JP3688671B2 - Electrochemical multi-analyte analysis method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrochemical multi-analyte analysis method.
[0002]
[Prior art]
Conventionally, a multititer plate widely applied for the purpose of quantifying the concentration of an analyte contained in a test solution has a microwell with a volume of 3 ml to 100 μl, and continuously analyzes 12 to 96 specimens. Is possible. In this multititer plate analysis, absorption, fluorescence, and luminescence measurement are adopted as detection principles, and in any detection system, there is a device that continuously and fully automatically measures the concentration of the analyte in each container. It is commercially available. In the analysis using this multititer plate, it is a major premise that the concentration of the target substance of interest in the container is uniform.
[0003]
Among the analytical methods using multititer plates, there are many methods that employ electrochemical detection. However, in this method, each of the consecutive containers is independent, and not only the detection electrode (working electrode) but also the reference electrode / counter electrode is generally arranged for each container. Is complicated. In the case of electrochemical detection, a concentration gradient is formed between the sample solution and the electrode surface. This is the concentration gradient formed when the detection current is acquired, and the offshore concentration of the target substance in the container is uniform. This is still a major premise. For analysis systems that combine a relatively small volume container and electrochemical detection for the purpose of high-sensitivity analysis, there is already known a method for obtaining the output of electricity when all target substances existing in the container are reacted. Yes.
[0004]
The present invention is completely different from the concept of the analysis system described above, and the present invention relates to the concentration and concentration gradient of each container arranged in a large number in a pair of electrochemical measurement systems (working electrode for measuring minute current and reference electrode / counter electrode). Provide a continuous measurement method.
[0005]
The present invention also relates to an oxygen consumption measurement method using a well (container) having a unique shape without using a “sample holding device” which is a part of the device configuration in Patent Document 1 below.
[0006]
The present invention makes it possible to easily carry out measurement even by a person who is not familiar with the operation of the apparatus. Furthermore, the container can hold a plurality of samples, and the time required for measurement per embryo can be greatly shortened.
[0007]
A scanning electrochemical microscope is a device that enables measurement of a concentration gradient in the vicinity of a sample surface using a microelectrode as a probe. However, there are currently only a limited number of cases where the mass balance rate between the sample and the solution can be determined from the measurement of the concentration gradient of the target substance. For example, for a sample having a small size (radius of about 100 μm or less) and a simple shape such as a sphere or a circle, the spherical diffusion equation can be applied, and the mass balance rate between the sample and the solution can be quantified. In the present invention, the concentration gradient formed in the container is measured, and the mass transfer rate between the sample and the solution is quantified.
[0008]
The analysis process is not bound by spherical diffusion assumptions and theoretical equations, and the sample need not be spherical. Further, it is not necessary to bring the electrode into close contact with the sample during measurement. Furthermore, the measurement and analysis process can be applied to other than the oxygen consumption measurement of the mammalian embryo, and in that case, the measurement and analysis process is not limited to Patent Document 1.
[0009]
Patent Document 1 discloses a non-invasive quality evaluation method and apparatus for mammalian embryos, and in particular, the normality of an embryo is determined based on the oxygen consumption in each embryo.
[0010]
Non-Patent Document 1 describes a measurement technique related to the concentration distribution of a solution (details will be described later).
Incidentally, an electrochemical analysis apparatus and method using a cell by the present inventors have already been proposed as Japanese Patent Application No. 2002-269651.
[0011]
[Patent Document 1]
JP 2002-122568, pp. 4-6 [Non-Patent Document 1]
Juju et al., Electrochemistry, 69, 806-810 (2001).
[0012]
[Problems to be solved by the invention]
The respiration measurement process in Patent Document 1 uses a manipulator for sample holding and electrode positioning, and complicated processes are inevitable. In addition, there is a problem that the time required for the measurement greatly depends on the training level of the person who performs the measurement.
[0013]
In the present invention, when mass transfer occurs between the sample to be measured arranged in the container and the surrounding solution and a concentration gradient is formed in the solution, the substance between the sample and the solution is determined from the concentration gradient in the container. Quantify travel speed.
[0014]
When the present invention is applied to the respiratory measurement process of Patent Document 1 that evaluates the quality of a mammalian embryo based on the measurement of respiratory volume, improvement can be expected in the following three points, and the measurement can be performed more efficiently. to enable.
[0015]
(1) A measurement well which is not listed in various devices constituting the “electrochemical microscope measurement device for measuring oxygen consumption of a single mammalian embryo” in Patent Document 1 was newly designed. That is, a special device such as a manipulator is not used at all, and the sample is manually placed at a target position. Moreover, the positioning accuracy when the sample is placed in the well is about 10 μm.
