Capillary array electrophoresis device

Abstract

The invention provides a capillary array electrophoresis device. The laser irradiation units of N capillary tubes having capillary tube numbers n = 1, 2,..., N are arranged on the same plane, and when the laser irradiation intensity of each capillary tube is set as L (n) and the output intensity of each capillary tube obtained by a computer when a light-emitting substance having the same concentration is present in each capillary tube is set as H (n), the laser irradiation intensity of each capillary tube is set as L (n) and the output intensity of each capillary tube is set as H (n). The absolute value of the average value of the second derivative of H (n) is made smaller than the absolute value of the average value of the second derivative of L (n) for any refractive index (n3) on the basis of digital correction obtained by a computer that varies in accordance with the refractive index (n3) of the separation medium (1.33 < = n3 < = 1.41) (fig. 11).

Description

[0001] This application is a divisional application: the parent application was filed on December 18, 2020, with application number "2020801071760" and invention title "Capillary Array Electrophoresis Device". Technical Field

[0002] This disclosure relates to a capillary array electrophoresis apparatus. Background Technology

[0003] Capillary array electrophoresis devices are widely used, employing multiple quartz glass capillaries filled with electrolyte solutions or electrolyte solutions containing polymer gels or other electrophoretic separation media for parallel electrophoretic analysis. Compared to existing capillary electrophoresis devices using a single capillary, capillary array electrophoresis devices not only increase analytical throughput but also reduce the cost per sample. The most widely used capillary array electrophoresis devices are the Thermo Fisher Scientific 3500 series and 3730 series gene analyzers. The 3500 series gene analyzer can perform parallel electrophoretic analysis with 8 or 24 capillaries, while the 3730 series gene analyzer can perform parallel electrophoretic analysis with 48 or 96 capillaries. In either case, the laser irradiation sections (the parts of the capillary array that are irradiated by the laser) of the multiple capillaries are arranged in the same plane with the polyimide coating removed. The same plane is called the alignment plane, and the arrangement of multiple capillaries is called a capillary array. When the capillary array consists of N capillaries, each capillary is numbered from 1 to N according to the arrangement order starting from its end. During electrophoresis, a laser beam is introduced from the side of the alignment plane to simultaneously irradiate multiple capillaries, and the emitted fluorescence is dispersed from each capillary and detected simultaneously. The method of simultaneously irradiating multiple capillaries by introducing a laser beam from the side of the alignment plane is called the multifocal method, which is described in detail in Patent Document 1. In the multifocal method, each capillary acts as a convex lens, and the laser beam is repeatedly focused along the alignment plane and advanced within the capillary array, enabling simultaneous irradiation of multiple capillaries. Thus, the DNA sequence or DNA fragment analysis of a sample equal to the number of capillaries can be performed in parallel. As described in Patent Document 1, in a laser irradiation section with multiple capillaries, when the outer radius of the capillary is set to R (outer diameter is 2R), the inner radius is set to r (inner diameter is 2r), the refractive index of the raw material of the capillary is set to n2, the refractive index of the medium outside the capillary is set to n1, the refractive index of the medium inside the capillary (separation medium) is set to n3, and the distance between the incident position of the laser beam and the arrangement plane is set to x (≤r) and set to x = r / 2, the refraction angle of the laser beam when it passes through a capillary is expressed by Equation (1).

[0004] Formula 1

[0005]

[0006] Each capillary acts as a concave lens when Δθ > 0 and as a convex lens when Δθ < 0. The condition Δθ < 0 enables the multifocal system to function, allowing simultaneous irradiation of multiple capillaries using a laser beam. Conversely, if Δθ > 0, the multifocal system does not function, and the laser beam diverges from the plane of arrangement, thus preventing simultaneous irradiation of multiple capillaries using a laser beam. Generally, the raw material for the capillaries is quartz glass, and n2 = 1.46 is a fixed value. According to equation (1), to enhance the convex lens effect of each capillary (weaken the concave lens effect), the smaller n1 and the larger n3, the better. Conversely, the larger n1 and the smaller n3, the stronger the concave lens effect of each capillary.

[0007] Even when multifocal focusing is used, the intensity of the laser beam decreases as it travels through the capillary array due to reflection losses at the interfaces between the external medium and the capillary, and between the internal medium and the capillary. Consequently, the fluorescence intensity also decreases. If the fluorescence intensity varies significantly between capillaries, it is unsuitable because multiple samples cannot be analyzed under identical conditions (in the following embodiments, fluorescence intensity is used as a representative signal intensity, but other signal intensities, such as scattering intensity or absorbance, can also be used). Therefore, in the 3500 series and 3730 series gene analyzers, a laser beam excited from a single laser source is split into two beams, which are then incident from opposite sides of the array plane, and each beam is used for multifocal focusing. This results in a more uniform sum of the intensity of the laser beam incident from one side of the array plane and the intensity of the laser beam incident from the other side. A structure where a laser beam enters from only one side of the array plane is called single-sided illumination, and a structure where a laser beam enters from both sides of the array plane is called double-sided illumination. Whether single-sided or double-sided illumination is used, the multifocal function remains the same. When the capillary array consists of N capillaries, in the case of single-sided illumination, the capillary at the end of the side receiving the laser beam is numbered 1, and the capillary at the end of the side emitting the laser beam is numbered N. In the case of double-sided illumination, the capillary at either end is numbered 1, and the capillary at the opposite end is numbered N.

[0008] In DNA sequence or fragment analysis using the 3500 series and 3730 series gene analyzers, since the DNA fragments in the sample are separated by electrophoresis in a single-stranded state, a polymer solution containing a high concentration of urea as a denaturing agent is used as the separation medium. In fact, the separation media POP-4, POP-6, and POP-7, sold for use with the 3500 series and 3730 series gene analyzers, all contain 8M urea. Compared to the refractive index of water (1.33), the refractive index of the aforementioned polymer solution containing 8M urea increases to n3 = 1.41. This enhances the convex lensing effect of each capillary, creating conditions favorable for multifocal focusing.

[0009] Based on the structure in Patent Document 1, the 3500 series gene analyzer has a laser irradiation section with multiple capillaries having an outer diameter of 2R = 323 μm and an inner diameter of 2r = 50 μm, arranged in air. That is, n1 = 1.00. At this time, according to the above formula (1), Δθ = -1.3°, and each capillary acts as a convex lens. Therefore, the multi-focal function can be performed, enabling simultaneous irradiation of 8 or 24 capillaries using a laser beam. However, in this structure, due to the large reflection loss of the laser beam at the interface between the air layer outside the capillary and the capillary (quartz glass), the maximum number of capillaries that can be irradiated simultaneously is about 24.

[0010] Therefore, based on the structure shown in Patent Document 2, the structure that increases the number of capillary roots that can be irradiated simultaneously is the following 3730 series gene analyzer. In the 3730 series gene analyzer, a laser irradiation section with multiple capillary roots having an outer diameter of 2R = 126 μm and an inner diameter of 2r = 50 μm is arranged in a fluorine solution with a refractive index of n1 = 1.29. At this time, according to the above formula (1), Δθ = -0.69°, each capillary acts as a convex lens, and the multifocal function is realized. Furthermore, since the reflection loss of the laser beam at the interface between the fluorine solution layer outside the capillary and the capillary (quartz glass) is reduced, the number of capillary roots that can be irradiated simultaneously increases. Therefore, it is possible to simultaneously irradiate 48 or 96 capillary roots using a laser beam.

[0011] The structure shown in Non-Patent Document 1 further increases the number of capillaries capable of simultaneous irradiation. In this structure, a laser irradiation section with multiple capillaries having an outer diameter of 2R = 126 μm and an inner diameter of 2r = 50 μm is arranged in a matching solution with a refractive index of n1 = 1.46. Furthermore, among the arranged capillaries, the odd-numbered capillaries from one end are used for analysis (hereinafter referred to as analytical capillaries), and the even-numbered capillaries are used as rod-shaped lenses (hereinafter referred to as lens capillaries). That is, analytical capillaries and lens capillaries are arranged alternately. The refractive index of the medium (separation medium) inside the analytical capillaries is set to n3 = 1.41, and the refractive index of the medium inside the lens capillaries is set to n4 = 1.53. The raw material for all capillaries is quartz glass with n2 = 1.46. Furthermore, since the reflection loss of the laser beam at the interface between the matching solution layer outside the capillary and the capillary (quartz glass) is zero, the number of capillary roots that can be simultaneously irradiated is further increased. Moreover, in Non-Patent Document 1, from page 2874 to page 2875, a definition of the maximum number of capillary roots that can be simultaneously irradiated by a laser beam is described. When irradiation is set to one side and the incident intensity is set to 100%, the maximum number of capillary roots that can be simultaneously irradiated is twice the number of capillary roots whose laser beam intensity attenuates to 50%. When the capillary array having this number of capillary roots is set to irradiate from both sides, it is to achieve uniformity of the irradiation intensity for each capillary. According to this definition, the maximum number of capillary roots in the structure of Patent Document 2 is 150, and the maximum number of capillary roots in the structure of Non-Patent Document 1 is 550.

[0012] Existing technical documents

[0013] Patent documents

[0014] Patent Document 1: Japanese Patent No. 3654290

[0015] Patent Document 2: Japanese Patent No. 5039156

[0016] Patent Document 3: Japanese Patent No. 6113549

[0017] Non-patent literature

[0018] Non-patent literature 1: Electrophoresis 2006, 27, 2869-2879 Summary of the Invention

[0019] The problem that the invention aims to solve

[0020] In all the aforementioned known techniques, the separation medium contains a high concentration of urea, with a refractive index of n3 = 1.41. However, in capillary electrophoresis apparatus using a single capillary tube, the separation medium is not limited to containing a high concentration of urea; various other separation media are used. For example, the separation medium used to electrophoretically separate DNA fragments in a double-stranded state does not contain urea and has the same refractive index as water, n3 = 1.33. In other words, generally speaking, the refractive index of the separation medium used in capillary electrophoresis can reach various values ​​from 1.33 ≤ n3 ≤ 1.41. In recent years, to achieve high throughput or low cost in electrophoretic analysis using such various separation media, there has been a demand for using such various separation media in capillary array electrophoresis apparatuses.

[0021] However, in any of the structures described above in the known techniques, if n3 = 1.33, the convex lens effect of each capillary is lost, the concave lens effect becomes stronger, and the multifocal function is not effective. That is, parallel electrophoretic analysis using multiple capillary tubes cannot be performed. Specifically, as follows.

[0022] In the 3500 series gene analyzer based on Patent Document 1, if n3 = 1.33, then according to Equation (1), Δθ = +1.3°, and each capillary acts as a concave lens. Therefore, the multifocal function is not effective, and simultaneous irradiation of 8 or 24 capillaries using a laser beam is not possible.

[0023] In the 3730 series gene analyzer based on patent document 2, if n3 = 1.33, then according to equation (1), Δθ = +2.9°, and each capillary acts as a concave lens. Therefore, the multifocal function is not effective, and simultaneous irradiation of 48 or 96 capillaries using a laser beam is not possible.

[0024] In the structure based on non-patent literature 1, if n3 = 1.33, then according to equation (1), the refraction angle of one analytical capillary is Δθ. A = +6.6°, on the other hand, the refraction angle of a lens capillary is Δθ B = -3.0°. At this time, due to Δθ A +Δθ B = +3.6°, therefore, a set of one analytical capillary and one lens capillary demonstrates the function of a concave lens, and the multifocal structure does not function. Non-Patent Document 1, p. 2875, describes the main idea that the structure of Non-Patent Document 1 also functions advantageously when n3 = 1.33. However, according to the definition of the maximum number of capillary roots in Non-Patent Document 1 above, from Non-Patent Document 1... Figure 11 It can be seen that the maximum number of capillary tubes when n3 = 1.33 is only about 8. Therefore, when n3 = 1.33, the structure in Non-Patent Document 1 does not function.

[0025] On the other hand, Patent Document 3 discloses the following correction method: In a capillary array electrophoresis apparatus, the fluorescence intensity of each capillary (hereinafter referred to as the corrected fluorescence intensity) is obtained by multiplying the measured fluorescence intensity of each capillary (hereinafter referred to as the measured fluorescence intensity) by the correction coefficient of each capillary recorded in advance on the computer, thereby making the fluorescence intensity on the appearance uniform. When the capillary numbers n of the N capillary array are sequentially set from the end to n = 1, 2, ..., N, the measured fluorescence intensity of the capillary number n is set to I(n), the I(n) during calibration is set to I0(n), and the capillary number with the largest I(n) is set to m, the correction coefficient of the capillary number n is taken as k(n) and obtained by Equation (2).

[0026] Formula 2

[0027]

[0028] Furthermore, the corrected fluorescence intensity J(n) of the capillary number n is obtained by equation (3).

[0029] Formula 3

[0030]

[0031] Here, calibration refers to the process of analyzing a standard sample of a certain concentration using all capillaries before analyzing the actual sample. Based on the above correction method, the biased measured fluorescence intensity is converted into a uniform corrected fluorescence intensity using each capillary in the capillary array. In equation (2), k(n) is set to be equal to the fluorescence intensity of each capillary and the fluorescence intensity of the maximum number of capillaries. This correction method assumes that k(n) is stable. For example, when multiple calibrations are performed, it is necessary to ensure that the obtained k(n) does not change significantly.

[0032] In view of this situation, this disclosure proposes a technique that enables electrophoretic analysis in a capillary array electrophoresis apparatus, even when using various separation media with arbitrary refractive indices in the range of 1.33≤n3≤1.41 (and of course, separation media with refractive indices outside the range of 1.33≤n3≤1.41).

[0033] Solution for solving the problem

[0034] To address the aforementioned issues, this disclosure provides, for example, a capillary array electrophoresis apparatus comprising: a laser source emitting a laser beam; a capillary array in which the laser irradiation portions of N capillaries, which are simultaneously irradiated by the laser beam, are arranged substantially on the same plane, wherein N is an integer greater than or equal to 2; an optical system measuring the light emitted from the N capillaries; and a computer applying predetermined processing to the light intensity measured by the optical system and outputting the result, wherein the outer radius of the N capillaries in the laser irradiation portion is defined as R, the inner radius as r, the refractive index of the external medium as n1, the refractive index of the raw material as n2, and the refractive index of the internal medium as n3. Starting from one end of the arrangement, the N capillaries in the laser irradiation section are labeled with capillary numbers n = 1, 2, ..., N. The average value of the second derivative of the convexity of L(n), which is a function of n and represents the intensity of the laser irradiation light of capillary number n, is set as A. The average value of the second derivative of the convexity of H(n), which is a function of n and represents the intensity of the output light of capillary number n obtained by the computer, is set as B when there is an equal concentration of luminescent material inside the N capillaries in the laser irradiation section. At this time, there is at least an analytical mode for capillary electrophoresis using a separation medium with a refractive index n3 < 1.36, and |A| > |B|.

[0035] Further features relating to this disclosure will become clear from the description and drawings in this specification. Furthermore, this disclosure is achieved and implemented through elements, combinations of elements, the detailed description below, and the claims.

[0036] It should be understood that the descriptions in this specification are merely typical examples and are not intended to limit the scope of the claims or applications of this disclosure in any way.

[0037] The effects of the invention are as follows.

[0038] According to the technology disclosed herein, capillary array electrophoresis apparatus can perform electrophoretic analysis using various separation media with arbitrary refractive indices in the range of 1.33 ≤ n3 ≤ 1.41. In particular, it can perform capillary electrophoretic analysis using separation media with low refractive indices, such as 1.33 (the same as or close to the refractive index of water). This significantly expands the application range of capillary array electrophoresis apparatuses, enabling increased analytical throughput and reduced analysis costs per sample. Attached Figure Description

[0039] Figure 1 This is a diagram showing an example of the configuration of a capillary array electrophoresis apparatus.

[0040] Figure 2 This is a diagram illustrating an example of the optical system configuration of a capillary array electrophoresis apparatus.

[0041] Figure 3 This is a diagram illustrating an example of the collaboration between a sensor and a computer.

[0042] Figure 4 The figure shows the structure of the capillary array based on Patent Document 1 and the results of laser beam ray tracing.

[0043] Figure 5 The figure shows the emission fluorescence intensity distribution and the measured fluorescence intensity distribution of the capillary array based on Patent Document 1.

[0044] Figure 6 This is a diagram illustrating the structure of the capillary array of this disclosure and the results of laser beam ray tracing.

[0045] Figure 7 This is a graph showing the emission fluorescence intensity distribution of the capillary array of this disclosure and the measured fluorescence intensity distribution.

[0046] Figure 8 This is a diagram used to illustrate the definition of the arrangement error ΔZ of a capillary array.

[0047] Figure 9 The graph shows the relationship between the arrangement error ΔZ of the capillary array and the emission fluorescence intensity distribution (n3 = 1.41).

[0048] Figure 10 The graph shows the relationship between the arrangement error ΔZ of the capillary array and the emission fluorescence intensity distribution (n3 = 1.33).

[0049] Figure 11 This is a graph showing the relationship between various fluorescence intensity distributions in a capillary array electrophoresis apparatus.

[0050] Figure 12 This is a graph showing the relationship between the arrangement error ΔZ of the capillary array and the output fluorescence intensity distribution (n3 = 1.41, correction reference: n3 = 1.41, ΔZ = 0 μm).

[0051] Figure 13 This is a graph showing the relationship between the arrangement error ΔZ of the capillary array and the output fluorescence intensity distribution (n3 = 1.33, correction reference: n3 = 1.33, ΔZ = 0 μm).

[0052] Figure 14 This is a graph showing the relationship between the arrangement error ΔZ of the capillary array and the output fluorescence intensity distribution (n3 = 1.41, correction reference: n3 = 1.41, ΔZ = 6 μm).

[0053] Figure 15The graph shows the relationship between the arrangement error ΔZ of the capillary array and the output fluorescence intensity distribution (n3 = 1.33, correction reference: n3 = 1.33, ΔZ = 6 μm).

[0054] Figure 16 This is a graph showing the relationship between the arrangement error ΔZ of the capillary array and the output fluorescence intensity distribution (n3 = 1.33, correction reference: n3 = 1.41, ΔZ = 0 μm).

[0055] Figure 17 This is a graph showing the relationship between the luminescence intensity distribution, output fluorescence intensity distribution, and quadratic coefficient of the output fluorescence intensity distribution for capillary arrays with various internal refractive indices n3.

