酸素還元電位である -0.5 V vs. Ag/AgCl を印加した。電流値の安定を待ち、一定電圧に対する電流値変化を測定するクロノアンペロメトリー測定を開始した。試料インレットに測定胚３を配置した後、図１４に示すように、シリンジポンプにより胚３を測定チャンバ内に導入し、電流値の変化を測定した。 As the measurement embryo 3, a two-cell embryo, a blastocyst and a fixed embryo were used. After filling the measurement solution ERAM-2 (Functional Peptide Laboratory Co., Ltd.) in the PDMS microchannel, the fertilized egg respiration measuring chip 11 is connected to a multipotentiostat, and the electrode 13 is an oxygen reduction potential. 0.5 V vs. Ag / AgCl was applied. Waiting for the current value to stabilize, chronoamperometry measurement was started to measure the change in current value for a constant voltage. After placing the measurement embryo 3 in the sample inlet, as shown in FIG. 14, the embryo 3 was introduced into the measurement chamber by a syringe pump, and the change in the current value was measured.
　図１５に示すように、測定胚３を測定チャンバ内に導入したところ、流路内の流れに起因するノイズの後、生存胚では胚の呼吸による電流値の減少が観察された。各作用電極１３における電流値の減少は、胚３に近いほど大きい値となった。電流値の変化から電極１３の近傍の酸素濃度を求め、フィックの第一法則から胚３の呼吸活性を算出したところ、固定胚および各発生ステージの呼吸活性は、固定胚が0.8×10-15 mol/s、２細胞胚が3.1×10-15 mol/s、胚盤胞が5.7×10-15 mol/sであった。このように、胚３の呼吸活性を指標とした生死判断および、胚発生ステージの進行による呼吸活性増加のモニタリングを行えることが確認された。 As shown in FIG. 15, when the measurement embryo 3 was introduced into the measurement chamber, a decrease in the current value due to the respiration of the embryo was observed in the viable embryo after noise caused by the flow in the flow path. The decrease in the current value at each working electrode 13 became larger as the distance to the embryo 3 was closer. The oxygen concentration in the vicinity of the electrode 13 was obtained from the change in the current value, and the respiratory activity of the embryo 3 was calculated from Fick's first law. The respiratory activity of the fixed embryo and each developmental stage was 0.8 × 10 −15 for the fixed embryo. mol / s, the 2-cell embryo was 3.1 × 10 −15 mol / s, and the blastocyst was 5.7 × 10 −15 mol / s. Thus, it was confirmed that life / death judgment using the respiratory activity of embryo 3 as an index and monitoring of increase in respiratory activity due to the progress of the embryonic development stage can be performed.
　図１６（ａ）に示すように、受精卵呼吸測定用チップ１１として、電気化学測定のための電極１３を石英ガラス基板上に集積化した測定部位を有する基板１２と、胚培養および呼吸測定時のサンプル操作のためのチャンバを有するPDMSマイクロ流路１５とで構成されるものを使用して測定を行った。図１６（ｂ）に示すように、培養時に受精卵呼吸測定用チップ１１は水平に保たれ、胚４は培養部位に配置されている。一方、測定時には受精卵呼吸測定用チップ１１の角度が水平から60°に変化することで、胚４は測定部位に誘導される。培養部位にサンプル胚４を配置した後、受精卵呼吸測定用チップ１１をマルチポテンショスタットに接続し、作用電極１３に酸素還元電位（-0.5 V vs. Ag/AgCl）を印加した。電位の安定を待ち、水平に静置した受精卵呼吸測定用チップ１１を水平から60°変化させることで、測定部位への胚４の導入を行った。呼吸による電極１３の近傍の酸素濃度変化を、クロノアンペロメトリーによりモニタリングした。 As shown in FIG. 16 (a), as a fertilized egg respiration measuring chip 11, a substrate 12 having a measurement site in which an electrode 13 for electrochemical measurement is integrated on a quartz glass substrate, and at the time of embryo culture and respiration measurement Measurements were made using a PDMS microchannel 15 having a chamber for sample manipulation. As shown in FIG. 16 (b), the fertilized egg respiration measuring chip 11 is kept horizontal at the time of culture, and the embryo 4 is arranged at the culture site. On the other hand, at the time of measurement, the angle of the fertilized egg respiration measuring chip 11 is changed from horizontal to 60 °, whereby the embryo 4 is guided to the measurement site. After placing the sample embryo 4 at the culture site, the fertilized egg respiration measuring chip 11 was connected to a multipotentiostat, and an oxygen reduction potential (−0.5 V Vs. V Ag / AgCl) was applied to the working electrode 13. The embryo 4 was introduced into the measurement site by changing the fertilized egg respiration measuring chip 11 placed horizontally and waiting 60 [deg.] From the horizontal while waiting for the potential to stabilize. Changes in oxygen concentration in the vicinity of the electrode 13 due to respiration were monitored by chronoamperometry.