[0016]
(2) In the conventional method, the oxygen concentration gradient in the surrounding aqueous solution due to the respiration of the sample is formed in a spherical shape in all directions with the center of the sample as the origin. On the other hand, when the present invention is used, the direction of the formation of the oxygen concentration gradient in the vicinity of the sample due to the respiration of the sample is remarkably narrowed, and the concentration change can be concentrated in a certain direction.
[0017]
(3) A plurality of samples can be arranged on the same container.
[0018]
In view of the above situation, the present invention is an electrochemical multi-analyte capable of quantifying the rate of mass transfer between a sample and a solution by measuring a non-uniform concentration distribution of a measurement object formed in a well. The purpose is to provide an analysis method.
[0019]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides
[1] In the electrochemical multi-analyte analysis method, when there is a mass balance between a sample placed in an inverted conical well formed on a cell base and a solution in the cell surrounding the sample. The mass transfer rate between the sample and the solution is quantified by measuring a non-uniform concentration distribution of the measurement object formed in the inverted conical well.
[0020]
[2] The electrochemical multi-analyte analysis method according to [1], wherein a cell having a plurality of wells is used when measuring the measurement object.
[0021]
[3] In the electrochemical multi-analyte analysis method according to [1], when measuring the measurement object, a concentration gradient of the measurement object exists only in the well, and mass transfer between the plurality of wells It is characterized by using a well constructed such that no is present.
[0022]
[4] The electrochemical multi-analyte analysis method according to [1], wherein the sample is a biological sample.
[0023]
[5] The electrochemical multi-analyte analysis method according to [4], wherein the biological sample is a mammalian embryo or an animal cell.
[0024]
[6] The electrochemical multi-analyte analysis method according to [4], wherein the biological sample is a protein or a nucleic acid.
[0025]
As described above, by using a cell in which a plurality of wells are arranged, a biological sample is arranged in the well, and the concentration distribution in the well is measured, whereby the amount of mass transfer between the sample and the solution can be quantified.
[0026]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
[0027]
FIG. 1 is a schematic view of an electrochemical multi-analyte analysis cell according to the present invention, FIG. 1 (a) is a plan view thereof, and FIG. 1 (b) is a cross-sectional view thereof taken along line AA. FIG. 2 is an enlarged sectional view of the well [FIG.
[0028]
In these figures, 1 is a cell, 2 is a base, 3 is a frame, 4 is a plurality of wells, and 5 is a measurement solution. Incidentally, the dimensions of each part are as follows. The length L ₁ of the base 2 is 80 mm, the width L ₂ of the base ₂ is 30 mm, the thickness L ₃ of the base 2 is 5 mm, the length of the frame 3 (distance between the outer wall and the outer wall) L ₄ is 60 mm, The length of the frame 3 (distance between the inner wall and the inner wall) L ₅ is 50 mm, the outer width L ₂ of the frame 3 is 30 mm, the inner width L ₆ of the frame 3 is 20 mm (therefore, the width L _{7 of the} frame 3 is 10 mm), the height L ₈ of the frame 3 is 5 mm, and the distance (pitch) L ₉ between the wells 4 is 6 mm. Further, as shown in FIG. 2, the shape of the plurality of wells 4 formed in the frame 3 is an inverted conical shape with a radius L ₁₁ and a depth L ₁₂ of 2 mm. The apex angle 2θ of the cone is 90 °.
[0029]
In this electrochemical multi-analyte analysis cell, a sample is placed in the well 4 and a measurement solution (electrically short-circuited by the measurement solution) 5 common to the plurality of wells 4 is provided. electrochemical measuring device system (working electrode and reference electrode / counter electrode for low current measurement) is provided.
[0030]
[A] Calculation of mass transfer rate based on the theoretical formula of spherical diffusion (conventional method)
When a mass balance exists between a spherical sample (radius r _s ) having a uniform concentration of reactive species on the sample surface and the surrounding solution, and the concentration gradient formed on the solution side is steady, the concentration distribution of the solution C (r) is expressed by the following equation with respect to the spherical coordinate axis r (see Non-Patent Document 1).
[0031]
l = (r + r _s )
C (r) = (C _s −C ^* ) r _s / l + C ^* (1)
Here, l is the distance between the electrode tip and the center of the sample, r _s is the sample radius, r is the distance from the sample surface, C _s is the surface concentration, and C ^* is the offshore concentration.