[0056] Figure 18 This is a graph showing the output fluorescence intensity distribution of a capillary array with various correction benchmarks and respect to the internal refractive index n3 of the capillary.

[0057] Figure 19 This is a graph showing the relationship between the arrangement error ΔZ of the capillary array, the relative fluorescence intensity, and the variation coefficient (n3 = 1.41).

[0058] Figure 20 The graph shows the relationship between the arrangement error ΔZ of the capillary array, the digitally corrected relative fluorescence intensity, and the variation coefficient (n3 = 1.41).

[0059] Figure 21 This is a graph showing the relationship between the arrangement error ΔZ of the capillary array, the relative fluorescence intensity, and the coefficient of variation (n3 = 1.33).

[0060] Figure 22 The graph shows the relationship between the arrangement error ΔZ of the capillary array, the digitally corrected relative fluorescence intensity, and the coefficient of variation (n3 = 1.33).

[0061] Figure 23 This is a diagram showing the structure of the capillary array (second one) of this disclosure and the results of laser beam ray tracing.

[0062] Figure 24 The figure shows the emission fluorescence intensity distribution, measured fluorescence intensity distribution, and output fluorescence intensity distribution (n3 = 1.41) of the capillary array (second one).

[0063] Figure 25 The figure shows the distribution of optical system correction coefficients and digital correction coefficients (n3 = 1.41) of the capillary array (second one).

[0064] Figure 26The figure shows the emission fluorescence intensity distribution, measured fluorescence intensity distribution, and output fluorescence intensity distribution (n3 = 1.33) of the capillary array (second one).

[0065] Figure 27 The figure shows the distribution of optical system correction coefficients and digital correction coefficients (n3 = 1.33) of the capillary array (second one).

[0066] Figure 28 This is a diagram illustrating a configuration example of a pattern of polymer A filled in a dual polymer block capillary array electrophoresis apparatus.

[0067] Figure 29 This is a diagram illustrating a configuration example of a pattern of polymer B filled in a dual polymer block capillary array electrophoresis apparatus.

[0068] Figure 30 This is a diagram showing a modified example of a dual polymer block capillary array electrophoresis apparatus. Detailed Implementation

[0069] The technology disclosed herein relates to a capillary array electrophoresis apparatus in which, during electrophoresis using multiple capillaries, a laser beam is simultaneously irradiated onto multiple capillaries, and the fluorescence emitted from each capillary is simultaneously detected, thereby simultaneously analyzing multiple samples.

[0070] (A) Technical Summary of this Disclosure

[0071] This disclosure primarily proposes the following technology: a separation medium with a low refractive index, either equal to or less than 1.36, similar to that of water (1.33). When using such a low-refractive-index separation medium, even when employing the technology disclosed in any of the known examples (Patent Documents 1 to 3 and non-patent documents), multifocal methods do not function, and simultaneous irradiation of multiple capillaries with a laser beam is difficult.

[0072] Furthermore, this disclosure also proposes the following technique: not only using the aforementioned low-refractive-index separation medium, but also using high-refractive-index separation media, typically with a refractive index of 1.36 or higher and 1.42 or lower, for capillary electrophoresis analysis. The more capillaries that can be simultaneously irradiated, the better; it can be set to 8 or more, and depending on the situation, it can be set to 24 or more. The higher the minimum irradiation intensity and fluorescence intensity of each capillary within the same capillary array, the better. Empirically, when the desired fluorescence intensity is set to 1 when irradiating the interior of a capillary with the total intensity of the laser beam excited from the laser source, a practical sensitivity is obtained if the minimum fluorescence intensity MIN is ≥ 0.2. Furthermore, the smaller the deviation in irradiation intensity and fluorescence intensity between multiple capillaries within the same capillary array, the better. Empirically, if the coefficient of variation (CV) of irradiation intensity and fluorescence intensity is ≤ 15%, preferably ≤ 10%, different samples can be analyzed under the same conditions.

[0073] Regarding each capillary in the capillary array, when the outer diameter of the capillary is 2R = 126 μm, the inner diameter of the capillary is 2r = 50 μm, the outside of the capillary is air and n1 = 1.00, the raw material of the capillary is quartz glass and n2 = 1.46, and the inside of the capillary is a separation medium and n3 = 1.33, according to equation (1), since Δθ = -3.2°, each capillary exhibits a convex lens effect, and the multifocal function is realized. In the 3500 series gene analyzer based on Patent Document 1, the difference from the case of n3 = 1.33 is that the outer diameter 2R of the capillary is reduced from 323 μm to 126 μm. Thus, the concave lens effect of each capillary is converted into a convex lens effect. Upon further examination, it can be seen that if the outer diameter 2R of the capillary is below 220 μm, then Δθ < 0, and the convex lens function is realized. Generalizing beyond the case where the capillary inner diameter is 2r = 50 μm, it can be seen that when R / r ≤ 4.4, Δθ < 0, and the convex lens functions. Low refractive index separating media with n3 = 1.33 were not examined in Patent Document 1. That is, this is a condition discovered for the first time using the technology of this disclosure.

[0074] When the capillary outer diameter is 2R = 126 μm and the capillary inner diameter is 2r = 50 μm, and the separation medium inside the capillary has a low refractive index of n3 = 1.34 and 1.35, according to equation (1), Δθ = -3.5° and -3.8°, and each capillary still exhibits the function of a convex lens, with the multifocal structure functioning. Upon further examination, it is found that if the capillary outer diameter 2R is 240 μm or less and 264 μm or less, then Δθ < 0, and the convex lens functions. Generalizing, it is found that when R / r ≤ 4.8 and 5.3, Δθ < 0, and the convex lens functions. Such a low refractive index separation medium was not examined in Patent Document 1. That is, this is a condition first discovered by the technology of this disclosure.

[0075] When experiments were conducted under the aforementioned convex lens conditions, the multifocal function functioned, enabling simultaneous irradiation of multiple capillaries using a laser beam, satisfying the practical performance requirements of MIN≥0.2 and CV≤15%. However, it was clarified that CV≤10% was not met. The fluorescence intensity variation coefficient was suppressed to a lower level under bilateral irradiation compared to unilateral irradiation, but it did not achieve CV≤10%, and depending on the situation, CV≤15% was sometimes not met either. Therefore, in order to reduce CV, a known correction method represented in Patent Document 3 was applied, but the influence caused by the random deviation of the fluorescence intensity of each capillary within the capillary array was clarified: there were cases where the uniformity of fluorescence intensity of each capillary did not function, and conversely, the degree of deviation of fluorescence intensity of each capillary increased.

[0076] Under lateral illumination, the fluorescence intensity distribution, represented by a line graph, shows a convex-concave shape that increases with the increase of the capillary number, and bulges downwards around the center of the capillary array (the capillary array consists of N capillaries; when N is even, the capillary number is around N / 2 or N / 2+1; when N is odd, the capillary number is around (N+1) / 2). (Refer to...) Figure 9 and Figure 10Generally, the greater the degree of unevenness in the former and the greater the degree of downward convexity in the latter, the greater the coefficient of variation (CV). However, in fluorescence intensity distribution, the unevenness of the distribution and the overall downward convexity are observed to be mixed together, making it impossible to distinguish them and derive their degree. On the other hand, according to the review using ray tracing simulation, it has been clarified that the greater the arrangement error ΔZ of the capillary array, the greater the degree of unevenness in the fluorescence intensity distribution; conversely, the smaller n3, the greater the degree of downward convexity in the overall fluorescence intensity distribution. Here, in this specification, the arrangement error ΔZ is defined as follows: First, the Z-axis is set in the direction perpendicular to the arrangement plane, and the arrangement plane is located at Z = 0 μm. Furthermore, the median value of each Z-coordinate of the central axis of the multiple capillaries in the laser irradiation section is zero. Moreover, the absolute value of the Z-coordinate of the central axis of the capillary farthest from the arrangement plane is set as ΔZ. At this time, the Z-coordinates of each central axis of each capillary are dispersed within the range of ±ΔZ. It can be seen that the above-mentioned case of not satisfying CV ≤ 15% occurs when ΔZ is large. Of course, the main reasons for the increased degree of convexity and concavity of the former include not only the arrangement error ΔZ of the capillary array, but also various other experimental errors.

[0077] Based on the above complex situation, this disclosure proposes the following technique: allowing for unevenness in the fluorescence intensity distribution, and modifying the degree of reduction or elimination of the downward convex shape as a whole in the fluorescence intensity of each capillary, thereby most effectively reducing CV. Unevenness in fluorescence intensity is allowed because ΔZ is difficult to determine in each capillary array; furthermore, even if ΔZ is known, the specific shape of the unevenness will randomly vary due to various main reasons. This is for the same reason why the aforementioned known modification method (Patent Document 3) does not function. Furthermore, since this disclosure clarifies that the degree of downward convex shape as a whole in the fluorescence intensity distribution varies according to n3, this disclosure also proposes a technique to dynamically change the correction coefficient according to n3. Such a modification method is not disclosed in known modification methods represented by Patent Document 3. Hereinafter, various embodiments of this disclosure will be described in detail. Each embodiment will be described separately below, and the techniques shown in each embodiment are not exclusive and can be appropriately combined with each other.

[0078] (B) First Implementation Method

[0079] <Example of the configuration of a capillary array electrophoresis apparatus>

[0080] Figure 1 This diagram illustrates an example of the configuration of a capillary array electrophoresis apparatus. In this capillary array electrophoresis apparatus, in addition to DNA sequence and single-stranded DNA fragment analysis performed using conventional capillary array electrophoresis apparatus, double-stranded DNA fragment analysis is also performed. In this embodiment, 24 capillaries are used (but... Figure 1 (Only four capillaries are shown in the diagram). First, DNA sequences of different samples are processed in each capillary. Then, double-stranded DNA fragment analysis of different samples is performed in each capillary. The DNA sequence samples contain single-stranded DNA fragments of various lengths labeled by four fluorophores corresponding to four bases. The electrophoresis separation medium filled into each capillary during DNA sequencing is a polymer solution containing 8M urea as a denaturant, with a refractive index of n3 = 1.41. On the other hand, the double-stranded DNA fragment analysis samples contain double-stranded DNA fragments of various lengths labeled by two fluorophores. The double-stranded DNA fragment labeled by one fluorophore is a PCR product, and the double-stranded DNA fragment labeled by the other fluorophore is a size marker. The electrophoresis separation medium filled into each capillary during double-stranded DNA fragment analysis is a polymer solution without urea as a denaturant, with a refractive index of n3 = 1.33. One analysis session is performed through the following steps (i) to (vi).

[0081] (i) First, the sample injection ends 2 of the 24 capillaries 1 are immersed in the cathode-side buffer solution 6, and the sample dissolution ends 3 are connected to the anode-side buffer solution 7 via the polymer block 9. Here, the 24 sample dissolution ends 3 are bundled together for easy connection to the polymer block 9.

[0082] (ii) Next, the valve 10 of the polymer block 9 is closed, and the piston of the syringe 11 connected to the polymer block 9 is pressed, thereby pressurizing the polymer solution inside and filling the inside of each capillary 1 from the sample dissolution end 3 toward the sample injection end 2.

[0083] (iii) Next, valve 10 is opened, and after different samples are injected into the electric field of each capillary 1 from the sample injection end 2, a high voltage is applied between the cathode 4 and the anode 5 by the power supply 8, thereby initiating capillary electrophoresis. DNA fragments labeled by various fluorescent agents are electrophoresed from the sample injection end 2 toward the sample dissolution end 3.

[0084] (iv) The position of each capillary 1 after electrophoresis at a certain distance from the sample injection end 2 is used as the laser irradiation section 14. The laser beam 13 excited from the laser source 12 is irradiated into the laser irradiation section 14 by means of a multi-focus method. Here, the coating of each capillary 1 near the laser irradiation section 14 is removed in advance. The capillary 1 near the laser irradiation section 14 is arranged on the arrangement plane. After the laser beam 13 is focused, it is injected from the side of the arrangement plane along the arrangement plane. Figure 1 In the simplified depiction, the laser beam 13 is shown as irradiating one side, but in reality, the laser beam 13 is divided into two parts for irradiating both sides.

[0085] (v) Then, the DNA fragments labeled with various fluorophores undergo electrophoresis inside each capillary 1. When passing through the laser irradiation section 14, the labeled fluorophores are excited by the laser beam 13 and emit fluorescence. In other words, various fluorophores emit fluorescence from 24 light-emitting points (laser irradiation sections), and the fluorescence intensity changes constantly during electrophoresis.

[0086] (vi) Finally, the samples injected into each capillary are analyzed by performing multicolor detection on the fluorescence emitted from each luminescent point and analyzing the obtained time series data.

[0087] The steps (i) to (vi) above are applicable to both DNA sequencing and double-stranded DNA fragment analysis, with appropriate changes to the polymer solution and buffer solution. Furthermore, the analysis session consisting of steps (i) to (vi) can be repeated multiple times. For example, by setting the analysis session to analyze samples 1-24 in the first session, samples 25-48 in the second session, and so on, multiple different samples can be analyzed. In this case, the same polymer solution and buffer solution can be used repeatedly for DNA sequencing, or the analysis can be switched to double-stranded DNA fragment analysis midway. Any application can be selected in any analysis session.

[0088] <Example of the configuration of an optical system for fluorescence detection>

[0089] Figure 2 This is a cross-sectional view showing an example of the configuration of an optical system for fluorescence detection in a capillary array electrophoresis apparatus. This optical system is located in... Figure 1 The inner side of the laser irradiation section 14. With Figure 1 same, Figure 2 The image depicts unilateral irradiation of 4 capillary arrays, but in reality, unilateral irradiation of 24 capillary arrays is performed.

[0090] Because the laser beam 13 utilizes multi-focal irradiation, it simultaneously irradiates each capillary 1 arranged on the array plane. The laser irradiation section 14 of each capillary 1 becomes a fluorescent emission point 20. The emitted fluorescence 21 from each emission point 20 is collimated by a condenser lens 15, the laser beam is cut off by a laser scribing filter 16, and transmitted through a transmission-type diffraction grating 17, thereby dispersing the wavelength along the central axis of each capillary. An imaging lens 18 then images each of these points onto a sensor 19, forming an imaging point 22. The sensor 19 can be a CCD, CMOS, or a photodiode array, etc., capable of simultaneously measuring multiple imaging points 22. Each imaging point 22 is actually located in... Figure 2 Wavelength dispersion along the depth direction, while Figure 2 In the figure, the single wavelength portion of each imaging point 22 is schematically depicted.

[0091] In such an optical system, as the light-emitting point 20 moves away from the optical axis 23 of the optical system, the light-gathering efficiency of the light emitted from the light-emitting point 20 decreases. This is because: Figure 2 As shown, the focusing angle of the emitted fluorescence 21 from the emission point 20, which is located away from the optical axis 23, decreases due to the vignetting effect of the optical system. Therefore, even if fluorescence of equal intensity is emitted from each emission point 20, the fluorescence intensity of the corresponding imaging point 22 decreases as the emission point 20 moves away from the optical axis 23. The degree of vignetting effect is determined by the optical system, i.e., the optical system correction coefficient based on the vignetting effect, and can be investigated by calculation or experiment. The fluorescence intensity of each imaging point 22 can be calculated from the fluorescence intensity of each emission point 20 using the optical system correction coefficient based on the vignetting effect.

[0092] <System Configuration Example for Data Parsing and Device Control>

[0093] Figure 3 This illustrates an example of the collaboration between a sensor and a computer. The optical system is part of the capillary array electrophoresis apparatus, and the sensor is part of the optical system. The computer is connected to the capillary array electrophoresis apparatus. The computer performs not only data analysis but also controls the capillary array electrophoresis apparatus. Conditions for data analysis and capillary array electrophoresis apparatus control are set via a touch panel, keyboard, mouse, etc., which serve as input devices. The timing raw data of the signals output from the sensor is sequentially stored in memory. Furthermore, the analysis parameter information stored in a database located inside the HDD is also stored in memory. The CPU uses the analysis parameter information stored in memory to analyze the timing raw data stored in memory, derives timing analysis data, and sequentially stores it in memory, while simultaneously displaying it on a monitor serving as the display unit. Furthermore, the analysis results can be compared with information on the network via the Network Interface Function (NIF).

[0094] <Examples of existing capillary array configurations>

[0095] Figure 4 The upper part is a structural cross-sectional view of the capillary array of the 3500 series gene analyzer based on Patent Document 1. The laser irradiation sections of 24 capillaries with an outer diameter 2R = 323 μm and an inner diameter 2r = 50 μm are arranged on the same plane at intervals of 370 μm. The arrangement error is zero (ΔZ = 0 μm). The outside of the capillaries is air with n1 = 1.00, and the capillary material is quartz glass with n2 = 1.46. Figure 4The middle section shows the laser beam tracing results when a φ50μm laser beam is irradiated from the left side under the above conditions, with a high refractive index separating medium inside the capillary and n3 = 1.41. The multifocal configuration clearly functions effectively, irradiating the interior of all 24 capillaries. This corresponds to: according to equation (1), Δθ = -1.3°, each capillary exhibits a convex lens effect. In contrast, Figure 4 The lower part shows the same laser beam tracing results under the above conditions, where the capillary interior is a low-refractive-index separating medium and n3 = 1.33. Clearly, the multifocal configuration is ineffective; the laser beam diverges from the capillary array and cannot effectively illuminate the entire array. This corresponds to Δθ = +1.3° according to equation (1), where each capillary exhibits a concave lens effect.

[0096] <Based on existing ( Figure 4 The relative fluorescence intensity distribution formed by the capillary array of ) >

[0097] Figure 5 The upper part shows the rewriting under the condition of bilateral illumination. Figure 4 The middle and lower portions of the graph show the relative fluorescence intensities of each capillary under unilateral irradiation conditions. Figure 4 The leftmost capillary is designated as 1, and subsequent numbers are sequentially labeled towards the right. The relative fluorescence intensity is calculated based on the irradiation intensity of each capillary after adding the laser beam reflection loss, assuming a certain concentration of phosphor in the laser-irradiated section of each capillary. The expected fluorescence intensity is set to 1 when the total intensity of the laser beam excited from the laser source irradiates the interior of a single capillary. In the calculation for irradiation from both sides, half of the total laser beam intensity is assumed to be irradiated from both sides of the capillary array. With n3 = 1.41, the minimum relative fluorescence intensity of the 24 capillary tubes is MIN = 0.42, and the coefficient of variation (= standard deviation of relative fluorescence intensity / average value of relative fluorescence intensity) is CV = 11%, satisfying the practical performance requirements of MIN ≥ 0.2 and CV ≤ 15%. However, the more desirable condition of CV ≤ 10% is not satisfied. The downward convex distribution of the relative fluorescence intensity relative to the capillary number is unrelated to whether the multifocal system functions correctly, but rather because the intensity of the laser beam attenuates due to reflection loss as it travels within the capillary array. In contrast, with n3 = 1.33, we get MIN = 0.068 and CV = 74%, which means neither satisfies any practical performance.