　胚４は、長さ750μm のチャンバ内を約90～150secで沈降し、図１６（ｃ）に示すように、測定部位に再現よく導入された。図１７に、酸素還元電流の変化を示す。生存胚ではサンプルの呼吸による電流値の変化が確認され、固定胚では電流値の変化がみられなかった。また、半球面拡散理論を用いた解析から、胚４の呼吸量は、4.2×10-15 mol・s-1と算出された。以上の結果から、受精卵呼吸測定用チップ１１を用いた胚呼吸量測定の可能性が示された。この受精卵呼吸測定用チップ１１では、細胞に対して低ストレスな単一基板１２上の操作のみで、マウス受精卵を対象として、微小環境での胚培養および電気化学計測による呼吸評価の両方が可能である。 Embryo 4 settled in a chamber having a length of 750 μm in about 90 to 150 seconds, and was reproducibly introduced into the measurement site as shown in FIG. 16 (c). FIG. 17 shows changes in the oxygen reduction current. In live embryos, changes in the current value due to respiration of the sample were confirmed, and in fixed embryos, no change in current value was observed. From the analysis using hemispherical diffusion theory, the respiration rate of embryo 4 was calculated to be 4.2 × 10 −15 mol · s −1 . From the above results, the possibility of embryo respiration measurement using the fertilized egg respiration measuring chip 11 was shown. In this fertilized egg respiration measurement chip 11, both the embryo culture in a microenvironment and the respiration evaluation by electrochemical measurement are performed on a mouse fertilized egg only by an operation on a single substrate 12 with low stress on the cells. Is possible.
　本発明の実施の形態の受精卵の呼吸活性測定装置および受精卵の呼吸活性測定方法によれば、受精卵呼吸測定チップ１１を使用することにより、電極１３の操作工程を省き、胚を「置くだけ」で測定を行うことができる。実施例１～５において、その受精卵呼吸測定チップ１１の機能評価およびマウス胚の呼吸活性評価を行っている。 According to the apparatus for measuring the respiratory activity of a fertilized egg and the method for measuring the respiratory activity of a fertilized egg according to an embodiment of the present invention, by using the fertilized egg respiration measuring chip 11, the operation step of the electrode 13 is omitted, and the embryo is placed. You can make measurements with just " In Examples 1 to 5, the function evaluation of the fertilized egg respiration measuring chip 11 and the respiratory activity of the mouse embryo are evaluated.
　これまで、体外培養技術の改良や培養培地、培養環境の研究が行われたことを受け、様々な哺乳動物に対して体外受精(IVF, in vitro fertilization)-胚移植が行われ、成功が報告されている。日本においては、高級品種である黒毛和種の効率的な生産などに用いられ、年間５万件以上の実施例が報告されている。また、医療分野においても、1978年にD. C. Steptoe とR. G. Edwards による体外受精児の誕生成功以来、生殖補助技術（ART, assisted reproductive technology）として応用され、不妊症治療の新たなステップとして期待されている。 So far, in vitro fertilization (IVF) -embryo transfer and embryo transfer have been reported to various mammals due to improvements in in vitro culture technology and research on culture media and culture environment. Has been. In Japan, it is used for efficient production of high-grade Japanese black varieties, and more than 50,000 examples are reported annually. Also in the medical field, since the birth of IVF by D. 児 C. Steptoe and R. G. Edwards in 1978, it has been applied as a assisted reproduction technology (ART) and a new treatment for infertility. Expected as a step.