[0032]
From the above equation (1), the probe electrode is scanned from the nearest ground point of the sample to the offshore direction along the horizontal line passing through the center of the sample, and the concentration gradient in the solution is directly measured. Concentration difference (ΔC = C _s −C ^* ) is obtained as the slope of the C (r) vsr _s / (r + r _s ) plot. At this time, the total flux (F ^direct [mols ⁻¹ ]) moving from the entire sample to the solution and the flux density (f _s [molcm ⁻² s ⁻¹ ]) on the sample surface are:
F ^direct = 4πr _s DΔC (2)
f _s = DΔC / r _s (3)
It is represented by
[0033]
In the same experimental system, as shown in FIG. 3, when the probe electrode is scanned in the vertical direction (z-axis direction) from the closest ground point (point A) of the sample, the distance l between the electrode tip and the center of the sample is ^{, l = (z 2 + r} s 2) 0.5
The concentration gradient C (z) in the z-axis direction is
C (z) = ΔCr _s / l + C ^* (1) ′
At this time, ΔC is obtained as the slope of the C (z) vsr _s / l plot.
[0034]
Hereinafter, the above formulas (2) and (3) can be similarly applied.
[0035]
Even when a spherical sample is allowed to stand at the bottom of the inverted conical well, the linearity of the C (z) vsr _s / l plot is good, and ΔC ^well can be obtained.
[0036]
[B] Method for Quantifying Sample Mass Transfer Rate from Concentration Gradient Formed in Well FIG. 3 shows the measurement process. Assuming that a spherical sample (radius r _s ) is placed in the inverted conical well 4, xyz coordinates with the center of the sample as the origin O are set as shown in FIG. Let the apex angle of the inverted cone be 2θ. The distance h between the vertex of the inverted cone and the sample center O is
h = (1 / sin θ) r _s
It is. At z = z ₀ , the surface area S _{z =} z _{0 of the} part of the sphere whose radius is the distance from the apex of the inverted cone (h + z ₀ ) that fits inside the well is:
S _{z =} z ₀ = 2π (1-cos θ) (h + z ₀ ) ²
It is. When an inverted conical well having an apex angle 2θ = 90 ° is used, h = √2r _s , S _{z = z0} = 2π (1-1 / √2) (h + z ₀ ) ² . There is a steady mass balance from offshore to the sample / solution interface, and the total flux is represented by F [mols ⁻¹ ].
[0037]
The total flux F _{z = z0} passing through the spherical surface S _{z = z0} is
F _{z = z0} = ∫f _{z = z0} (x, y, z) dS
Here, S _{z = z0} = ∫dS, f _{z = z0} (x, y, z) is a flux density [molcm ⁻² s ^{− at} an arbitrary coordinate (x, y, z) of the spherical surface S _{z = z0.} ¹ ].
[0038]
From Fick's first law,
f _{z = z0} (x, y, z) = D (∂C / ∂x + ∂C / ∂y + ∂C / ∂z)
Here, D and C are the diffusion coefficient [cm ⁻² s ⁻¹ ] and the concentration [molcm ⁻³ ] of the substance of interest. If f _{z = z0} is uniform in the sphere,
F _{z = z0} = f _{z = z0} S _{z = z0}
It can be expressed. When the amount of material supplied offshore is equal to the amount of material consumed by the sample, the concentration gradient and flux do not change depending on time and converge to a steady state.
[0039]
[C] Concentration gradient in the well (measurement example 1)
Here, alga honey protoplasts were selected as spherical samples. The radius of the honeycomb was 154 μm, the measurement was at room temperature, the solution was seawater, the oxygen diffusion coefficient D = 2.1 × 10 ⁻⁵ cm ² mol ⁻¹ , and the offshore oxygen concentration C ^* = 0.204 × 10 ⁻⁶ molcm ⁻³ .
[0040]
The oxygen concentration gradient in the well was measured by scanning the microelectrode. The local oxygen concentration was determined by scanning the Pt disk electrode (electrode radius 1 μm) with the electrode potential fixed at −0.6 V vs Ag / AgCl and recording the oxygen reduction current.
[0041]
FIG. 4 is a diagram in which points having the same oxygen concentration are plotted in the container.
[0042]
Here, the bold line represents S _z (concentric circle centered on the apex of the well), and the solid line represents S ′ _z (concentric circle centered on the origin).
[0043]
From this figure, it can be seen that the concentration gradient of the well spreads in a spherical shape. The surface area S _z of the part of the sphere centered on the apex of the well that fits inside the well can be expressed by the following equation.