[0098] Here, with multifocal operation and zero arrangement error, a simpler method is used to derive the relative fluorescence intensity distribution of the capillary array. This method is performed for the first time in this disclosure. To approximate the transmittance of the laser beam after considering reflection losses, the incident angle of the laser beam incident on the interface of two media with different refractive indices is assumed to be 0°. The reflectance when light is incident at an incident angle of 0° on the interface between a medium with refractive index n1 and a medium with refractive index n2 is ref = {(n1 - n2) / (n1 + n2)}. 2 The transmittance is tra = 1 - ref. Therefore, the transmittance T of the laser beam when it passes through a capillary can be approximately calculated by the following equation (4).

[0099] Formula 4

[0100]

[0101] exist Figure 4 Under the conditions of the 3500 series gene analyzer based on Patent Document 1 shown in the middle part, since n1 = 1.00, n2 = 1.46, and n3 = 1.41, T = 93% is calculated according to Equation (4). In reality, the transmittance of the component containing the laser beam with an incident angle of not 0° is a value slightly smaller than that of Equation (4). Therefore, Equation (4) shows the ideal transmittance. In the case of unilateral irradiation, when the laser irradiation intensity of the capillary with capillary number n = 1 is set to 1, the laser irradiation intensity L(n) of the capillary with capillary number n is expressed by the following Equation (5).

[0102] Formula 5

[0103]

[0104] In other words, in the aforementioned 3500 series gene analyzer, when the number of capillaries is N=24, the laser irradiation intensity decreases by 93% for each capillary in the capillary array, and the laser irradiation intensity of the capillary with n=24 decreases to 0.19. On the other hand, under the condition of irradiation from both sides, when the laser irradiation intensity of the capillary with capillary number n=1 and n=N is set to 0.5 respectively, the laser irradiation intensity L(n) of the capillary with capillary number n is expressed by the following formula (6).

[0105] Formula 6

[0106]

[0107] Unlike the case of unilateral illumination, the uniformity of laser irradiation intensity in each capillary is improved due to the attenuation and cancellation of the laser beam intensity incident from both sides of the array plane, and the minimum laser irradiation intensity is increased. Specifically, the capillaries located at the ends of the capillary array (n=1 and n=N) have the highest laser irradiation intensity, while the capillaries located in the center of the array (n=(N+1) / 2 when N is odd, and n=N / 2 and n=N / 2+1 when N is even) have the lowest laser irradiation intensity. That is, as... Figure 5 The relative fluorescence intensity distribution in the upper part of the graph with n3 = 1.41 is shown, and when a line graph is formed with the horizontal axis n and the vertical axis L(n), it becomes a downward convex distribution. Under the conditions of the 3500 series gene analyzer described above, when the number of capillaries is N = 24, according to equation (6), the laser irradiation intensity of the capillaries (n = 1 and n = 24) at both ends of the capillary array is 0.60, and the laser irradiation intensity of the capillaries (n = 12 and n = 13) in the center of the capillary array is 0.44 (MIN = 0.44), which satisfies the practical performance requirement of MIN ≥ 0.2. Furthermore, the coefficient of variation of the laser irradiation intensity of the 24 capillaries is 11% (CV = 11%), which satisfies the practical performance requirements of CV ≤ 20% and CV ≤ 15%. The above results are consistent with the above... Figure 5 The results for the upper part, where n3 = 1.41, show MIN = 0.42 and CV = 11%, which are roughly consistent. That is, with the multifocal function working and with zero arrangement error, the laser irradiation intensity distribution L(n) can be obtained using the simple method described above.

[0108] Figure 5 The lower part shows the... Figure 4 The results from the upper part, after incorporating the vignetting effect of the 3500 series gene analyzer's optical system, were multiplied by an optical system correction factor based on the vignetting effect to obtain the optical system-corrected relative fluorescence intensity for each capillary. With n3 = 1.41, the downward convex distribution of relative fluorescence intensity relative to the capillary number cancels out the optical system correction distribution, resulting in a flattened optical system-corrected relative fluorescence intensity distribution. As a result, the minimum fluorescence intensity MIN = 0.42 remains unchanged, but the coefficient of variation is significantly reduced to CV = 0.76%. Furthermore, it satisfies the practical performance requirements of MIN ≥ 0.2 and CV ≤ 15%, further satisfying CV ≤ 10%. In contrast, with n3 = 1.33, MIN = 0.066 and CV = 61% remain essentially unchanged, and the failure to meet practical performance requirements remains unaffected.

[0109] <Example of the configuration of the capillary array in this embodiment>

[0110] Figure 6The upper part is a structural cross-sectional view of the capillary array based on this embodiment. The laser irradiation sections of 24 capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm are arranged on the same plane at intervals of 155 μm. The arrangement error is zero (ΔZ = 0 μm). The outside of the capillaries is air with n1 = 1.00, and the capillary material is quartz glass with n2 = 1.46. Figure 6 The middle section shows the laser beam tracing results when a φ50μm laser beam is irradiated from the left side under the above conditions, with a high refractive index separating medium inside the capillary and n3 = 1.41. The multifocal function is clearly effective, efficiently irradiating the interior of all 24 capillaries. This corresponds to Δθ = -5.8° according to equation (1), where each capillary acts as a convex lens.

[0111] In contrast, Figure 6 The lower part shows the same laser beam tracing results under the above conditions, where the inside of the capillary is a low-refractive-index separation medium and n3 = 1.33. Similarly, in this case, the multifocal function is clearly effective, efficiently illuminating the interior of all 24 capillaries. This corresponds to Δθ = -3.2° according to equation (1), where the capillary acts as a convex lens. Thus, regardless of whether it is a high-refractive-index separation medium (n3 ≥ 1.36) or a low-refractive-index separation medium (n3 < 1.36), each capillary acts as a convex lens, and the multifocal function is achieved—something that cannot be achieved in any known example, but is achieved for the first time by the technology of this disclosure. That is, in the capillary array electrophoresis apparatus of this embodiment, the multifocal function is effective in both the analysis mode where n3 < 1.36 and the analysis mode where n3 ≥ 1.36. Furthermore, in each analysis mode, it is effective to appropriately change the conditions of the electrophoretic analysis according to each purpose. The conditions for electrophoretic analysis that can be modified include the capillary control temperature, the electric field strength during electrophoresis, the electric field strength and sample injection time, the laser irradiation intensity, and the sensor exposure time. For example, the capillary temperature can be adjusted to 30°C in one analysis mode and to 60°C in other analysis modes. It is effective to change the capillary control temperature in each analysis mode.

[0112] According to this embodiment ( Figure 6 The relative fluorescence intensity distribution formed by the capillary array of ) >

[0113] Figure 7 The upper part shows the rewriting under the condition of bilateral illumination. Figure 6The middle and lower portions show the relative fluorescence intensities of each capillary under unilateral irradiation conditions. With n3 = 1.41, the minimum relative fluorescence intensity obtained for 24 capillaries is MIN = 0.42, with a coefficient of variation (CV) of 11%, indicating that the practical performance requirements of MIN ≥ 0.2 and CV ≤ 15% are met. However, compared with... Figure 5 The upper part is the same, and the CV ≤ 10% does not satisfy the more preferred condition. On the other hand, when n3 = 1.33, it is the same as... Figure 5 The upper part is different; MIN = 0.40 and CV = 12%, indicating that the practical performance is satisfied. However, in this case, CV ≤ 10% is not satisfied either.

[0114] Figure 7 The lower part shows the... Figure 7 The results from the upper part of the dataset, after incorporating the vignetting effect of the 3500 series gene analyzer's optical system, are multiplied by an optical system correction factor based on the vignetting effect to obtain the optical system-corrected relative fluorescence intensity for each capillary. The optical system correction used here is the same as that used in... Figure 5 The optical system corrections used are the same. The results of the optical system corrections are as follows: when n3 = 1.41, MIN = 0.42 and CV = 9.0%; when n3 = 1.33, MIN = 0.40 and CV = 10%, satisfying the practical performance requirements of MIN ≥ 0.2 and CV ≤ 15%, and further satisfying CV ≤ 10%.

[0115] according to Figure 7 ,and Figure 5 In contrast, the relative fluorescence intensity did not change significantly with or without optical system correction. This is because: Figure 4 The full width of the capillary array is 370μm spacing × (24 segments - 1 segment) = 8.5mm. In contrast, Figure 6 The capillary array has a relatively narrow full width of 155μm × (24 capillaries - 1 capillary) = 3.6mm, meaning that each capillary is relatively close to the optical axis, resulting in a smaller vignetting effect in the optical system. Consequently, in Figure 4 In the capillary array of the 3500 series gene analyzer shown, with n3 = 1.41, optical system correction based on the vignetting effect of the optical system significantly reduced the CV from 11% to 0.76%. In contrast, Figure 6 In the capillary array of this disclosure, when n3 = 1.41, the CV decreases only from 11% to 9% through optical system correction based on the vignetting effect of the optical system. Similarly, in the capillary array of this disclosure, when n3 = 1.33, the CV decreases only from 12% to 10% through optical system correction based on the vignetting effect of the optical system.

[0116] However, it is clear from the structure of this embodiment that when a separation medium with an arbitrary refractive index of n3 ≥ 1.33 is used while n3 = 1.41 is included, each capillary acts as a convex lens, and the multifocal function is achieved. Furthermore, as a variation of this structure, for any capillary with R / r ≤ 4.4, for example, when the inner diameter is fixed at 2r = 50 μm, and an arbitrary capillary with an outer diameter of 2R ≤ 220 μm is used, under the condition that n3 ≥ 1.33, each capillary acts as a convex lens, thus enabling the multifocal function to be achieved.

[0117] (C) Second Implementation

[0118] In the first embodiment, the results of the examination were shown when the arrangement error of the capillary array was zero (ΔZ = 0 μm). However, in reality, ΔZ = 0 μm is not always the case. Therefore, in this embodiment, the relationship between arrangement error, multifocal performance, and the relative fluorescence intensity of each capillary is systematically examined. This examination is a first of its kind using the technology of this disclosure.

[0119] <Definition of Arrangement Error of Capillary Array>

[0120] Figure 8 This is a diagram illustrating the definition of the arrangement error ΔZ. Figure 8 The upper part shows a cross-sectional view of a 24-capillary array with zero alignment error (ΔZ = 0 μm). This structure is similar to... Figure 6 The upper part is the same. Taking the position of the central axis of the leftmost capillary (capillary number 1) as the origin, the X-axis is set along the arrangement plane, and the Z-axis is set in a direction perpendicular to the arrangement plane. Furthermore, the Y-axis is set along the central axis of the leftmost capillary. The central axis of each capillary lies on the X-axis, and the Z-coordinate is zero. In contrast, Figure 8 The lower part shows this cross-sectional view under the condition of arrangement error (ΔZ≠0μm). The X coordinate of the central axis of each capillary is shown in the figure. Figure 8 The situation is the same for the upper part, but the Z-coordinates deviate randomly upwards and downwards from the X-axis (Z = 0 μm). Here, ΔZ is set as the maximum absolute value of each Z-coordinate. That is, the distance between the central axis of the capillary furthest from the X-axis and the X-axis is set as ΔZ. At this time, the Z-coordinates of the central axes of each capillary are randomly dispersed within the range of ±ΔZ. ΔZ is an indicator that quantitatively shows the magnitude of the arrangement error.

[0121] <Relative fluorescence intensity under the condition of arrangement error of capillary array>

[0122] Figure 9 It is shown that Figure 6The upper part of the diagram shows a 24-capillary array with n3 = 1.41 as a baseline. The relative fluorescence intensities of each capillary are obtained by irradiation from both sides under the following conditions: (a) ΔZ = 0 μm, (b) ΔZ = 3 μm, (c) ΔZ = 6 μm, (d) ΔZ = 9 μm, and (e) ΔZ = 12 μm. For ΔZ = 0 μm, the relative fluorescence intensity was calculated for one group of capillary arrays. For ΔZ other than 0 μm, the relative fluorescence intensity was calculated for 10 randomly arranged capillary arrays. The results are displayed overlapping. Figure 9 (a) (ΔZ = 0 μm) shows the relationship with Figure 7 The upper part of n3 = 1.41 yields the same result. Figure 9 In (f), the average relative fluorescence intensities of each capillary with respect to ΔZ = 0 μm and the average relative fluorescence intensities of the aforementioned 10 groups of capillary capillary capillary capillary capillaries with respect to ΔZ other than 0 μm are shown in overlapping view. It can be seen that as ΔZ increases, the average and minimum values ​​of the relative fluorescence intensities decrease, and the deviation of the relative fluorescence intensities increases. The minimum values ​​of the relative fluorescence intensities for ΔZ = 0 μm, 3 μm, 6 μm, 9 μm, and 12 μm are MIN = 0.42, 0.40, 0.33, 0.22, and 0.066, respectively. Furthermore, the coefficients of variation of the relative fluorescence intensities for ΔZ = 0 μm, 3 μm, 6 μm, 9 μm, and 12 μm are CV = 11%, 11%, 12%, 17%, and 28%, respectively. Here, the minimum values ​​and coefficients of variation for the relative fluorescence intensity of each capillary at ΔZ = 0 μm and for the aforementioned 10 groups of capillary ...

[0123] Figure 10 It is shown that Figure 6 The upper part of the diagram shows a graph of the relative fluorescence intensities of each capillary obtained by irradiation from both sides, based on a structure of n3 = 1.33 in a 24-capillary array, under the conditions of (a) ΔZ = 0 μm, (b) ΔZ = 3 μm, (c) ΔZ = 6 μm, (d) ΔZ = 9 μm, and (e) ΔZ = 12 μm. For ΔZ = 0 μm, the relative fluorescence intensity was calculated for one group of capillary arrays. For ΔZ other than 0 μm, the relative fluorescence intensity was calculated for 10 randomly arranged capillary arrays, and the results are displayed overlapping. Figure 10 (a) (ΔZ = 0 μm) shows the relationship with Figure 7 The upper part of n3 = 1.33 yields the same result. Figure 10 In (f), the average relative fluorescence intensities of each capillary with respect to ΔZ = 0 μm and the average relative fluorescence intensities of the aforementioned 10 groups of capillary capillary capillary capillary capillaries with respect to ΔZ other than 0 μm are shown in overlapping pairs. It can be seen that as ΔZ increases, both the average and minimum values ​​of the relative fluorescence intensities decrease, and the deviation of the relative fluorescence intensities increases. The minimum values ​​of the relative fluorescence intensities for ΔZ = 0 μm, 3 μm, 6 μm, 9 μm, and 12 μm are MIN = 0.40, 0.39, 0.30, 0.25, and 0.058, respectively. Furthermore, the coefficients of variation of the relative fluorescence intensities for ΔZ = 0 μm, 3 μm, 6 μm, 9 μm, and 12 μm are CV = 12%, 12%, 14%, 16%, and 28%, respectively. Here, the minimum values ​​and coefficients of variation for the relative fluorescence intensity of each capillary at ΔZ = 0 μm and for the aforementioned 10 groups of capillary ...

[0124] Figure 9 and Figure 10 The distribution of relative fluorescence intensity for each capillary number shown exhibits a downward convex shape. Furthermore, the degree of downward convexity increases with increasing ΔZ. Conversely, the degree of variation in the relative fluorescence intensity for each capillary number also increases with increasing ΔZ.

[0125] The above Figure 9 and Figure 10 The results are the same as those of the first embodiment, showing that the reduction effect of the coefficient of variation of relative fluorescence intensity obtained by optical system correction based on the vignetting effect of the optical system is insufficient. If the alignment error ΔZ increases, this situation becomes more pronounced, failing to meet practical performance requirements. Therefore, this embodiment proposes the following technique: in addition to, or instead of, optical system correction based on the vignetting effect of the optical system, by applying digital correction obtained by a computer to the relative fluorescence intensity, the coefficient of variation of relative fluorescence intensity taking into account the alignment error ΔZ is reduced, achieving a CV ≤ 10% that meets practical performance requirements.

[0126] <The process of obtaining a practical output fluorescence intensity distribution for electrophoretic analysis>

[0127] Figure 11 This diagram illustrates the process of obtaining a practical output fluorescence intensity distribution for electrophoretic analysis. Specifically, Figure 11 This describes a process from the concentration of any type of phosphor present in the laser irradiation section of each capillary constituting the capillary array to reaching the fluorescence intensity of the phosphor in that capillary, as output by a computer. Furthermore, Figure 11 In this embodiment, a phosphor and fluorescence performance are used, but this embodiment is not limited to the application of a phosphor and fluorescence. Therefore, it is possible to... Figure 11 The phosphor and fluorescence are replaced by light emitting body and light emission, scattering body and scattered light, or absorber and absorbance.

[0128] The following is a detailed explanation. Figure 11 Furthermore, starting from the end, the capillaries in the capillary array consisting of N capillaries are sequentially numbered as n = 1, 2, ..., N. Figure 11 The functions C(n), L(n), I(n), J(n), M(n), K(n), and H(n) appearing in the equation hold true for any time and any type of fluorophore. In other words, these functions are also functions of time and the type of fluorophore (including functions whose changes are relatively small due to time and fluorophore type), but... Figure 11 For simplicity, it is shown as a function of only n. Furthermore, the value of the function refers to its relative value with respect to n, i.e., the distribution of function values ​​with respect to n, not its absolute value. Therefore, coefficients used to derive the absolute value from the relative value are omitted. Moreover, when the distribution of the function values ​​of any function with respect to n is approximately constant or sufficiently flat, regardless of n, the function and its value can be replaced with 1. Therefore, based on... Figure 11 In the review, MIN≥0.2 is not considered for practical performance, and CV≤15% and CV≤10% are the subjects of review.