　このような状況の中で、畜産分野および臨床医療分野において、胚の活性を定量的に評価する方法が求められている。そこで、本発明の実施の形態の受精卵の呼吸活性測定装置および受精卵の呼吸活性測定方法により、呼吸活性を根拠とする胚の活性評価を、電気化学的手法を用いた非浸襲的かつ定量的な手法を用いて行った。さらに、微細加工技術を用いて電極１３を基板１２上に集積し、従来の測定装置よりも簡易でコンパクトな受精卵呼吸測定チップ１１の開発を行い、胚の呼吸活性評価を行った。本発明により、呼吸活性を指標とした小型測定システムの開発が可能であると考えられ、畜産分野および医療分野などへの応用が期待される。 Under such circumstances, there is a need for a method for quantitatively evaluating embryo activity in the field of livestock and clinical medicine. Therefore, by using the apparatus for measuring the respiratory activity of a fertilized egg and the method for measuring the respiratory activity of a fertilized egg according to an embodiment of the present invention, the evaluation of the activity of an embryo based on the respiratory activity is performed in a non-invasive manner using an electrochemical technique. A quantitative method was used. Furthermore, the electrode 13 was integrated on the board | substrate 12 using the fine processing technique, the simple and compact fertilized egg respiration measuring chip | tip 11 was developed rather than the conventional measuring apparatus, and the respiratory activity evaluation of the embryo was performed. According to the present invention, it is considered possible to develop a small measurement system using respiratory activity as an index, and application to the livestock field and the medical field is expected.
　１　　球状サンプル
　２，３，４　　胚
　１１　受精卵呼吸測定用チップ
　１２　基板
　１３　電極
　１４　PDMSウェル
　１５　PDMSマイクロ流路
  1 Spherical sample 2, 3, 4 Embryo 11 Fertilized egg respiration measuring chip 12 Substrate 13 Electrode 14 PDMS well 15 PDMS microchannel

Claims (10)

  1. 　基板上に電極を配置して成るチップと、
    　前記電極に一定の電位を印加した状態で、前記電極の近傍に動物の胚を導入したときの、前記胚の導入前の電流値と導入後の電流値を測定する電流測定部と、
    　前記電流測定部で測定された前記胚の導入前の電流値と導入後の電流値に基づいて、前記胚の酸素消費量を求める解析部とを、
    　有することを特徴とする受精卵の呼吸活性測定装置。     A chip comprising electrodes arranged on a substrate;
    In a state where a constant potential is applied to the electrode, when an animal embryo is introduced in the vicinity of the electrode, a current measurement unit that measures a current value before introduction of the embryo and a current value after introduction,
    Based on the current value before the introduction of the embryo measured by the current measurement unit and the current value after the introduction, an analysis unit for obtaining the oxygen consumption of the embryo,
    A device for measuring the respiratory activity of a fertilized egg, comprising:
  2. 　前記電極は、前記電極の酸素還元反応に伴う酸素消費量が前記胚の酸素消費量に与える影響を無視できるよう、前記電極の酸素還元反応に伴う酸素消費量と前記胚の酸素消費量とに基づいて、酸素還元電流値の上限値が定められることを、特徴とする請求項１記載の受精卵の呼吸活性測定装置。 The electrode has an oxygen consumption accompanying the oxygen reduction reaction of the electrode and an oxygen consumption of the embryo so that the influence of the oxygen consumption accompanying the oxygen reduction reaction of the electrode on the oxygen consumption of the embryo can be ignored. 2. An apparatus for measuring the respiratory activity of a fertilized egg according to claim 1, wherein an upper limit value of the oxygen reduction current value is determined based on the oxygen reduction current value.
  3. 　前記電極は酸素還元電流値の上限値が１ｎＡであることを、特徴とする請求項１または２記載の受精卵の呼吸活性測定装置。 3. An apparatus for measuring the respiratory activity of a fertilized egg according to claim 1 or 2, wherein the upper limit of the oxygen reduction current value of the electrode is 1 nA.
  4. 　前記基板は石英ガラス基板から成り、
    　前記チップは少なくとも前記電極が露出するよう、前記基板の表面がＳｉＯ２蒸着絶縁膜により覆われていることを、
    　特徴とする請求項１、２または３記載の受精卵の呼吸活性測定装置。     The substrate comprises a quartz glass substrate;
    The chip is such that the surface of the substrate is covered with a SiO 2 vapor-deposited insulating film so that at least the electrode is exposed.
    The apparatus for measuring respiratory activity of a fertilized egg according to claim 1, 2, or 3.
  5. 　前記チップは前記電極の近傍に前記胚を導入するためのウェルまたは流路構造を有することを、特徴とする請求項１、２、３または４記載の受精卵の呼吸活性測定装置。 The fertilized egg respiratory activity measuring device according to claim 1, 2, 3 or 4, wherein the chip has a well or a channel structure for introducing the embryo in the vicinity of the electrode.