[0044]
S _z = 2π (1-1 / √2) (h + z) ²
On the other hand, the surface area S ′ _z of the part of the sphere centered at the origin that fits inside the well is
S ′ _z = 2π (1-cos ψ) z ²
sinψ = (z−h) / z
(Ψ is the angle of the cone axis and the intersection of sphere S and well)
It can be seen that the iso-concentration surface inside the well agrees well with the spherical surface S _z rather than the spherical surface S ′ _{z when} z <2r _s .
[0045]
The oxygen concentration gradient is recorded in the + z direction with the point B at the coordinates (x, y, z) = (0, 0, r _s ) as the scanning start point (see FIG. 3). As shown in FIG. At the point, the relationship between z and the flux density f _{z = z0} , the sphere area S _{z = z0 of the well} , and the total flux F _{z = z0} passing through the spherical surface was obtained. In FIG. 5, FIG. 5A shows a dimensionless concentration gradient in the z-axis direction from point B in FIG. 3, and FIG. 5B shows z and flux density f _{z = z0 at} several points. FIG. 5C is a diagram illustrating the relationship between z and the sphere area S _z , and FIG. _{5D is} a characteristic diagram of the total flux F _{z = z 0} passing through z and the spherical surface.
[0046]
As is clear from these figures, as z increases, f _z decreases and S _z increases. The total flux F _z agrees well regardless of z. That is, the total flux F _{z = z0} can be obtained by measuring the gradient in the z-axis direction.
[0047]
[D] Comparison of measurement using the conventional method and the wells of the present invention For the honey and bovine embryos having various actual sizes, each single sample was placed in a flat bottom container and an inverted conical well, and the oxygen consumption was reduced. Quantified. First, the oxygen concentration difference ΔC ^direct of the sample placed in the flat bottom container was compared with the concentration difference ΔC ^well obtained by transferring the same sample to the well and similarly quantifying it (FIG. 6).
[0048]
In FIG. 6, FIG. 6 (A) is a diagram showing the relationship between the concentration difference ΔC ^direct quantified in the conventional flat-bottom vessel for ^honey and the concentration difference ΔC ^well quantified in the inverted conical well according to the present invention. (B) is a diagram showing the relationship between the concentration difference ΔC ^direct quantified in a conventional flat bottom container for bovine embryos and the concentration difference ΔC ^well quantified in an inverted conical well in the present invention, where ΔC ^direct is the above formula. Obtained from (1). ΔC ^well was obtained from equation (1) ′ by scanning the probe electrode in the vertical direction (z-axis direction) from point A in FIG. As a result, both honey and bovine embryos, regardless of size,
ΔC ^well = 2 ・ ΔC ^direct
Was obtained. In other words, it was found that when the inverted conical well is used, the change in the oxygen concentration of the sample can be observed after being amplified by about 2 compared to the conventional method.
[0049]
Next, the oxygen consumption amount F ^direct of the sample placed in the flat bottom container was compared with the oxygen consumption amount F _z calculated in accordance with the above-mentioned process by transferring the same sample to the well (FIG. 7). The sample used was a bovine embryo (embryo cultured for 1, 5, 6, 8 days after in vitro fertilization). In FIG. 7, ● represents the case where the probe electrode was scanned in the vertical direction from the point B in FIG. 3, and ○ represents the case where the probe electrode was scanned in the vertical direction from the point A in FIG. 3.
[0050]
Result of comparison F _z and F ^direct, regardless of the differences in size and the scanning start point of the sample,
F _z = 0.8 ・ F ^direct
Was obtained. The angle of the oxygen supply is limited in the inverted conical well as compared with the flat bottom container, and it is considered appropriate that F _z / F ^direct is smaller than 1.
[0051]
The following was found from the above measurement examples.
[0052]
When a sample of the order of 10 to several hundred μm is placed in an inverted conical well or similar container and the mass balance speed between the sample and the solution around it is quantified, [in this case, the reverse of radius 2 mm, depth 2 mm In the case of installing a honeybee or bovine embryo in a conical well, and quantifying the respiratory activity (oxygen consumption rate) of the sample], at a distance relatively far from the sample, the presence of flux density and mass transfer distributed in the container The total mass balance between the solution and the sample can be determined by the product of the cross-sectional area to be measured. Here, "relatively large distance from a sample", for example, in the case of a spherical sample of radius r _s, may be a z> 2r _s.
[0053]
Further, it can be seen from FIG. 4 that the concentration gradient between the sample and the solution exists only inside the well, and there is no mass transfer between the individual wells. Therefore, each well maintains independence without being disturbed by adjacent wells. The enzyme immunoassay is usually carried out using a 96-well plate (12 cm × 8 cm). However, the present invention can be expected to reduce the size of the plate and the amount of sample / reagent used.