[0129] (i) Distribution of luminescence intensity

[0130] The fluorescence intensity distribution is the intensity distribution of fluorescence obtained from the capillary array. Let C(n) be the concentration of phosphors in the laser-irradiated section of capillary n, and C(n) for n is called the phosphor concentration distribution. C(n) represents the concentration of phosphors in capillary n at a certain time, and is a function of time. On the other hand, let L(n) be the irradiation intensity of the laser beam from capillary n, and L(n) for n is called the laser irradiation intensity distribution. L(n) is a function of the laser irradiation intensity of capillary n at a certain time. The fluorescence intensity emitted from the emission point of capillary n can then be expressed as I(n) = L(n) × C(n), and I(n) for n is called the fluorescence intensity distribution. Although repeated, the coefficients usually multiplied with the right side of the above equation are omitted. The above C(n), L(n), and I(n) illustrate the phenomena inside the capillary array and each capillary.

[0131] (ii) Measured fluorescence intensity distribution

[0132] The measured fluorescence intensity distribution is the fluorescence intensity distribution actually detected by the sensor, showing the distribution of fluorescence intensity from the capillary array passing through a predetermined optical system and detected by the sensor (that is, the fluorescence intensity distribution obtained by measuring the fluorescence after correction by the optical system). This will be based on... Figure 2 The optical system correction coefficient for the capillary n in the vignetting effect of the optical system described in the text is denoted as J(n). J(n) for n is called the optical system correction coefficient distribution. Even with any optical system structure, the light passing through it is physically altered, but the optical system correction coefficient mathematically (as a numerical value) represents the physical phenomenon caused by the optical system. Generally, the optical system correction coefficient is smaller for capillaries farther from the optical axis than for capillaries closer to it. In this case, the fluorescence intensity of the imaging point formed by the light emission point of capillary n on the sensor, i.e., the measured fluorescence intensity obtained by the sensor, is M(n) = J(n) × I(n). M(n) for n is called the measured fluorescence intensity distribution. The above J(n) and M(n) illustrate the internal phenomena of the optical system. The optical system correction coefficient is a function of the capillary number n, and also... Figure 8 The x-axis coordinate x is shown as a function. For example... Figure 8 As shown, when the X-coordinate of the central axis of the capillary with n=1 is set to x=0 and the arrangement interval of the capillary is set to p, n and x can be transformed according to x=(n-1)×p.

[0133] (iii) Distribution of output fluorescence intensity

[0134] The output fluorescence intensity distribution is the fluorescence intensity distribution obtained by applying the predetermined digital correction processing of this embodiment to the measured fluorescence intensity distribution. The digital correction coefficient for capillary n applied to the measured fluorescence intensity output by the sensor on the computer is set as K(n), and K(n) for n is called the digital correction coefficient distribution. At this time, the output fluorescence intensity of capillary n output by the computer based on the digital correction is H(n) = K(n) × M(n), and H(n) for n is called the output fluorescence intensity distribution.

[0135] based on Figure 11 To reiterate Figure 5 , Figure 7 , Figure 9 ,as well as Figure 10 . Figure 5 The upper part Figure 7 The upper part Figure 9 ,as well as Figure 10 This shows the fluorescence intensity distribution I(n) = L(n) under the condition that the phosphor concentration distribution is constant, i.e., C(n) = 1. In contrast, Figure 5 The lower part and Figure 7 The lower part shows the measured fluorescence intensity distribution M(n) = J(n) × L(n) after applying optical system correction to the fluorescence intensity distribution. J(n) used here is the distribution of optical system correction coefficients derived experimentally in the optical system of the 3500 series gene analyzer. Furthermore, no digital correction is applied here, K(n) = 1, therefore it can also be said that... Figure 5 The lower part and Figure 7 The lower part shows the output fluorescence intensity distribution H(n) = J(n) × L(n).

[0136] based on Figure 11 For use in reducing Figure 9 and Figure 10 The numerical corrections to the variation coefficients of the luminescent fluorescence intensity distribution I(n) = L(n) shown are reviewed. As mentioned above, since the optical system correction in this structure is sufficiently small, it is set to J(n) = 1 in this review. If J(n) is not considered to be 1, the following derived K(n) can be replaced with K(n) / J(n). Figure 9 and Figure 10In this context, since C(n) = 1, H(n) = K(n) × L(n). That is, we examine K(n) for an output fluorescence intensity distribution H(n) that is nearly flat. If the laser irradiation intensity distribution L(n) is very stable, we set K(n) = 1 / L(n) and H(n) = 1, thus reducing the coefficient of variation of the relative fluorescence intensity. For example, we can pre-calculate I(n) = L(n) in a calibration experiment, set its reciprocal as K(n), and apply it to the relative fluorescence intensity obtained in subsequent experiments. This is the same numerical correction method as in Patent Document 3. However, as... Figure 9 and Figure 10 As shown, I(n) = L(n) is actually completely unstable. Figure 9 and Figure 10 Regarding the deviation in relative fluorescence intensity shown, the refractive index n3 of the medium inside the capillary and the arrangement error ΔZ of the capillary array are the main causes, but they are not limited to these. It is known that it can also be caused by minor deformations of the structural components of the device due to changes in ambient temperature and humidity, or vibration, load, etc., and it varies randomly. A reference is needed for digital correction. In Patent Document 3, the fluorescence intensity distribution obtained during calibration is used as the reference. Figure 9 and Figure 10 The diagram shows a total of 92 fluorescence intensity distribution curves, but it is unclear which one should be chosen as the baseline. If an inappropriate fluorescence intensity distribution is chosen as the baseline, the coefficient of variation of the output fluorescence intensity distribution may sometimes increase due to digital correction, thus making the digital correction have the opposite effect.

[0137] <An example of digital correction for fluorescence intensity distribution>

[0138] Based on the above, in this embodiment, as a correction method, the following is applied: Figure 9 (a) and Figure 10 The digital correction is explained based on the emission fluorescence intensity distribution with ΔZ = 0 μm in (a). Wherein, Figure 9 The case where n3 = 1.41 and Figure 10 When n3 = 1.33, the distribution of the numerical correction factor is changed. More generally, the value of n3 is used to change the distribution of the numerical correction factor. This is because it was found that as n3 decreases, the downward convexity of the emission fluorescence intensity distribution increases. On the other hand, it is permissible that as ΔZ increases, the downward convexity of the emission fluorescence intensity distribution and the degree of variation in the relative fluorescence intensity with respect to capillary number also increase.

[0139] Figure 12 (a) to (f) show the results for n3 = 1.41. Figure 9The fluorescence intensity distributions I(n) = L(n) shown in (a) to (f) were subjected to [f] . Figure 9 The digitally corrected output fluorescence intensity distribution H(n) = K(n) × L(n) is based on the luminescent fluorescence intensity distribution with ΔZ = 0 μm shown in (a). When... Figure 9 When the fluorescence intensity distribution with ΔZ = 0 μm in (a) is set to L0(n), the digital correction factor distribution K(n) used here is K(n) = 1 / L0(n). Therefore, of course, Figure 12 The output fluorescence intensity distribution of (a) is H(n) = 1. On the other hand, although the same digital correction coefficient distribution K(n) is used as described above. Figure 12 The output fluorescence intensity distribution of (b) to (e) and Figure 9 The fluorescence intensity distributions of (b) to (e) are also relatively flat. As a result, for ΔZ = 0 μm, 3 μm, 6 μm, 9 μm, and 12 μm, Figure 9 The coefficients of variation for the relative fluorescence intensity were CV = 11%, 11%, 12%, 17%, and 28%, respectively. Figure 12 The digital correction significantly reduces the coefficient of variation of relative fluorescence intensity to CV = 0%, 0.4%, 3%, 8%, and 19%. Therefore, with this digital correction, any capillary array with ΔZ ≤ 9 μm satisfies the practical performance requirement of CV ≤ 10%.

[0140] Figure 13 (a) to (f) show the results for n3 = 1.33. Figure 10 The fluorescence intensity distributions I(n) = L(n) shown in (a) to (f) were subjected to [f] . Figure 10 The digitally corrected output fluorescence intensity distribution H(n) = K(n) × L(n) is based on the luminescent fluorescence intensity distribution with ΔZ = 0 μm shown in (a). When... Figure 10 When the fluorescence intensity distribution with ΔZ = 0 μm in (a) is set to L0(n), the digital correction factor distribution K(n) used here is K(n) = 1 / L0(n). This L0(n) is related to the above... Figure 9 The fluorescence intensity distribution at ΔZ = 0 μm differs in (a), which is a key feature of the technology disclosed herein. Therefore, of course, Figure 13 The output fluorescence intensity distribution of (a) is H(n) = 1. On the other hand, although the same digital correction coefficient distribution K(n) is used as described above, Figure 13 The output fluorescence intensity distribution of (b) to (e) and Figure 10 The fluorescence intensity distributions of (b) to (e) are also relatively flat. As a result, for ΔZ = 0 μm, 3 μm, 6 μm, 9 μm, and 12 μm, Figure 10The coefficients of variation for the relative fluorescence intensity were CV = 12%, 12%, 14%, 16%, and 28%, respectively. Figure 13 The digital correction significantly reduces the coefficient of variation of relative fluorescence intensity to CV = 0%, 0.5%, 3%, 6%, and 18%. Therefore, with this digital correction, any capillary array with ΔZ ≤ 9 μm satisfies the practical performance requirement of CV ≤ 10%.

[0141] The above Figure 9 The fluorescence intensity distribution of (a) with ΔZ = 0 μm is set as L0(n) and the ... Figure 10 The fluorescence intensity distribution of (a) with ΔZ = 0 μm is set as L0(n) and can be easily obtained using the above equations (4) and (6). Here, in Figure 9 In case (a), we can set n1 = 1.00, n2 = 1.46, n3 = 1.41, and N = 24. Figure 10 In case (a), we can set n1 = 1.00, n2 = 1.46, n3 = 1.33, and N = 24. Thus, the calculation of the numerical correction coefficient distribution K(n) = 1 / L0(n) is accomplished for the first time in this disclosure.

[0142] In this embodiment, assuming the optical system correction coefficient distribution is J(n) = 1, the optimal digital correction coefficient distribution K(n) that reduces the variation coefficient of the output fluorescence intensity distribution H(n) is derived. As described above, without considering J(n) = 1, the derived K(n) can be replaced with K(n) / J(n). That is, by using digital correction and optical system correction, i.e., K(n) × J(n), the desired luminescent fluorescence intensity distribution I(n) with ΔZ = 0 μm at n3 is made as flat as possible. Therefore, it is effective to change J(n) according to n3, even if it is not K(n).

[0143] The above, although regarding the use of laser beams to... Figure 6 The upper part and Figure 8 The structure shown, which uses a 24-capillary array for bilateral irradiation, was reviewed, but the same effect can be obtained in other structures as well.

[0144] (D) Third implementation method

[0145] In the second embodiment, it is shown that, in order to flatten the output fluorescence intensity distribution, it is most preferable to perform a digital correction on the emitted fluorescence intensity distribution based on an emitted fluorescence intensity distribution with a ΔZ = 0 μm relative to the same n3. However, the technique disclosed herein is not limited to an emitted fluorescence intensity distribution with a ΔZ = 0 μm, and is effective when other values ​​are used as a reference. Therefore, in this embodiment, as an example, a digital correction based on an average emitted fluorescence intensity distribution with a ΔZ = 6 μm is described. In this embodiment, similar to the second embodiment, although the laser beam is used for... Figure 6 The upper part and Figure 8 The structure shown, with 24 capillary arrays irradiated from both sides, is reviewed, but other structures are also possible.

[0146] <Example of digital correction for fluorescence intensity distribution: based on an average emission fluorescence intensity distribution of ΔZ = 6 μm>

[0147] Figure 14 (a) to (f) show the results for n3 = 1.41. Figure 9 The fluorescence intensity distributions I(n) = L(n) shown in (a) to (f) were subjected to [f] . Figure 9 The digitally corrected output fluorescence intensity distribution H(n) = K(n) × L(n) is based on the average luminescent fluorescence intensity distribution with ΔZ = 6 μm shown in (f). Figure 9 When the average luminescence intensity distribution of (f) with ΔZ = 6 μm is set to L0(n), the digital correction factor distribution K(n) used here is K(n) = 1 / L0(n). Therefore, of course, Figure 14 The average output fluorescence intensity distribution of (f) with ΔZ = 6 μm is H(n) = 1.

[0148] On the other hand, although the same numerical correction coefficient distribution K(n) is used as described above, Figure 14 The output fluorescence intensity distribution of (a) to (e) and Figure 9 The fluorescence intensity distributions of (a) to (e) are also relatively flat. As a result, for ΔZ = 0 μm, 3 μm, 6 μm, 9 μm, and 12 μm, Figure 9 The coefficients of variation for the relative fluorescence intensity were CV = 11%, 11%, 12%, 17%, and 28%, respectively. Figure 14 The digital correction coefficients of relative fluorescence intensity were significantly reduced to CV = 1%, 1%, 3%, 7%, and 18%.

[0149] Therefore, with this numerical correction, any capillary array with ΔZ ≤ 9 μm satisfies a practical performance CV ≤ 10%. Compared to the case where the emission fluorescence intensity distribution with ΔZ = 0 μm is used as a reference in the second embodiment, the CV for ΔZ = 0 μm to 3 μm increases. In summary, from the viewpoint of reducing the coefficient of variation of relative fluorescence intensity, the case where the emission fluorescence intensity distribution with ΔZ = 0 μm is used as a reference is superior in terms of overall performance, but the case where the average emission fluorescence intensity distribution with ΔZ = 6 μm is also effective.

[0150] Figure 15 (a) to (f) show the results for n3 = 1.33. Figure 10 The fluorescence intensity distributions I(n) = L(n) shown in (a) to (f) were subjected to [f] . Figure 10 The digitally corrected output fluorescence intensity distribution H(n) = K(n) × L(n) is based on the average luminescent fluorescence intensity distribution with ΔZ = 6 μm shown in (f). Figure 10 When the average luminescence intensity distribution of (f) with ΔZ = 6 μm is set to L0(n), the digital correction factor distribution K(n) used here is K(n) = 1 / L0(n). Similar to the second embodiment, this L0(n) is the same as described above. Figure 9 The average luminescence intensity distribution of (f) with ΔZ = 6 μm is different, which is one of the important features of the technology disclosed herein. Therefore, of course, Figure 15 The average output fluorescence intensity distribution of (f) with ΔZ = 6 μm is H(n) = 1.

[0151] On the other hand, although the same numerical correction coefficient distribution K(n) is used as described above, Figure 15 The output fluorescence intensity distribution of (a) to (e) and Figure 10 The fluorescence intensity distributions of (a) to (e) are also relatively flat. As a result, for ΔZ = 0 μm, 3 μm, 6 μm, 9 μm, and 12 μm, Figure 10 The coefficients of variation for the relative fluorescence intensity were CV = 12%, 12%, 14%, 16%, and 28%, respectively. Figure 15 The digital correction coefficients of relative fluorescence intensity were significantly reduced to CV = 2%, 2%, 2%, 5%, and 16%.

[0152] Therefore, with this numerical correction, any capillary array with ΔZ ≤ 9 μm satisfies a practical performance CV ≤ 10%. Compared to the case where the emission fluorescence intensity distribution with ΔZ = 0 μm is used as a reference in the second embodiment, the CV for ΔZ = 0 μm to 3 μm increases. In summary, from the viewpoint of reducing the coefficient of variation of relative fluorescence intensity, the case where the emission fluorescence intensity distribution with ΔZ = 0 μm is used as a reference is superior in terms of overall performance, but the case where the average emission fluorescence intensity distribution with ΔZ = 6 μm is also effective.

[0153] <Example of digital correction for fluorescence intensity distribution: based on a fluorescence intensity distribution with ΔZ = 0 μm>

[0154] Next, the digital corrections based on the emission fluorescence intensity distribution with ΔZ = 0 μm for different n3 are reviewed. Figure 16 (a) to (f) show the results for n3 = 1.33. Figure 10 The fluorescence intensity distributions I(n) = L(n) shown in (a) to (f) were subjected to [f] . Figure 9 The digitally corrected output fluorescence intensity distribution H(n) = K(n) × L(n) is based on the luminescent fluorescence intensity distribution with ΔZ = 0 μm shown in (a) for n3 = 1.41. Figure 9 When the fluorescence intensity distribution with ΔZ = 0 μm in (a) is set to L0(n), the digital correction factor distribution K(n) used here is K(n) = 1 / L0(n). As mentioned above, this L0(n) is related to... Figure 10 The fluorescence intensity distribution at ΔZ = 0 μm in (a) differs from that in this disclosure, which is one of the key features of the technology. Therefore, compared with Figure 13 The results of (a) are different. Figure 16 The output fluorescence intensity distribution of (a) is not H(n) = 1, but has a slightly downward convex shape.

[0155] same, Figure 16 The output fluorescence intensity distributions of (a) to (f) also exhibit a downward convex shape, but are different from those of (a) to (f). Figure 10 Compared to the fluorescence intensity distributions of (a) to (f), the distributions all become flatter. As a result, for each ΔZ = 0 μm, 3 μm, 6 μm, 9 μm, and 12 μm, Figure 10 The coefficients of variation for the relative fluorescence intensity were CV = 12%, 12%, 14%, 16%, and 28%, respectively. Figure 16 The digital correction coefficients of relative fluorescence intensity were significantly reduced to CV = 1%, 1%, 4%, 7%, and 19%.

[0156] Therefore, with this numerical correction, any capillary array with ΔZ ≤ 9 μm satisfies a practical performance CV ≤ 10%. However, compared with... Figure 13 Compared to the numerical correction based on the fluorescence intensity distribution with ΔZ = 0 μm at n3 = 1.33, the overall coefficient of variation increases. In summary, from the perspective of reducing the coefficient of variation of relative fluorescence intensity, the case based on the fluorescence intensity distribution with the same n3 is superior overall, but the case based on the average fluorescence intensity distribution with different n3s is also effective.

[0157] (E) Fourth Implementation

[0158] In this embodiment, the third embodiment is examined in greater detail, and the relationship between the reference for digital correction and the result of digital correction is examined in detail for various n3. Similar to the second embodiment, although the use of a laser beam for... Figure 6 The upper part and Figure 8 The structure shown, with 24 capillary arrays irradiated from both sides, is reviewed, but other structures are also possible.