  6. 　前記解析部は、前記胚の導入前の電流値と導入後の電流値に基づいて、球面拡散理論を用いて、前記胚の表面の酸素濃度を求め、その酸素濃度から前記胚の酸素消費量を求めることを、特徴とする請求項１、２、３、４または５記載の受精卵の呼吸活性測定装置。 The analysis unit obtains the oxygen concentration on the surface of the embryo using the spherical diffusion theory based on the current value before the introduction of the embryo and the current value after the introduction, and the oxygen consumption of the embryo from the oxygen concentration The apparatus for measuring the respiratory activity of a fertilized egg according to claim 1, 2, 3, 4 or 5.
  7. 　前記解析部は、あらかじめ前記胚として酸素消費量が既知の試料胚を用いて酸素消費量を求め、その求めた酸素消費量と前記試料胚の既知の酸素消費量とに基づいて補正値を求めておき、前記試料胚とは異なる胚について求めた酸素消費量を前記補正値により補正することを、特徴とする請求項１、２、３、４、５または６記載の受精卵の呼吸活性測定装置。 The analysis unit obtains an oxygen consumption amount in advance using a sample embryo whose oxygen consumption is known as the embryo, and obtains a correction value based on the obtained oxygen consumption amount and the known oxygen consumption amount of the sample embryo. The respiratory activity measurement of a fertilized egg according to claim 1, wherein the oxygen consumption determined for an embryo different from the sample embryo is corrected by the correction value. apparatus.
  8. 　基板上に配置された電極に一定の電位を印加した状態で、前記電極の近傍に動物の胚を導入したときの、前記胚の導入前の電流値と導入後の電流値とに基づいて、前記胚の酸素消費量を求めることを、特徴とする受精卵の呼吸活性測定方法。 Based on the current value before the introduction of the embryo and the current value after the introduction when the animal embryo is introduced in the vicinity of the electrode with a constant potential applied to the electrode arranged on the substrate, A method for measuring the respiratory activity of a fertilized egg, characterized by determining the oxygen consumption of the embryo.
  9. 　前記胚の導入前の電流値と導入後の電流値に基づいて、球面拡散理論を用いて、前記胚の表面の酸素濃度を求め、その酸素濃度から前記胚の酸素消費量を求めることを、特徴とする請求項８記載の受精卵の呼吸活性測定方法。 Based on the current value before the introduction of the embryo and the current value after the introduction, using the spherical diffusion theory, to determine the oxygen concentration of the surface of the embryo, to determine the oxygen consumption of the embryo from the oxygen concentration, The method for measuring the respiratory activity of a fertilized egg according to claim 8.
  10. 　あらかじめ前記胚として酸素消費量が既知の試料胚を用いて酸素消費量を求め、その求めた酸素消費量と前記試料胚の既知の酸素消費量とに基づいて補正値を求めておき、前記試料胚とは異なる胚について求めた酸素消費量を前記補正値により補正することを、特徴とする請求項８または９記載の受精卵の呼吸活性測定方法。
          The oxygen consumption is obtained in advance using a sample embryo whose oxygen consumption is known as the embryo, a correction value is obtained based on the obtained oxygen consumption and the known oxygen consumption of the sample embryo, and the sample The method for measuring the respiratory activity of a fertilized egg according to claim 8 or 9, wherein the oxygen consumption determined for an embryo different from an embryo is corrected by the correction value.
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EP3199637A4 (en) * 2014-09-25 2017-10-04 Panasonic Intellectual Property Management Co., Ltd. Electrochemical measuring method and electrochemical measuring device
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US10619179B2 (en) 2014-09-25 2020-04-14 Panasonic Intellectual Property Management Co., Ltd. Electrochemical measuring method and electrochemical measuring device
CN106661605B (en) * 2014-09-25 2020-10-20 松下知识产权经营株式会社 Electrochemical measurement method and electrochemical measurement device
US10837936B2 (en) 2016-03-11 2020-11-17 Panasonic Intellectual Property Management Co., Ltd. Electrochemical measurement system, electrochemical measurement device, and electrochemical measurement method
WO2018105454A1 (en) * 2016-12-07 2018-06-14 パナソニックＩｐマネジメント株式会社 Electrochemical measuring method and electrochemical measuring device
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