[0054]
FIG. 8 is a schematic plan view of a cell when 96 wells to which the present invention can be applied are arranged.
[0055]
In this figure, 96 wells 13 are arranged in the cell 10. Incidentally, the length L ₂₁ of the base 11 is 80 mm, the width L _{22 of the} base 11 is 50 mm, the length of the frame 12 (distance between the outer wall and the outer wall) L ₂₃ is 70 mm, and the length of the frame 12 (with the inner wall) the inner wall of the distance) L ₂₄ is 60 mm, the outer width L ₂₂ of the frame 12 is 50 mm, inner width L ₂₅ of the frame 12 is 40 mm.
[0056]
In the above embodiment, algal honey and bovine embryos have been described as biological samples, but they are widely applied to biological samples such as mammalian embryos, animal cells, proteins (antigens, antibodies, enzymes) and nucleic acids (DNA, RNA). Needless to say, you can.
[0057]
In addition, this invention is not limited to the said Example, A various deformation | transformation is possible based on the meaning of this invention, and these are not excluded from the scope of the present invention.
[0058]
【The invention's effect】
As described above in detail, according to the present invention, the following effects can be obtained.
[0059]
(A) When there is a mass balance between a sample arranged in a plurality of wells and a solution surrounding the sample, the sample- The mass transfer rate between solutions can be quantified.
[0060]
(B) It is possible to continuously measure each of a large number of measurement object containers arranged one by one.
[0061]
(C) The shape of the measurement target (simple shape such as a sphere or a circle) is not limited and has a wide application range.
[0062]
(D) Furthermore, when measuring respiration of a mammalian embryo, the operability can be improved, and the time required for measurement per embryo can be greatly shortened.
[0063]
According to the present invention, full automation of multi-sample / multi-item measurement can be expected for the first time in an analytical chemistry approach based on a scanning electrochemical microscope. In addition to respiratory measurement of mammalian embryos, application to enzyme immunoassay can be expected.
[Brief description of the drawings]
FIG. 1 is a schematic view of an electrochemical multi-analyte analysis cell according to the present invention.
FIG. 2 is an enlarged cross-sectional view of a well of an electrochemical multi-analyte analysis cell according to the present invention.
FIG. 3 is an explanatory diagram of a method for quantifying a mass transfer rate of a sample from a concentration gradient formed in a well according to the present invention.
FIG. 4 is a diagram in which points having the same oxygen concentration are plotted in a container according to the present invention.
FIG. 5 is a diagram showing the relationship between z, flux density f _{z = z0} , well sphere area S _{z = z0} , and total flux F _{z = z0} passing through a spherical surface at several points according to the present invention. .
FIG. 6 is a diagram showing the relationship between a concentration difference ΔC ^direct quantified in a conventional flat bottom container for ^honey and a concentration difference ΔC ^well quantified in an inverted conical well in the present invention, and a bovine embryo in a conventional flat bottom container. a concentration difference [Delta] C ^direct quantified, is a diagram showing the relationship between quantitative the concentration difference [Delta] C ^well in inverted conical well according to the present invention.
FIG. 7 is a graph showing the relationship between the oxygen consumption F ^direct quantified in a conventional flat bottom container and the oxygen consumption F _z quantified in an inverted conical well according to the present invention for each single sample of a bovine embryo. It is.
FIG. 8 is a schematic plan view of a cell when 96 wells to which the present invention can be applied are arranged.
[Explanation of symbols]
1,10 cell 2,11 base 3,12 frame 4,13 multiple wells 5 measuring solution

Claims (6)

If the material balance exists between the solution in the cell which surrounds the sample and the sample placed in the well of the inverted cone shape formed on the base of the cell, which is formed in the well of the reverse conical shape An electrochemical multi-analyte analysis method characterized in that the mass transfer rate between the sample and the solution is quantified by measuring a non-uniform concentration distribution of a measurement object.   2. The electrochemical multi-analyte analysis method according to claim 1, wherein a cell having a plurality of wells is used when measuring the measurement object.   2. The electrochemical multi-analyte analysis method according to claim 1, wherein when measuring the measurement object, a concentration gradient of the measurement object exists only in the well, and mass transfer between a plurality of wells does not exist. A method for electrochemical multi-analyte analysis, comprising using a well constructed as described above.   2. The electrochemical multi-analyte analysis method according to claim 1, wherein the sample is a biological sample.   5. The electrochemical multi-analyte analysis method according to claim 4, wherein the biological sample is a mammalian embryo or an animal cell.   5. The electrochemical multi-analyte analysis method according to claim 4, wherein the biological sample is a protein or a nucleic acid.

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