[0159] <Examples of digital correction factors for relative fluorescence intensity corresponding to each refractive index at ΔZ = 0 μm>

[0160] Figure 17 (a) is a graph showing the changes in relative fluorescence intensity for 13 different capillary numbers when the refractive index n3 of the medium inside the capillary is varied from 1.30 to 1.42 in increments of 0.01, with an arrangement error of ΔZ = 0 μm in the capillary array. That is, Figure 17 (a) shows Figure 11 Let the fluorescence intensity distribution be I(n) = L(n) when C(n) = 1. Figure 17 In (a), the fluorescence intensity distribution I(n) = L(n) is shown as a solid line when the second decimal place of n3 is even, and as a dashed line when it is odd. The fluorescence intensity distributions for n3 = 1.41 and n3 = 1.33 are respectively... Figure 9 (a) and Figure 10 The fluorescence intensity distribution shown in (a) is the same. Among them, in Figure 17 In (a), the scale of the vertical axis is enlarged. It can be seen that as n3 decreases, the degree of downward convexity of the luminescence intensity distribution L(n), that is, the curvature of the downward convex curve, monotonically increases. Figure 17 (b) shows that Figure 17 The graph shows five numerical correction coefficients for capillary numbering, with n3 = 1.30, 1.33, 1.36, 1.39, and 1.42, respectively, as the baseline. That is, Figure 17(b) shows Figure 11 The distribution of the digital correction coefficients K(n). In this embodiment, it is also assumed that... Figure 11 The optical correction coefficients are distributed as J(n) = 1. Each K(n) is the reciprocal of the corresponding L(n), K(n) = 1 / L(n).

[0161] Figure 17 The fluorescence intensity distributions I(n) = L(n) of (a) can also be easily obtained using equations (4) and (6) above. Here, we set n1 = 1.00, n2 = 1.46, n3 = 1.30~1.42, and N = 24. Thus, the calculation of the digital correction coefficient distribution K(n) = 1 / L(n) is accomplished for the first time in this disclosure.

[0162] <Example of digitally corrected output fluorescence intensity distribution H(n)>

[0163] Figure 18 (a) shows the... Figure 17 The 13 luminescence intensity distributions I(n) = L(n) shown in (a) were implemented using... Figure 17 Figure (b) shows the digitally corrected output fluorescence intensity distribution H(n) = K(n) × L(n) for the digital correction coefficient distribution K(n) of n3 = 1.30. Similarly, Figure 18 of (b) Figure 18 (c) Figure 18 (d) and Figure 18 (e) respectively show the pair of pairs. Figure 17 The 13 luminescence intensity distributions I(n) = L(n) shown in (a) were implemented using... Figure 17 The figure shown in (b) is a graph of the digitally corrected output fluorescence intensity distribution H(n) = K(n) × L(n) for the digital correction coefficient distributions K(n) of n3 = 1.33, 1.36, 1.39, and 1.42. Figure 18 The distributions of all output fluorescence intensity H(n) shown are as follows: Figure 17The fluorescence intensity distribution L(n) shown in (a) is relatively flat. The output fluorescence intensity distribution H(n) becomes flattest when the reference value n3 of the digital correction is the same as the target value n3 of the digital correction. It can be seen that when the reference value n3 of the digital correction is recorded as n3 (correction reference) and the target value n3 of the digital correction is recorded as n3 (correction target), the output fluorescence intensity distribution H(n) = 1 when n3 (correction reference) = n3 (correction target), the output fluorescence intensity distribution H(n) shows an upward convex curve when n3 (correction reference) < n3 (correction target), and the output fluorescence intensity distribution H(n) shows a downward convex curve when n3 (correction reference) > n3 (correction target). Furthermore, the greater the difference between n3 (correction reference) and n3 (correction target), the greater the degree of upward or downward convexity. For example, as... Figure 18 As shown in (a), when a numerical correction of H(n) = 1 is applied for n3 = 1.30, the relative fluorescence intensity is shown when the same correction is applied to H(n) for each refractive index. H(n) becomes flat when n3 = 1.30, but for other refractive indices, the larger the difference between the value and 1.30, the less appropriate the correction is. From Figure 18 As can also be seen from (a), the correction was excessive when n3 = 1.42.

[0164] <Changes in the quadratic coefficients when the output fluorescence intensity distribution H(n) before and after digital correction is approximated using a quadratic function>

[0165] The degree of upward and downward convexity of the output fluorescence intensity distribution H(n) can be represented by the quadratic coefficients when each output fluorescence intensity distribution H(n) is approximated by a quadratic function. In this embodiment, the output fluorescence intensity distribution is approximated by a quadratic function, but other functions or methods can also be used to represent the degree of upward and downward convexity of H(n).

[0166] Figure 17 (c) shows the expression of the refractive index n3 of the medium inside the capillary using a quadratic function. Figure 17 The fluorescence intensity distribution I(n) before digital correction of (a) and Figure 18 The graph shows the variation of the quadratic coefficients when the digitally corrected output fluorescence intensity distribution H(n) is approximated based on various correction benchmarks. When the quadratic coefficients, as shown on the vertical axis, are zero, the output fluorescence intensity distribution H(n) = 1, which is completely flat. In contrast, as the quadratic coefficients become larger than zero, the downward bulge of the output fluorescence intensity distribution H(n) increases. On the other hand, as the quadratic coefficients become smaller than zero, the upward bulge of the output fluorescence intensity distribution H(n) increases. The uncorrected quadratic coefficients gradually decrease along with n3. This is consistent with... Figure 17 This corresponds to the case in (a) where the downward bulge of the fluorescence intensity distribution L(n) weakens as n3 increases. The corrected quadratic coefficients are all smaller than the original quadratic coefficients. Furthermore, the absolute values ​​of the corrected quadratic coefficients are all smaller than the absolute values ​​of the original quadratic coefficients. The above demonstrates that any numerical correction can flatten the fluorescence intensity distribution.

[0167] Figure 17 In (c), the five curves shown using the corrected n3 = 1.30 (corrected reference), n3 = 1.33 (corrected reference), n3 = 1.36 (corrected reference), n3 = 1.39 (corrected reference), and n3 = 1.42 (corrected reference) are respectively compared with... Figure 18 of (a) Figure 18 of (b) Figure 18 (c) Figure 18 (d) and Figure 18 (e) corresponds to this. In any variation of the quadratic coefficient of the output fluorescence intensity distribution based on the five correction bases, the quadratic coefficient is zero when n3 (correction base) = n3 (correction target), negative when n3 (correction base) < n3 (correction target), and positive when n3 (correction base) > n3 (correction target). Here, Figure 17 The horizontal axis of (c) represents n3 (the correction object). Furthermore, the greater the difference between n3 (the correction reference) and n3 (the correction object), the larger the absolute value of the quadratic coefficient. This is consistent with the above... Figure 18 The changes in the degree of upward and downward convexity are shown.

[0168] In summary, when implementing digital correction, setting n3 (correction reference) = n3 (correction target) is most effective. Alternatively, minimizing the difference between n3 (correction reference) and n3 (correction target) is most effective. When n3 (correction reference) < n3 (correction target), digital correction is excessive, and the output fluorescence intensity distribution becomes an upward-convex curve. Conversely, when n3 (correction reference) > n3 (correction target), digital correction is insufficient, and the output fluorescence intensity distribution becomes a downward-convex curve. In these cases, the curve becomes flatter than the downward-convex curve of the fluorescence intensity distribution before correction, thus being effective. When using separation media with various n3 values ​​at different times, it is effective to perform digital correction only that matches the n3 used at each time. That is, the correction reference and the distribution of digital correction coefficients are changed to match the n3 used. These are the characteristics of digital correction in this embodiment.

[0169] The above discussion addressed the case where the capillary array arrangement error is ΔZ = 0 μm. However, the same method yields the same result when ΔZ ≠ 0 μm. For example, if ΔZ = 6 μm is set as a common condition, the result is similar to... Figure 17 The same result as (c). That is, each correction reference is obtained under the condition of ΔZ = 6 μm, and the fluorescence intensity distribution that is the object of correction is also obtained under the condition of ΔZ = 6 μm. On the other hand, some deformation occurs when the ΔZ of each correction reference is different from the ΔZ of the fluorescence intensity distribution that is the object of correction. Figure 17 In (c), if n3 = 1.33 (correction reference) for ΔZ = 0 μm is replaced with n3 = 1.33 (correction reference) for ΔZ = 6 μm, the corresponding curve shifts to the lower left. That is, when n3 (correction object) on the horizontal axis is 1.33, if a digital correction based on n3 = 1.33 (correction reference) for ΔZ = 0 μm is applied, the quadratic coefficient on the vertical axis is zero; however, if a digital correction based on n3 = 1.33 (correction reference) for ΔZ = 6 μm is applied, the quadratic coefficient on the vertical axis is negative. Although this curve shift occurs, the overall trend is the same. Regardless of the reference on which the digital correction is based, when n3 (correction object) on the horizontal axis is set to a larger value, such as n3 = 1.42, the quadratic coefficient on the vertical axis is negative; on the other hand, when n3 (correction object) on the horizontal axis is set to a smaller value, such as n3 = 1.30, the quadratic coefficient on the vertical axis is positive.

[0170] (F) Fifth Implementation

[0171] In the fifth embodiment, the effect of digitally correcting capillary arrays with various ΔZ values ​​becomes clear, and ΔZ values ​​that satisfy practical performance (MIN ≥ 0.2 and CV ≤ 15% or CV ≤ 10%) become well-defined. In this embodiment, the use of a laser beam to... Figure 6 The upper part and Figure 8 The structure shown, with 24 capillary arrays irradiated from both sides, is reviewed, but other structures are also possible.

[0172] <Results after implementing optical and digital corrections>

[0173] (i) The case where the refractive index n3 = 1.41

[0174] Figure 19 The upper part shows the relative fluorescence intensity of each capillary obtained by irradiation from both sides when n3 = 1.41 and the ΔZ = 0.0, 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, 12.0, 13.5, and 15.0 μm. The lower part shows its variation coefficient. Figure 19 The right side of each of the upper and lower parts is a magnified view of the left side. For ΔZ = 0.0 μm, one set of capillary arrays is used; for each ΔZ ≠ 0.0 μm, 100 sets of randomly arranged capillary arrays are used respectively. (The last sentence appears to be incomplete and possibly refers to a separate process.) Figure 19 For the upper part of the line graph, 24 relative fluorescence intensity data points were used for ΔZ = 0.0 μm, and 24 × 100 groups = 2400 relative fluorescence intensity data points were used for each ΔZ ≠ 0.0 μm. Furthermore, in this line graph, black circles represent the average value, error bars represent the ± standard deviation, black triangles represent the maximum value, and black squares represent the minimum value. From... Figure 19 As shown in (a), with the increase of ΔZ, the mean, maximum and minimum values ​​all decrease, and the standard deviation increases.

[0175] In making Figure 19 In the lower part of the line graph, for ΔZ = 0.0 μm, a set of relative fluorescence intensity variation coefficients is used; for each ΔZ ≠ 0.0 μm, 100 sets of relative fluorescence intensity variation coefficients are used. Furthermore, in this line graph, black circles indicate the average value, error bars indicate ± standard deviation, black triangles indicate the maximum value, and black squares indicate the minimum value. From... Figure 19 As shown in (c), with the increase of ΔZ, the mean, maximum, and minimum values ​​all increase, and the standard deviation increases.

[0176] It can be seen that: when Figure 19 When the minimum relative fluorescence intensity of the upper part is set to MIN, in order to satisfy the practical performance requirement of MIN≥0.2, it can be set to ΔZ≤7.2μm. On the other hand, it can be seen that when... Figure 19 When the average value plus standard deviation of the relative fluorescence intensity of the lower part is set as CV, in order to meet the practical performance requirement of CV≤15%, it can be set to ΔZ≤6.4μm. On the other hand, it is impossible to meet the requirement of CV≤10%.

[0177] Figure 20 It shows the... Figure 19 The graph shows the results of optical and digital corrections for the fluorescence intensity distributions I(n) = L(n) of 1001 capillary arrays. The optical correction coefficient distribution J(n) is the distribution measured using the optical correction coefficient distribution of the 3500 series gene analyzer. J(n) reaches a maximum of 1 at the center of the capillary array, between n=12 and n=13, and decreases as it moves away from the center. Therefore, the absolute values ​​of the fluorescence intensity distribution I(n) and the measured fluorescence intensity distribution M(n) can be compared.

[0178] On the other hand, setting the benchmark for digital correction to be relative to Figure 7The upper part of the fluorescence intensity distribution I(n) = L(n) with ΔZ = 0 μm and n3 = 1.41 was modified according to the above optical system. Figure 7 The measured fluorescence intensity distribution M(n) = J(n) × L(n) for the lower part of ΔZ = 0 μm and n3 = 1.41. At this time, similar to the second and third embodiments, the digital correction coefficient distribution can also be set to K(n) = 1 / M(n), while in this embodiment it is set to K(n) = α × [{n - (N + 1) / 2} × p]. 2 +1. Here, α is a coefficient and α = -0.074, N is the total number of capillaries and N = 24, and p is the spacing between the capillaries and p = 0.155 mm. K(n) reaches its maximum value of 1 at the center of the capillary array, i.e., between n = 12 and n = 13, and decreases as it moves away from the center. Therefore, the absolute values ​​of the measured fluorescence intensity distribution M(n) and the output fluorescence intensity distribution H(n) can be compared. At this time, when this digital correction coefficient distribution K(n) is applied... Figure 7 When the measured fluorescence intensity distribution M(n) of the lower part is n3 = 1.41, the output fluorescence intensity distribution H(n) = K(n) × M(n) becomes the flattest. Figure 7 The coefficient of variation for the fluorescence intensity distribution in the upper part is CV = 11%. In contrast, Figure 7 The coefficient of variation (CV) for the measured fluorescence intensity distribution in the lower part is 9%. Furthermore, the coefficient of variation for the output fluorescence intensity distribution after applying digital correction is reduced to 0.6%.

[0179] Figure 20 This shows the results of applying the above optical system corrections and digital corrections not only to the case of ΔZ = 0 μm, but also to the emission and fluorescence intensity distributions for all ΔZ values. It can be seen that when... Figure 20 The upper part and Figure 19 When comparing the upper part, the minimum value of the relative fluorescence intensity distribution remains unchanged, but the maximum and average values ​​decrease, as do the difference between the maximum and minimum values ​​and the standard deviation. On the other hand, it can be seen that when... Figure 20 The lower part and Figure 19 When comparing the lower part, not only is ΔZ = 0 μm, but the variation coefficients of all ΔZ values ​​are significantly reduced. When... Figure 20 When the minimum relative fluorescence intensity of the upper part is set as MIN, the condition for practical performance (MIN≥0.2) is ΔZ≤7.2μm. Figure 19 The results for the upper part are the same. On the other hand, it can be seen that when... Figure 20 When the average value plus standard deviation of the relative fluorescence intensity of the lower part is set as CV, to meet the practical performance requirement of CV ≤ 15%, ΔZ ≤ 9.0 μm is sufficient. On the other hand, it can be seen that to meet the requirement of CV ≤ 10%, ΔZ ≤ 7.8 μm is sufficient.

[0180] The allowable range of ΔZ above is related to Figure 19 Compared to the previous method, the range of ΔZ required to meet practical performance requirements has been significantly expanded. This means that the numerical corrections in this disclosure have substantially increased the permissible range of ΔZ.

[0181] (ii) The case where the refractive index n3 = 1.33

[0182] Figure 21 The upper part shows the relative fluorescence intensity of each capillary obtained by irradiation from both sides when n3 = 1.33 varies with ΔZ = 0.0, 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, 12.0, 13.5, and 15.0 μm, and the lower part shows its variation coefficient. Figure 21 The right side of each of the upper and lower parts is an enlarged view of the left side. For ΔZ = 0.0 μm, one set of capillary arrays is used; for each ΔZ ≠ 0.0 μm, capillary arrays composed of 100 randomly arranged groups are used respectively. In the fabrication... Figure 21 For the upper part of the line graph, 24 relative fluorescence intensity data points were used for ΔZ = 0.0 μm, and 24 × 100 groups = 2400 relative fluorescence intensity data points were used for each ΔZ ≠ 0.0 μm. In this line graph, black circles indicate the average value, error bars indicate the ± standard deviation, black triangles indicate the maximum value, and black squares indicate the minimum value. From Figure 21 As shown in (a), with the increase of ΔZ, the mean, maximum and minimum values ​​all decrease, and the standard deviation increases.

[0183] In making Figure 21 In the lower part of the line graph, a set of relative fluorescence intensity variation factors is used for ΔZ = 0.0 μm, and 100 sets of relative fluorescence intensity variation factors are used for each ΔZ ≠ 0.0 μm. In this line graph, black circles indicate the average value, error bars indicate the ± standard deviation, black triangles indicate the maximum value, and black squares indicate the minimum value. From Figure 21 From (c), we can see that as ΔZ increases, the mean, maximum, and minimum values ​​all increase, and the standard deviation increases. Therefore, when... Figure 21 When the minimum relative fluorescence intensity of the upper part is set to MIN, in order to meet the practical performance requirement of MIN≥0.2, it can be set to ΔZ≤7.9μm.

[0184] On the other hand, it can be seen that: when... Figure 21 When the average value plus standard deviation of the relative fluorescence intensity of the lower part is set as CV, in order to meet the practical performance requirement of CV≤15%, ΔZ≤5.7μm is sufficient. However, CV≤10% cannot be satisfied.

[0185] Figure 22 It shows the... Figure 21 The graph shows the results of optical system correction and digital correction for the fluorescence intensity distribution I(n) = L(n) of 1001 capillary arrays. The optical system correction coefficient distribution J(n) is the distribution of the optical system correction coefficient measured on the 3500 series gene analyzer, compared with... Figure 20 The same distribution is used. J(n) reaches a maximum of 1 at the center of the capillary array, i.e., between n=12 and n=13, and decreases as it moves away from the center. Therefore, the absolute values ​​of the luminescent fluorescence intensity distribution I(n) and the measured fluorescence intensity distribution M(n) can be compared with each other.

[0186] On the other hand, setting the benchmark for digital correction to be relative to Figure 7 The upper part of the fluorescence intensity distribution I(n) = L(n) with ΔZ = 0 μm and n3 = 1.33 was modified according to the above optical system. Figure 7 The measured fluorescence intensity distribution M(n) = J(n) × L(n) for the lower part of ΔZ = 0 μm and n3 = 1.33. Similar to the second and third embodiments, the digital correction coefficient distribution can also be set as K(n) = 1 / M(n), while in this embodiment it is set as K(n) = α × [{n - (N + 1) / 2} × p]. 2 +1. Here, α is a coefficient and α = -0.081, N is the total number of capillaries and N = 24, and p is the spacing between capillaries and p = 0.155 mm. That is to say, compared with... Figure 20 The only difference in the digital correction applied is that α changes from -0.074 to -0.081. This difference corresponds to the case where n3 changes from 1.41 to 1.33. K(n) reaches a maximum of 1 at the center of the capillary array, i.e., between n=12 and n=13, and decreases as it moves away from the center. Therefore, the absolute values ​​of the measured fluorescence intensity distribution M(n) and the output fluorescence intensity distribution H(n) can be compared. At this point, when this digital correction coefficient distribution K(n) is applied... Figure 7 When the measured fluorescence intensity distribution M(n) of the lower part is n3 = 1.33, the output fluorescence intensity distribution H(n) = K(n) × M(n) becomes the flattest. Figure 7 The coefficient of variation for the fluorescence intensity distribution in the upper part is CV = 12%, relative to this, Figure 7 The coefficient of variation for the measured fluorescence intensity distribution in the lower part is CV = 10%. Furthermore, the coefficient of variation for the output fluorescence intensity distribution after applying digital correction is reduced to CV = 0.8%.

[0187] Figure 22 This shows the results of applying the above optical system corrections and digital corrections not only to the case of ΔZ = 0 μm, but also to the emission and fluorescence intensity distributions for all ΔZ values. Figure 22The upper part and Figure 21 When comparing the upper part, the minimum value of the relative fluorescence intensity distribution remains unchanged, but the maximum and average values ​​decrease, as do the difference between the maximum and minimum values ​​and the standard deviation. On the other hand, when... Figure 22 The lower part and Figure 21 When comparing the lower part, not only is ΔZ = 0 μm, but the variation coefficients of all ΔZ values ​​are significantly reduced. When... Figure 22 When the minimum relative fluorescence intensity of the upper part is set as MIN, the condition for practical performance (MIN≥0.2) is ΔZ≤7.9μm. Figure 21 The results for the upper part are the same. On the other hand, it can be seen that when... Figure 22 When the average value plus standard deviation of the relative fluorescence intensity of the lower part is set as CV, to meet the practical performance requirement of CV ≤ 15%, ΔZ ≤ 10.1 μm is sufficient. On the other hand, to meet CV ≤ 10%, ΔZ ≤ 8.5 μm is sufficient. The above allowable range of ΔZ is related to... Figure 21 Compared to the previous method, the range of ΔZ required to meet practical performance requirements has been significantly expanded. This means that the numerical corrections in this disclosure have substantially increased the permissible range of ΔZ.

[0188] (G) Sixth Implementation Method

[0189] In the aforementioned non-patent document 1, the refractive index of the medium inside the analytical capillary (separation medium) is set to n3 = 1.41, and the refractive index of the medium inside the lens capillary is set to n4 = 1.53. The raw material of the capillary is quartz glass and n2 = 1.46. At this time, according to the above equation (1), the refraction angle of one analytical capillary is Δθ. A = +2.4°, while the refraction angle of a lens capillary is Δθ. B = -3.0°. At this time, due to Δθ A +Δθ B = -0.61°, therefore, a set of analytical capillary tubes and lens capillary tubes demonstrates the function of a convex lens, with multifocal focusing. Thus, through Δθ A +Δθ B A method for evaluating the presence or absence of multi-focus functionality is discovered for the first time in the art disclosed herein.

[0190] <Analysis of an example of a capillary array with alternating arrangements of capillary tubes and lens capillary tubes>

[0191] Figure 23The upper part shows a configuration example of the capillary array of this disclosure, capable of simultaneously irradiating multiple capillaries. 192 capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm are arranged in the same plane at intervals of 155 μm. The arrangement error is zero (ΔZ = 0 μm). The outer surface of the capillaries is a low-refractive-index solution with n1 = 1.25, and the capillary material is quartz glass with n2 = 1.46. For example, Fluorinert from 3M can be used as the aforementioned low-refractive-index solution. The odd-numbered 96th capillaries from the left side of the capillary array are designated as analytical capillaries, and the refractive index of the internal medium is set to n3. Furthermore, each analytical capillary is sequentially labeled with capillary numbers 1 to 96 from the left side. On the other hand, the even-numbered 96th capillaries from the left side of the capillary array are designated as lens capillaries, and the refractive index of the internal medium is set to n4 = 1.46. The capillary tubes of each lens are not labeled with capillary numbers.

[0192] Figure 23 The middle section shows the laser beam tracing results when a φ50μm laser beam is irradiated from the left side under the above conditions of n3 = 1.41. At this time, the multifocal system functions effectively, irradiating the interior of all 192 capillaries. According to equation (1) above, the refraction angle of one analyzed capillary is Δθ. A = -1.4°, the angle of refraction of a lens capillary is Δθ B = -3.3°. At this time, due to Δθ A +Δθ B = -4.7°, so a set of one analytical capillary and one lens capillary demonstrates the function of a convex lens, and the multifocal function is realized.

[0193] Figure 23 The lower part shows the laser beam tracing results when a φ50μm laser beam is irradiated from the left side under the above conditions of n3 = 1.33. At this time, the multifocal system functions effectively, irradiating the interior of all 192 capillaries. According to equation (1) above, the refraction angle of one analyzed capillary is Δθ. A = +2.0°, the angle of refraction of a lens capillary is Δθ B = -3.3°. At this time, due to Δθ A +Δθ B = -1.3°, so a set of one analytical capillary and one lens capillary shows the function of a convex lens, and the multifocal function is realized.

[0194] <Examples of relative fluorescence intensity with n3 = 1.41, optical system-corrected relative fluorescence intensity, and digitally corrected and optical system-corrected relative fluorescence intensity>

[0195] Figure 24(a) shows the rewrite under bilateral illumination. Figure 23 The results of unilateral irradiation shown in the middle section, with n3 = 1.41, are the relative fluorescence intensity, i.e., the luminescence fluorescence intensity distribution, I(n) = L(n) for capillary numbers 1 to 96. The relative fluorescence intensity of the lens capillary is not shown. Regarding the 96 analytical capillary numbers, the minimum relative fluorescence intensity is MIN = 0.30, and the coefficient of variation is CV = 20%. This satisfies the practical performance requirement of MIN ≥ 0.2, but does not satisfy CV ≤ 15% and CV ≤ 10%.

[0196] Figure 25 (a) shows the distribution of optical system correction coefficients J(n) after incorporating the vignetting effect of the optical system of the 3730 series gene analyzer used in this embodiment. Furthermore, the optical system of the 3730 series gene analyzer differs from that of the 3500 series gene analyzer, resulting in different vignetting effects. Figure 25 The optical system correction coefficient distribution shown in (a) is in J(x) = a + (b - a) × exp{-1 × x 2 / (2×c 2 In the case of}, a = -0.25635, b = 1, c = 11.736815. Here, according to x = {n - (N + 1) / 2} × p, the capillary number n is associated with the spatial coordinate x. N is the total number of analyzed capillaries and N = 96, p is the arrangement interval of the analyzed capillaries and p = 0.155 mm × 2 = 0.310 mm. x is along the Figure 8 The spatial coordinates of the X-axis shown are different from those of the X-axis. Figure 8 Unlike other methods, the origin of the X-axis is located in the center of the capillary array, between n=48 and n=49. For example... Figure 25 As shown in (a), J(n) reaches a maximum of 1 at the center of the capillary array and decreases as it moves away from the center. Therefore, the absolute values ​​of the luminescent fluorescence intensity distribution L(n) and the measured fluorescence intensity distribution M(n) can be compared with each other.

[0197] Figure 24 (b) shows that Figure 25 J(n) of (a) and Figure 24 The measured fluorescence intensity distribution M(n) is obtained by multiplying (a) and L(n). Figure 24 The measured fluorescence intensity distribution of (b) and Figure 7 The measured fluorescence intensity distribution in the lower part of the array differs, exhibiting an upward-convex curve. This is because the full width of the capillary array is relatively wide, resulting in a larger correction by the optical system. Figure 24The minimum relative fluorescence intensity in the measured fluorescence intensity distribution of (b) is MIN = 0.17, and the coefficient of variation is CV = 14%. Therefore, it does not meet the practical performance requirement of MIN ≥ 0.2, but it does meet the requirement of CV ≤ 15%, and does not meet the requirement of CV ≤ 10%.

[0198] Figure 25 (b) shows the method for reducing Figure 24 The digital correction coefficient distribution K(n) of the variation coefficient of the measured fluorescence intensity distribution in (b). This digital correction coefficient distribution is consistent with... Figure 17 The numerical correction coefficients in (b) have a different distribution, exhibiting a downward-convex curve. This is to... Figure 24 The measured fluorescence intensity distribution of (b) with its upward convexity was flattened. Figure 25 The distribution of the numerical correction coefficients shown in (b) is based on K(x) = 1 / [a + (b - a) × exp{-1 × x] 2 / (2×c 2 In the formula, a = -0.25635, b = 1, and c = 16.8 are set. Here, according to x = {n - (N + 1) / 2} × p, the capillary number n is associated with the spatial coordinate x. N is the total number of analyzed capillaries and N = 96, p is the arrangement interval of the analyzed capillaries and p = 0.155 mm × 2 = 0.310 mm. x is the distance along the […]. Figure 8 The spatial coordinates of the X-axis shown are different from those of the X-axis. Figure 8 Unlike other methods, the origin of the X-axis is located in the center of the capillary array, between n=48 and n=49. For example... Figure 25 As shown in (b), K(n) reaches a maximum of 1 at both ends of the capillary array, i.e., n=1 and n=96, and decreases as it approaches the center. Therefore, the absolute values ​​of the measured fluorescence intensity distribution M(n) and the output fluorescence intensity distribution H(n) can be compared with each other.

[0199] Figure 24 (c) shows that Figure 25 The distribution of the digital correction coefficients K(n) in (b) and Figure 24 The output fluorescence intensity distribution H(n) is obtained by multiplying the measured fluorescence intensity distribution M(n) of (b) by itself. This gives H(n) = K(n) × M(n). It can be seen that: [The text abruptly ends here, so the translation stops as well.] Figure 24 Compared to the measured fluorescence intensity distribution M(n) of (b), Figure 24 The output fluorescence intensity distribution H(n) of (c) is flattened. Figure 24The minimum relative fluorescence intensity in the output fluorescence intensity distribution of (c) is MIN = 0.17, with a variation coefficient of CV = 2%. Therefore, it does not satisfy the practical performance requirement of MIN ≥ 0.2, but on the other hand, it satisfies both CV ≤ 15% and CV ≤ 10%. In this embodiment, the laser output intensity is increased by 50%, thereby effectively setting MIN = 0.26, satisfying the practical performance requirement of MIN ≥ 0.2. In summary, all practical performance requirements are met.

[0200] Figure 26 (a) shows the rewrite under bilateral illumination. Figure 23 The lower part shows the results of unilateral irradiation, with n3 = 1.33, and the relative fluorescence intensity, i.e., the luminescence fluorescence intensity distribution, I(n) = L(n) for capillary numbers 1 to 96. The relative fluorescence intensity of the lens capillary is not shown. Regarding the 96 analytical capillary numbers, the minimum relative fluorescence intensity is MIN = 0.23, and the coefficient of variation is CV = 27%, satisfying the practical performance requirement of MIN ≥ 0.2, but not satisfying CV ≤ 15% and CV ≤ 10%.

[0201] <Examples of relative fluorescence intensity with n3 = 1.33, optical system-corrected relative fluorescence intensity, and digitally corrected and optical system-corrected relative fluorescence intensity>

[0202] Figure 27 (a) shows the distribution of optical system correction coefficients J(n) after incorporating the vignetting effect of the optical system of the 3730 series gene analyzer used in this embodiment, and... Figure 25 The J(n) shown in (a) is the same. Figure 26 (b) shows that Figure 27 J(n) of (a) and Figure 26 The measured fluorescence intensity distribution M(n) is obtained by multiplying (a) and L(n). Figure 26 The measured fluorescence intensity distribution of (b) and Figure 24 (b) also presents an upward convex curve, but with Figure 24 (b) is flatter. Figure 26 The minimum relative fluorescence intensity in the measured fluorescence intensity distribution of (b) is MIN = 0.17, and the coefficient of variation is CV = 9%. Therefore, it does not meet the practical performance requirement of MIN ≥ 0.2, but on the other hand, it meets both CV ≤ 15% and CV ≤ 10%.

[0203] Figure 27 (b) shows the method for further reduction Figure 26 The digital correction coefficient distribution K(n) of the variation coefficient of the measured fluorescence intensity distribution in (b). This digital correction coefficient distribution is consistent with... Figure 25The curve is the same as (b), and it convexes downwards. It is located at K(x) = 1 / [a + (b - a) × exp{-1 × x] 2 / (2×c 2 In the formula [], we set a = -0.25635, b = 1, and c = 21.8. Here, based on x = {n - (N + 1) / 2} × p, we associate the capillary number n with the spatial coordinate x. Figure 25 The only difference between K(n) and (b) is the value of c mentioned above.

[0204] Figure 26 (c) shows that Figure 27 The distribution of the digital correction coefficients K(n) in (b) and Figure 26 The output fluorescence intensity distribution H(n) is obtained by multiplying the measured fluorescence intensity distribution M(n) of (b) by itself. This gives H(n) = K(n) × M(n). It can be seen that: [The text abruptly ends here, so the translation stops as well.] Figure 26 Compared to the measured fluorescence intensity distribution M(n) of (b), Figure 26 The output fluorescence intensity distribution H(n) of (c) is further flattened. Figure 26 The minimum relative fluorescence intensity in the output fluorescence intensity distribution of (c) is MIN = 0.17, and the coefficient of variation is CV = 3%. Therefore, it does not satisfy the practical performance requirement of MIN ≥ 0.2, but on the other hand, it satisfies both CV ≤ 15% and CV ≤ 10%. In this embodiment, the laser output intensity is increased by 50%, thereby effectively setting MIN = 0.26, which satisfies the practical performance requirement of MIN ≥ 0.2. In summary, all practical performance requirements are met.

[0205] (H) Seventh Implementation Method

[0206] The seventh embodiment discloses an example of the configuration of the improved capillary array electrophoresis apparatus.

[0207] <Example of the configuration of a capillary array electrophoresis apparatus>

[0208] Figure 28 and Figure 29 This diagram illustrates an example of the configuration of a capillary array electrophoresis apparatus capable of switching between polymer solution 25 (A) and polymer solution 28 (B) as two separation media on the device. By applying the structure of the capillary array electrophoresis apparatus of this embodiment, the same capillary array can be used, and electrophoretic analysis based on multiple polymer solutions can be performed without attaching or removing the capillary array from the apparatus.

[0209] Figure 28 Mode A is shown, in which a polymer solution 25 is filled into a capillary tube 1 and capillary electrophoresis is performed using the polymer solution 25. Figure 29The diagram shows a B mode in which a polymer B solution 28 is filled into a capillary tube 1 and capillary electrophoresis is performed using the polymer B solution 28. When repeatedly performing an analytical session consisting of steps (1) to (6) as shown in the first embodiment, any mode of A and mode B can be selected in each analytical session. For example, analytical sessions of mode B can be repeatedly performed after multiple analytical sessions of mode A, or analytical sessions of mode A and mode B can be performed alternately. The above settings can be configured by… Figure 3 The input is processed through the computer's input section, and confirmation can be made on the display section. A separation medium having a refractive index of 1.33 ≤ n3 < 1.36 is designated as a low-refractive-index separation medium, and a separation medium having a refractive index of 1.36 ≤ n3 ≤ 1.42 is designated as a high-refractive-index separation medium. Polymer solution A 25 and polymer solution B 28 can also be either high-refractive-index separation media or low-refractive-index separation media, but in this embodiment, polymer solution A 25 is a high-refractive-index separation medium, and polymer solution B 28 is a low-refractive-index separation medium. Figure 28 and Figure 29 Most of the and Figure 1 Similarly, objects shown by the same symbols in each diagram have the same function resulting from the same construction. The following will be used in conjunction with... Figure 1 The different parts will be explained in detail. The number of capillaries is not limited to the 24 in the first embodiment, but can be arbitrary. In the laser irradiation unit 14, the analytical capillaries and lens capillaries can be arranged alternately (with...). Figure 23 (Same arrangement). In this case, the object represented below is only the capillary for analyzing capillary.

[0210] Furthermore, in this disclosure, in a single analysis mode, it is assumed that the interiors of all capillaries constituting a single capillary array are filled with the same separation medium having the same refractive index. However, in a single analysis mode, the interiors of different capillaries constituting a single capillary array can also be filled with different separation media having different refractive indices. In such cases, according to the structure of this disclosure, it is also possible to efficiently and simultaneously irradiate all capillaries constituting a single capillary array using a laser beam.

[0211] exist Figure 28 and Figure 29 In the capillary electrophoresis apparatus shown, a dual polymer block 30 is used instead of Figure 1Polymer block 9. In the laser irradiation section 14, with the analytical capillary and lens capillary arranged alternately, the lens capillary is not connected to the dual polymer block 30; only the sample dissolution end 3 of the analytical capillary is bound to connect it to the dual polymer block 30. An A syringe 24 containing an A polymer solution 25 and a B syringe 27 containing a B polymer solution 28 are connected to the dual polymer block 30. When valve A 26 is open and anode tank valve 31 is open, the A polymer solution 25 inside the A syringe 24 is connected to the anode-side buffer solution 7 through the internal flow path of the dual polymer block 30. Similarly, when valve B 29 is open and anode tank valve 31 is open, the B polymer solution 28 inside the B syringe 27 is connected to the anode-side buffer solution 7 through the internal flow path of the dual polymer block 30. When cleaning valve 32 is open and anode tank valve 31 is open, the cleaning flow 33 used in the cleaning flow path is connected to the anode-side buffer solution 7 through the internal flow path of the dual polymer block 30. Furthermore, the sample dissolution end 3 is connected to the flow path between valve A 26 and anode tank valve 31, the flow path between valve B 29 and anode tank valve 31, and the flow path between cleaning valve 32 and anode tank valve 31.

[0212] <Analyze actions in mode A and mode B during a conversation, and the actions that switch between them>

[0213] [1] Regarding Mode A

[0214] First, let's explain pattern A. For example... Figure 28 As shown, the flow path between valve A 26 and anode tank valve 31 is filled with polymer A solution 25, meaning the sample dissolution end 3 is in contact with polymer A solution 25. At this time, valve A 26 is opened, valve B 29 is closed, anode tank valve 31 is closed, and cleaning valve 32 is closed. The piston of syringe A 24 is pressed to pressurize the polymer A solution 25 inside the dual polymer block 30, filling each capillary 1 with polymer A solution 25 from the sample dissolution end 3 towards the sample injection end 2. After filling with polymer A solution 25, anode tank valve 31 is opened, and different samples are injected into each capillary 1 from the sample injection end 2. Capillary electrophoresis is then performed by applying a high voltage between cathode 4 and anode 5 using power supply 8. At this time, valve A 26 can also be closed.

[0215] [2.1] Regarding the switching from mode A to mode B

[0216] Next, the method for switching from mode A to mode B will be explained. To perform the desired electrophoretic analysis, the polymer solution in contact with the sample dissolution end 3 within the dual polymer block 30 must not result in the following: a mixture of polymer solution A 25 and polymer solution B 28, or a diluted polymer solution A 25 or polymer solution B 28, or the presence of air bubbles in the polymer solution. Therefore, the switching from mode A to mode B is performed in the next step. Close valve A 26, open valve B 29, open anode tank valve 31, and close cleaning valve 32. Press the piston of syringe B 27 to discharge polymer solution A 25, which exists in the flow path between valve B 29 and the anode-side buffer 7, into the anode-side buffer 7, while... Figure 29 The flow path between valve B 29 and anode-side buffer 7 is replaced with polymer B solution 28, as shown. Once the replacement is complete, the piston of syringe B 27 is stopped. In this step, the amount (volume) of polymer B solution 28 expelled by syringe B 27 is greater than the internal volume of the flow path between valve B 29 and anode-side buffer 7, ensuring that the polymer solution contacting the sample dissolution end 3 is pure polymer B solution 28. Furthermore, since the amount of polymer A solution 25 discharged into anode-side buffer 7 is less than the amount of anode-side buffer 7, the polymer A solution 25 discharged into anode-side buffer 7 will not adversely affect electrophoresis. Alternatively, polymer A solution 25 can be discharged into a waste tank instead of anode-side buffer 7.

[0217] [2.2] Regarding a more efficient switch from mode A to mode B

[0218] Next, a more efficient method for switching from mode A to mode B will be described. (1) Close valve A 26, close valve B 29, open anode tank valve 31 and open cleaning valve 32, and inject pure water into the dual polymer block 30 by cleaning flow 33, thereby discharging the A polymer solution 25 present in the flow path between cleaning valve 32 and anode side buffer 7 into the waste liquid tank, while replacing the flow path between cleaning valve 32 and anode side buffer 7 with pure water. In this process, the anode side buffer tank is replaced with the waste liquid tank in advance. By making the amount (volume) of pure water injected by cleaning flow 33 greater than the internal volume of the flow path between cleaning valve 32 and anode side buffer 7, the A polymer solution 25 remaining in the flow path is brought close to zero. (2) Next, while keeping valve A 26 closed, valve B 29 closed, anode tank valve 31 open, and cleaning valve 32 open, air is injected into the dual polymer block 30 by the cleaning flow 33. This discharges the pure water present in the flow path between cleaning valve 32 and anode-side buffer 7 into the waste tank, while simultaneously replacing the flow path between cleaning valve 32 and anode-side buffer 7 with air. This ultimately prevents polymer B solution 28 from becoming diluted. (3) Afterwards, valve A 26 is closed, valve B 29 is opened, anode tank valve 31 is opened, and cleaning valve 32 is closed. The piston of syringe B 27 is pressed, thereby discharging the air present in the flow path between valve B 29 and anode-side buffer 7 into the waste tank while simultaneously replacing the flow path between valve B 29 and anode-side buffer 7 with polymer B solution 28. Finally, if... Figure 29 The waste liquid tank is replaced with an anode-side buffer tank, as shown, thus completing the transition to mode B. According to the above method, it is also possible to reduce the amount of polymer B solution 28 consumed during the switch from mode A to mode B. It is also possible to omit either the injection of pure water or the injection of air using the aforementioned cleaning flow 33.

[0219] [3] Regarding Mode B

[0220] Next, we will explain Mode B. For example... Figure 29 As shown, the flow path between valve B 29 and anode tank valve 31 is filled with polymer B solution 28, meaning the sample dissolution end 3 is in contact with polymer B solution 28. At this time, valve A 26 is closed, valve B 29 is opened, anode tank valve 31 is closed, and cleaning valve 32 is closed. The piston of syringe B 27 is pressed to pressurize the polymer B solution 28 inside the dual polymer block 30, filling each capillary 1 with polymer B solution 28 from the sample dissolution end 3 towards the sample injection end 2. After filling with polymer B solution 28, anode tank valve 31 is opened, and different samples are injected into each capillary 1 from the sample injection end 2. Capillary electrophoresis is then performed by applying a high voltage between cathode 4 and anode 5 using power supply 8. Valve B 29 can also be closed at this time.

[0221] [4] Regarding the switch from mode B to mode A

[0222] Finally, the method for switching from mode B to mode A is explained. (1) Close valve A 26, close valve B 29, open anode tank valve 31 and open cleaning valve 32, and inject pure water into the dual polymer block 30 by cleaning flow 33, thereby discharging the B polymer solution 28 present in the flow path between cleaning valve 32 and anode side buffer 7 to the waste liquid tank, while replacing the flow path between cleaning valve 32 and anode side buffer 7 with pure water. In this process, the anode side buffer tank is replaced with the waste liquid tank in advance. By making the amount (volume) of pure water injected by cleaning flow 33 greater than the internal volume of the flow path between cleaning valve 32 and anode side buffer 7, the B polymer solution 28 remaining in the flow path is brought close to zero. (2) Next, while keeping valve A 26 closed, valve B 29 closed, anode tank valve 31 open, and cleaning valve 32 open, air is injected into the dual polymer block 30 by the cleaning flow 33. This discharges the pure water in the flow path between cleaning valve 32 and anode-side buffer 7 to the waste tank while replacing the flow path between cleaning valve 32 and anode-side buffer 7 with air. This ultimately prevents polymer A solution 25 from becoming thin. (3) Afterward, valve A 26 is opened, valve B 29 is closed, anode tank valve 31 is closed, and cleaning valve 32 is opened. The piston of syringe A 24 is pressed, thereby discharging the air in the flow path between valve A 26 and cleaning valve 32 to the outside while replacing the flow path between valve A 26 and cleaning valve 32 with polymer A solution 25. (4) Next, open valve A 26, close valve B 29, open anode tank valve 31, close cleaning valve 32, and press the piston of syringe A 24. This expels air from the flow path between valve A 26 and anode-side buffer solution 7 into the waste tank while simultaneously replacing the flow path between valve A 26 and anode-side buffer solution 7 with polymer solution A 25. Finally, as... Figure 28 As shown, if the waste liquid tank is replaced with an anode-side buffer tank, the conversion to mode A is completed. According to the above method, it is also possible to reduce the amount of polymer A solution 25 consumed when switching from mode B to mode A. It is also possible to omit either the injection of pure water or the injection of air using the aforementioned cleaning flow 33.

[0223] <Structure of the dual polymer block 30>

[0224] In order to effectively or reliably implement the switching between mode A and mode B as described above, the dual polymer block 30 has the following characteristic structure.

[0225] Feature 1 is characterized in that: the sample dissolution end 3 of the capillary 1 is connected in the flow path between valve A 26 and anode tank valve 31, valve A 26 is the opening and closing mechanism of injector A 24 which is the pressurization mechanism for polymer A solution 25, and anode tank valve 31 is the opening and closing mechanism of the anode side buffer solution 7.

[0226] Feature 2 is that the sample dissolution end 3 of the capillary tube 1 is connected to the flow path between the B valve 29 and the anode tank valve 31. The B valve 29 is the opening and closing mechanism of the B syringe 27, which is the pressurization mechanism for the B polymer solution 28. The anode tank valve 31 is the opening and closing mechanism of the anode side buffer solution 7.

[0227] Feature 3 is characterized in that: the sample dissolution end 3 of the capillary tube 1 is connected to the flow path between the cleaning valve 32 and the anode tank valve 31; the cleaning valve 32 is the opening and closing mechanism closest to the injection port of the cleaning flow 33; and the anode tank valve 31 is the opening and closing mechanism closest to the anode side buffer solution 7.

[0228] Feature 4 is characterized in that: A valve 26 and B valve 29 are connected in the flow path between cleaning valve 32 and anode tank valve 31. Cleaning valve 32 is the opening and closing mechanism closest to the injection port of cleaning flow 33, anode tank valve 31 is the opening and closing mechanism closest to the anode side buffer solution 7, A valve 26 is the opening and closing mechanism closest to A syringe 24, which is the pressurization mechanism for A polymer solution 25, and B valve 29 is the opening and closing mechanism closest to B syringe 27, which is the pressurization mechanism for B polymer solution 28.

[0229] (I) Eighth Implementation Method

[0230] The eighth embodiment discloses another configuration example of the improved capillary array electrophoresis apparatus.

[0231] <Example of the configuration of a capillary array electrophoresis apparatus>

[0232] Figure 30 It is shown Figure 28 and Figure 29 The diagram shows another configuration example (modified example) of a capillary array electrophoresis apparatus that allows switching between polymer solution 25 (A) and polymer solution 28 (B) as two separation media. By employing this structure, the same capillary array can be used, and electrophoretic analysis based on multiple polymer solutions can be performed without detaching the capillary array from the apparatus.

[0233] exist Figure 30In the capillary array electrophoresis apparatus shown, a rotary valve 35 is inserted between the syringe 11 and the polymer block 9 and connected by tubing. The rotary valve 35 is also connected to a container containing polymer solution A 25, a container containing polymer solution B 28, and a container containing pure water 34. The rotary valve 35 can connect to any two of the following: the interior of the syringe 11, polymer solution A 25, polymer solution B 28, pure water 34, air, and the interior of the polymer block 9.

[0234] <Analyze actions in mode A and mode B during a conversation, and the actions that switch between them>

[0235] [1] Regarding Mode A

[0236] First, let's explain pattern A. For example... Figure 30 As shown, the flow path between the rotary valve 35 and the valve 10 is filled with polymer solution 25 (A), meaning the sample dissolution end 3 is in contact with polymer solution 25. At this time, the rotary valve 35 connects the polymer solution 25 inside the syringe 11 with the polymer solution 25 inside the polymer block 9. The valve 10 is closed, and the piston of the syringe 11 is pressed to pressurize the polymer solution 25 inside the polymer block 9, filling each capillary 1 with polymer solution 25 from the sample dissolution end 3 towards the sample injection end 2. After filling with polymer solution 25, the valve 10 is opened, and different samples are injected into each capillary 1 from the sample injection end 2. Then, a high voltage is applied between the cathode 4 and the anode 5 by the power supply 8 to perform capillary electrophoresis.

[0237] [2] Regarding the switch from mode A to mode B

[0238] Next, the method for switching from mode A to mode B will be explained. In order to perform the desired electrophoretic analysis, the polymer solution in contact with the sample dissolution end 3 inside the polymer block 9 should not produce the following conditions: the A polymer solution 25 and the B polymer solution 28 are mixed, or the A polymer solution 25 or the B polymer solution 28 becomes thin, or air bubbles are mixed in the polymer solution. Therefore, the switching from mode A to mode B is performed in the next step. (1) Rotate valve 35 to connect the A polymer solution 25 inside syringe 11 with the A polymer solution 25 inside polymer block 9, and open valve 10 and press the piston of syringe 11 to the bottom to discharge the full amount of the A polymer solution 25 inside syringe 11. The same amount of A polymer solution 25 as discharged is discharged from polymer block 9 into the waste tank. At this time, the flow path from the front end of syringe 11 to the boundary between polymer block 9 and the anode-side buffer 7 is filled with A polymer solution 25. (2) Next, rotate valve 35 to connect the inside of syringe 11 to pure water 34, and close valve 10 and pull the piston of syringe 11 up to the top to fill the inside of syringe 11 with pure water 34. (3) Then, rotate valve 35 to connect the pure water 34 inside syringe 11 to polymer solution 25 inside polymer block 9, and open valve 10 and press the piston of syringe 11 down to the bottom to fully discharge the pure water 34 inside syringe 11. The same amount of polymer solution 25 and pure water 34 as discharged is discharged from polymer block 9 into waste tank. At this time, the flow path from the front end of syringe 11 to the boundary between polymer block 9 and the anode-side buffer 7 is filled with pure water 34. Repeat steps (2) and (3) as needed to ensure that polymer solution 25 does not remain in the flow path. (4) Next, rotate valve 35 to connect the inside of syringe 11 to the external gas, and close valve 10 and pull up the piston of syringe 11 to the top to fill the inside of syringe 11 with air. (5) Then, rotate valve 35 to connect the air inside syringe 11 to the pure water 34 inside polymer block 9, and open valve 10 and press down the piston of syringe 11 to the bottom to fully expel the air from syringe 11. The same amount of pure water 34 and air is discharged from polymer block 9 into the waste tank. At this time, the flow path from the front end of syringe 11 to the boundary between polymer block 9 and the anode-side buffer solution 7 is filled with air. Repeat steps (4) and (5) as needed to prevent liquid from remaining in the flow path. (6) This time, rotate valve 35 to connect the inside of syringe 11 to polymer solution 28, and close valve 10 and pull up the piston of syringe 11 to the top to fill the inside of syringe 11 with polymer solution 28.(7) Finally, the rotary valve 35 connects the B polymer solution 28 inside the syringe 11 with the air inside the polymer block 9, and the valve 10 is opened and the piston of the syringe 11 is pressed to discharge all the air inside the polymer block 9 into the waste tank. During the stage when the flow path from the tip of the syringe 11 to the boundary between the polymer block 9 and the anode-side buffer 7 is filled with the B polymer solution 28, the descent of the piston of the syringe 11 is stopped, completing the conversion to B mode.

[0239] [3] Regarding Mode B

[0240] Next, the B mode will be explained. The flow path between rotary valve 35 and valve 10 is filled with polymer solution 28 (B), meaning the sample dissolution end 3 is in contact with polymer solution 28. At this time, rotary valve 35 connects the polymer solution 28 inside syringe 11 with the polymer solution 28 inside polymer block 9, and valve 10 is closed while the piston of syringe 11 is pressed, pressurizing the polymer solution 28 inside polymer block 9. The polymer solution 28 is then filled into each capillary 1 from sample dissolution end 3 towards sample injection end 2. After filling with polymer solution 28, valve 10 is opened, and different samples are injected into each capillary 1 from sample injection end 2. Then, a high voltage is applied between cathode 4 and anode 5 by power supply 8 to perform capillary electrophoresis.

[0241] [4] Regarding the switch from mode B to mode A

[0242] Finally, the method for switching from mode B to mode A is explained. (1) Rotate valve 35 to connect the B polymer solution 28 inside syringe 11 to the B polymer solution 28 inside polymer block 9, open valve 10 and press the piston of syringe 11 to the bottom to fully discharge the B polymer solution 28 inside syringe 11. The same amount of B polymer solution 28 as discharged is discharged from polymer block 9 to waste liquid tank. At this time, the flow path from the front end of syringe 11 to the boundary between polymer block 9 and anode-side buffer 7 is filled with B polymer solution 28. (2) Next, rotate valve 35 to connect the inside of syringe 11 to pure water 34, close valve 10 and pull up the piston of syringe 11 to the top to fill the inside of syringe 11 with pure water 34. (3) Then, rotate valve 35 to connect the pure water 34 inside syringe 11 to the B polymer solution 28 inside polymer block 9, open valve 10 and press the piston of syringe 11 to the bottom to fully discharge the pure water 34 inside syringe 11. The same amount of polymer B solution 28 and pure water 34 as the polymer block 9 are discharged into the waste liquid tank. At this time, the flow path from the tip of the syringe 11 to the boundary between the polymer block 9 and the anode-side buffer 7 is filled with pure water 34. Steps (2) and (3) are repeated as needed to ensure that polymer B solution 28 does not remain in the flow path. (4) Next, the rotary valve 35 connects the inside of the syringe 11 to the external gas, and the valve 10 is closed and the piston of the syringe 11 is pulled up to the top to fill the inside of the syringe 11 with air. (5) Then, the rotary valve 35 connects the air inside the syringe 11 to the pure water 34 inside the polymer block 9, and the valve 10 is opened and the piston of the syringe 11 is pressed down to the bottom to fully expel the air from the syringe 11. The same amount of pure water 34 and air as the polymer block 9 are discharged into the waste liquid tank. At this time, the flow path from the tip of the syringe 11 to the boundary between the polymer block 9 and the anode-side buffer 7 is filled with air. As needed, repeat steps (4) and (5) to ensure no liquid remains in the flow path. (6) This time, rotate valve 35 to connect the inside of syringe 11 to polymer solution 25, and close valve 10 and pull the piston of syringe 11 up to the top to fill the inside of syringe 11 with polymer solution 25. (7) Finally, rotate valve 35 to connect the inside of syringe 11 with the air inside polymer block 9, and open valve 10 and press down the piston of syringe 11 to discharge all the air inside polymer block 9 into the waste tank. During the stage when the flow path from the front end of syringe 11 to the boundary between polymer block 9 and the anode-side buffer 7 is filled with polymer solution 25, stop the descent of the piston of syringe 11 to complete the conversion to mode A.

[0243] As described above, the device is effective in effectively or reliably switching between mode A and mode B. The rotary valve 35 in the eighth embodiment combines the functions of valve A 26, valve B 29, and cleaning valve 32 in the seventh embodiment. Furthermore, the syringe 11 in the eighth embodiment combines the functions of syringe A 24, syringe B 27, and cleaning flow in the seventh embodiment. Additionally, the valve 10 in the eighth embodiment functions the same as the anode tank valve 31 in the seventh embodiment. When considered in this way, it can be said that the characteristic structures 1 to 4 described in the seventh embodiment also hold true in the eighth embodiment. Alternatively, it can be a specialized embodiment as follows: The characteristic structure is that the sample dissolution end 3 of the capillary 1 is connected to the flow path between the rotary valve 35 and the anode tank valve 10; the rotary valve 35 is the opening and closing mechanism closest to the syringe 11, which serves as a pressurization mechanism for any one of polymer solution A 25, polymer solution B 28, pure water 34, or air; and the anode tank valve 10 is the opening and closing mechanism closest to the anode-side buffer solution 7.

[0244] (J) Summary

[0245] (i) The optimal correction factor for the emission fluorescence intensity distribution varies depending on the refractive index n3 of the separation medium. Given the above, in the absence of a specified refractive index n3 (an unclear situation), it is unclear which correction factor is better, and without using the optimal correction factor, it may itself become a major cause of error. Originally, the refractive index n3 of the separation medium also varies depending on environmental conditions such as temperature. Under such conditions, it is not necessarily clear which correction is better. Therefore, in the technology disclosed herein, the relationship between the refractive index n3 of the separation medium introduced into the capillary array and the quadratic coefficient when the relative fluorescence intensity distribution is approximated using a quadratic function is ( Figure 17 (c)). For example Figure 17 As shown in (a), the distribution of relative fluorescence intensity before correction is entirely convex downwards, as... Figure 17As shown in (c), the quadratic coefficient is positive. The appropriateness of this correction depends on the refractive index n3 used as the reference. For example, when digital correction is performed using a refractive index n3 = 1.36 as the reference, in the case of a separation medium with a refractive index n3 = 1.36, the corrected relative fluorescence intensity distribution becomes flat, and the quadratic coefficient is zero. In contrast, in the case of a separation medium with a refractive index greater than 1.36, the corrected relative fluorescence intensity distribution is convex upwards, and the quadratic coefficient is negative. This indicates overcorrection. Furthermore, in the case of a separation medium with a refractive index less than 1.36, the corrected relative fluorescence intensity distribution is convex downwards, and the quadratic coefficient is positive. This indicates undercorrection. However, even in cases of overcorrection or undercorrection, the distribution is flattened compared to the relative fluorescence intensity distribution before correction, so the effect of the correction (the correction is effective) is considered to be expected. In other words, one of the features of this disclosure is that the correction reference and the distribution of the digital correction coefficients are changed in accordance with the refractive index n3 of the separation medium used.

[0246] In this disclosure, the average value of the second derivative of the convexity of the laser irradiation intensity distribution L(n) of the capillary number n (numbered n = 1, 2, ..., N in order of arrangement from one end of the capillary arrangement) of the N capillaries in the laser irradiation section 14, expressed as a function of n, is set as A (since this second derivative deviates slightly depending on the position of the capillary, the average value is used). A is a value uniquely determined under predetermined conditions. Furthermore, the average value of the second derivative of the convexity of the output fluorescence intensity distribution H(n) of the capillary number n, expressed as a function of n, when there is an equal concentration of luminescent material inside the N capillaries in the laser irradiation section 14, is set as B. At this time, the capillary electrophoresis apparatus is configured to have at least an analytical mode for capillary electrophoresis using a separation medium with a refractive index n3 < 1.36, and |A| > |B| holds. In other words, if |A| > |B|, then when using a separation medium with a refractive index of n3 < 1.36, the downward convex relative fluorescence intensity distribution is nearly flat, which can achieve effective correction.

[0247] (ii) More specifically, |A| > |B| can be achieved by performing at least one of optical correction based on the optical system or digital correction based on a computer. Even without specifically studying the structure of the optical system, |A| > |B| can be achieved through digital correction, but the structure of the optical system can also be modified to perform digital correction based on computer calculations. Furthermore, |A| > |B| can also be achieved by simply modifying the structure of the optical system.

[0248] For example, in the case of a separation medium with 24 capillaries and a high refractive index, such as Figure 5As shown, by applying only the correction factor J(n) of the actual optical system, the relative fluorescence intensity distribution becomes flat. It can be seen that in such cases (where the refractive index of the separation medium is high), no digital correction is needed. In contrast, when using a finer capillary (refer to...), Figure 6 When the refractive index is low, even with optical system corrections, the desired effect cannot be expected. This is because, due to the use of fine capillaries, each capillary is close to the central axis of the optical system, rendering optical corrections ineffective. For example, sometimes optical system corrections may slightly flatten the relative fluorescence intensity distribution, but not completely flatten it. In such cases, additional digital corrections can further improve flatness (see [reference]). Figure 11 In other words, digital corrections compensate for deficiencies in the optical system's corrections, and by using both optical and digital corrections together, the overall distribution becomes flat. A simple method, for example, is to optimize the digital correction coefficients to flatten the relative fluorescence intensity distribution according to the refractive index. On the other hand, depending on the situation, sometimes digital corrections are not performed, and the optical system is artificially modified (at least a portion of the optical system's structure is changed) to optimize the optical system, thereby achieving a flat relative fluorescence intensity distribution.

[0249] As described above, in the technology disclosed herein, a flat relative fluorescence intensity distribution can be achieved by modifying at least a portion of the structure of the optical system, or it can be achieved solely through digital correction. Alternatively, both of the above methods can be used.

[0250] Furthermore, specific methods for controlling the distribution of correction coefficients in an optical system are shown, for example, in U.S. Patent 7,477,381B and the aforementioned Patent Document 3 (Japanese Patent No. 6,113,549). The former patent shows that by… Figure 24 The illustrated cat-eye-shaped light blocking aperture 112 is inserted into the optical system to receive light emitted from each capillary with equal efficiency. Furthermore, the latter patent illustrates that by inserting a variable concentration filter (a filter that changes the light transmittance according to position) into the optical system, light emitted from each capillary can be received equally. Both documents demonstrate achieving uniform light reception efficiency, but this is only one example and does not necessarily ensure uniformity. The technique shown here allows control of the correction coefficient distribution of the optical system by varying the design of each device.

[0251] (iii) In this disclosure, predetermined digital correction coefficients are stored in memory corresponding to the values ​​(multiple) of the refractive index n3 of the separation medium used for analysis. The computer reads the correction coefficients from the memory (switching tables), and by applying these correction coefficients, the measured fluorescence intensity distribution M(n) can be appropriately corrected. Correction is usually performed through calibration, but since there are cases where correction is possible and cases where it is not, reading and using the correction coefficients is effective.

[0252] (iv) One feature of the technology disclosed herein is that when a separating medium with a refractive index greater or smaller than the intended n3 is introduced into the capillary, the value of B (showing the average value of the second derivative of the convexity of H(n) as a function of n, 1 ≤ n ≤ N) changes. For example, when the relative fluorescence intensity distribution is corrected based on a refractive index n3 = 1.36, from Figure 17 As shown in (c) (referring to the value on the vertical axis), B ≈ 0 when the refractive index n3 = 1.36. In this case, if a separation medium with a refractive index higher than n3 = 1.36 and a refractive index lower than n3 = 1.36 is introduced into the capillary, the degree of upward and downward bulging of the relative fluorescence intensity distribution will change. This observation is first discovered by the technology disclosed herein.

[0253] (v) Furthermore, one of the features of this disclosure is that, as the refractive index n3 of the separating medium increases sequentially, the average value B of the second derivative of the output fluorescence intensity distribution H(n), which is a function of n representing the output light intensity of the capillary number n, must change from positive to negative. This is a feature that is impossible to possess in the prior art. In this case, if a separating medium with a very high refractive index n3 (e.g., refractive index n3 = 1.42) is introduced into the capillary, the value of B is negative (the relative fluorescence intensity distribution bulges upward), and if a separating medium with a very low refractive index n3 (e.g., refractive index n3 = 1.30) is introduced into the capillary, the value of B is positive (the relative fluorescence intensity distribution bulges downward).

[0254] (vi) Furthermore, one of the features of this disclosure is the use of multiple separation media with a predetermined refractive index n3. Current products can use multiple separation media, but the refractive index n3 is 1.41 in any one of them. In contrast, in this disclosure, not only n3 = 1.41, but also separation media with n3 = 1.33, n3 = 1.36, etc. (multiple refractive index separation media) can be used for analysis. This concept is first discovered by the technology of this disclosure.

[0255] Symbol Explanation

[0256] 1—Capillary, 2—Sample injection end, 3—Sample dissolution end, 4—Cathode, 5—Anode, 6—Cathode-side buffer solution, 7—Anode-side buffer solution, 8—Power supply, 9—Polymer block, 10—Valve, 11—Injector, 12—Laser source, 13—Laser beam, 14—Laser irradiation section, 15—Focusing lens, 16—Laser cutoff filter, 17—Transmission diffraction grating, 18—Imaging lens, 19—Sensor, 20—Emitting point, 21—Fluorescence, 22—Imaging point, 23—Optical axis, 24—Injector A, 25—Polymer solution A, 26—Valve A, 27—Injector B, 28—Polymer solution B, 29—Valve B, 30—Dual polymer block, 31—Anode tank valve, 32—Cleaning valve, 33—Cleaning flow, 34—Pure water, 35—Rotary valve.

Claims

1. A capillary array electrophoresis apparatus, characterized in that, have: A laser source that emits a laser beam; A capillary array is constructed in which the laser irradiation parts of N capillaries that will be irradiated by the laser beam are arranged approximately on the same plane, wherein N is set to an integer greater than or equal to 2. An optical system that simultaneously measures the fluorescence emitted from the aforementioned N capillaries; and A computer applies a predetermined processing to the fluorescence intensity of the N capillaries measured by the aforementioned optical system and outputs the result. Let the outer radius of the N capillaries in the laser irradiation section be R, the inner radius be r, the refractive index of the external medium be n1, the refractive index of the raw material be n2, and the refractive index of the internal medium be n3. Starting from one end of the arrangement, label the N capillaries in the laser irradiation section with capillary numbers n = 1, 2, ..., N, according to their arrangement order. Compared to the downward bulging of the fluorescence intensity distribution I(n) of the luminescent fluorescence intensity of the capillary number n, which is a function of n, when there is an equal concentration of fluorescent material inside the N capillaries in the laser irradiation section, the downward bulging of the output fluorescence intensity distribution H(n) of the capillary number n obtained by the computer, which is a function of n, when there is an equal concentration of fluorescent material inside the N capillaries in the laser irradiation section, becomes smaller.

2. A capillary array electrophoresis apparatus, characterized in that, have: A laser source that emits a laser beam; A capillary array is constructed in which the laser irradiation parts of N capillaries that will be irradiated by the laser beam are arranged approximately on the same plane, wherein N is set to an integer greater than or equal to 2. An optical system that simultaneously measures the fluorescence emitted from the aforementioned N capillaries; and A computer applies a predetermined processing to the fluorescence intensity of the N capillaries measured by the aforementioned optical system and outputs the result. Let the outer radius of the N capillaries in the laser irradiation section be R, the inner radius be r, the refractive index of the external medium be n1, the refractive index of the raw material be n2, and the refractive index of the internal medium be n3. Starting from one end of the arrangement, label the N capillaries in the laser irradiation section with capillary numbers n = 1, 2, ..., N, according to their arrangement order. Compared to the downward bulge of the measured fluorescence intensity distribution M(n) of the measured fluorescence intensity of the capillary number n, which is a function of n, when there is an equal concentration of fluorescent material inside the N capillaries in the laser irradiation section, the downward bulge of the output fluorescence intensity distribution H(n) of the capillary number n obtained by the computer, which is a function of n, when there is an equal concentration of fluorescent material inside the N capillaries in the laser irradiation section, is smaller.

3. The capillary array electrophoresis apparatus according to claim 1 or 2, characterized in that, The above processing is a digital correction process as follows: multiply the above luminescent fluorescence intensity distribution I(n) or the above measured fluorescence intensity distribution M(n) by the digital correction coefficient distribution K(n) which is a function of n representing the above capillary number n, and derive the output fluorescence intensity distribution H(n).

4. The capillary array electrophoresis apparatus according to claim 3, characterized in that, The distribution of the numerical correction factor K(n) is changed according to the refractive index n3 of the internal medium.

5. The capillary array electrophoresis apparatus according to claim 3 or 4, characterized in that, It also has a memory for storing the aforementioned digital correction coefficient distribution K(n). The computer reads the digital correction coefficient distribution K(n) from the memory and performs the digital correction process.

6. The capillary array electrophoresis apparatus according to any one of claims 3 to 5, characterized in that, When the above-mentioned digital correction coefficient distribution K(n) is used to correct the refractive index n3 of the above-mentioned internal medium as the correction reference and the above-mentioned internal medium as the correction object, the degree of downward or upward bulging of the above-mentioned output fluorescence intensity distribution H(n) is minimized when n3 is the correction reference = n3 is the correction object.

7. The capillary array electrophoresis apparatus according to any one of claims 3 to 5, comprising: When the digital correction coefficient distribution K(n) is used to correct the refractive index n3 of the internal medium, and the digital correction process is performed on the actual refractive index n3 of the internal medium, the output fluorescence intensity distribution H(n) becomes an upward convex curve when n3 (correction reference) < n3 (correction object), and becomes a downward convex curve when n3 (correction reference) > n3 (correction object).

8. The capillary array electrophoresis apparatus according to any one of claims 3 to 5, characterized in that, When the above-mentioned digital correction coefficient distribution K(n) is used to correct the refractive index n3 of the above-mentioned internal medium as the correction reference, and the above-mentioned digital correction processing is performed on the actual refractive index n3 of the above-mentioned internal medium as the correction object, the greater the difference between n3 as the correction reference and n3 as the correction object, the greater the degree to which the above-mentioned output fluorescence intensity distribution H(n) bulges upward or downward.

9. A capillary array electrophoresis apparatus, characterized in that, have: A laser source that emits a laser beam; A capillary array is constructed in which the laser irradiation parts of N capillaries that will be irradiated by the laser beam are arranged approximately on the same plane, wherein N is set to an integer greater than or equal to 2. An optical system that simultaneously measures the fluorescence emitted from the aforementioned N capillaries; and A computer applies a predetermined processing to the fluorescence intensity of the N capillaries measured by the aforementioned optical system and outputs the result. Let the outer radius of the N capillaries in the laser irradiation section be R, the inner radius be r, the refractive index of the external medium be n1, the refractive index of the raw material be n2, and the refractive index of the internal medium be n3. Starting from one end of the arrangement, label the N capillaries in the laser irradiation section with capillary numbers n = 1, 2, ..., N, according to their arrangement order. When there is an equal concentration of fluorescent material inside the N capillaries in the laser irradiation section, the quadratic coefficient of the distribution of luminous fluorescence intensity I(n), which is expressed as a function of n for the luminous fluorescence intensity of the capillary number n, is set to A. When there is an equal concentration of fluorescent material inside the N capillaries in the laser irradiation section, the quadratic coefficient of the distribution of output fluorescence intensity H(n), which is expressed as a function of n for the output fluorescence intensity of the capillary number n obtained by the computer, is set to B. In this case, the computer performs processing that becomes |A| > |B|.

10. A capillary array electrophoresis apparatus, characterized in that, have: A laser source that emits a laser beam; A capillary array is constructed in which the laser irradiation parts of N capillaries that will be irradiated by the laser beam are arranged approximately on the same plane, wherein N is set to an integer greater than or equal to 2. An optical system that simultaneously measures the fluorescence emitted from the aforementioned N capillaries; and A computer applies a predetermined processing to the fluorescence intensity of the N capillaries measured by the aforementioned optical system and outputs the result. Let the outer radius of the N capillaries in the laser irradiation section be R, the inner radius be r, the refractive index of the external medium be n1, the refractive index of the raw material be n2, and the refractive index of the internal medium be n3. Starting from one end of the arrangement, label the N capillaries in the laser irradiation section with capillary numbers n = 1, 2, ..., N, according to their arrangement order. When there is an equal concentration of fluorescent material inside the N capillaries in the laser irradiation section, the quadratic coefficient of the measured fluorescence intensity distribution M(n), which is expressed as a function of n for the measured fluorescence intensity of the capillary number n, is set to A. When there is an equal concentration of fluorescent material inside the N capillaries in the laser irradiation section, the quadratic coefficient of the output fluorescence intensity distribution H(n), which is expressed as a function of n for the output fluorescence intensity of the capillary number n obtained by the computer, is set to B. In this case, the computer performs processing that becomes |A| > |B|.

11. The capillary array electrophoresis apparatus according to claim 9 or 10, characterized in that, The above processing is a digital correction process as follows: multiply the above luminescent fluorescence intensity distribution I(n) or the above measured fluorescence intensity distribution M(n) by the digital correction coefficient distribution K(n) which is a function of n representing the above capillary number n, and derive the output fluorescence intensity distribution H(n).

12. The capillary array electrophoresis apparatus according to claim 11, characterized in that, The distribution of the numerical correction factor K(n) is changed according to the refractive index n3 of the internal medium.

13. The capillary array electrophoresis apparatus according to claim 11 or 12, characterized in that, It also has a memory for storing the aforementioned digital correction coefficient distribution K(n). The computer reads the digital correction coefficient distribution K(n) from the memory and performs the digital correction process.

14. The capillary array electrophoresis apparatus according to any one of claims 11 to 13, characterized in that, When the above-mentioned digital correction coefficient distribution K(n) is used to apply the above-mentioned digital correction process to the actual refractive index n3 of the above-mentioned internal medium as the correction object, when n3 is the correction reference = n3 is the correction object, |B| becomes the minimum.

15. The capillary array electrophoresis apparatus according to any one of claims 11 to 13, characterized in that, When the above-mentioned digital correction coefficient distribution K(n) is used for the refractive index n3 of the above-mentioned internal medium as the correction reference, and the above-mentioned digital correction process is performed on the actual refractive index n3 of the above-mentioned internal medium as the correction object, when n3 is the correction reference < n3 is the correction object, B < 0, and when n3 is the correction reference > n3 is the correction object, B > 0.

16. The capillary array electrophoresis apparatus according to any one of claims 11 to 13, characterized in that, When the above-mentioned digital correction coefficient distribution K(n) is used for the refractive index n3 of the above-mentioned internal medium as the correction reference, and the above-mentioned digital correction process is performed on the actual refractive index n3 of the above-mentioned internal medium as the correction object, the larger the difference between n3 as the correction reference and n3 as the correction object, the larger |B| is.

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