Capillary array

Abstract

In a capillary array consisting of N analytical capillaries filled with a separation medium of refractive index n3 and used for electrophoretic analysis, and N±1 lens capillaries filled with a medium of refractive index n4 and not used for electrophoretic analysis, arranged alternately on the same plane, the outer diameter of each capillary is set to 2R, the inner diameter is set to 2r, the refractive index of the external medium is set to n1, and the refractive index of the raw material is set to n2. When the refractive index n3 takes any value of 1.33≤n3≤1.42, by making R, r, n1, n2, n3, and n4 satisfy the specified relationship, a laser beam incident from the side of the array plane can be used to efficiently and simultaneously irradiate the N analytical capillaries.

Description

Technical Field

[0001] This disclosure relates to capillary arrays. Background Technology

[0002] Capillary array electrophoresis (CAGE) devices, which use multiple quartz glass capillaries filled with electrolyte solutions or electrolyte solutions containing polymer gels or other electrophoretic separation media and perform electrophoretic analysis in parallel, are widely used. Compared to conventional capillary electrophoresis devices using a single capillary, CAGE devices not only increase analytical throughput but also reduce the cost per sample. The most widely used CAGE 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-irradiated portions (the parts of the capillary array that are irradiated by the laser) of the multiple capillaries are arranged on the same plane with the polyimide coating removed. The same plane is called the arrangement 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 sequence, starting from one end. During electrophoresis, a laser beam is introduced from the side of the arrangement plane to simultaneously irradiate multiple capillaries, and the fluorescence induced by this is dispersed and detected simultaneously. The method of simultaneously irradiating multiple capillaries by introducing a laser beam from the side of the arrangement 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, causing the laser beam to be repeatedly focused along the arrangement plane and travel in the capillary array, thereby enabling simultaneous irradiation of multiple capillaries. As a result, DNA sequence or DNA fragment analysis of the same number of samples as the number of capillaries can be performed side by side. As described in Patent Document 1, in the laser irradiation section of multiple capillaries, the outer radius of the capillaries 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 capillaries is set to n2, the refractive index of the medium outside the capillaries is set to n1, the refractive index of the medium inside the capillaries (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). When x = r / 2, the refraction angle of the laser beam passing through one capillary is expressed by the following formula (1).

[0003] [Number 1]

[0004]

[0005] Each capillary acts as a concave lens when Δθ > 0 and as a convex lens when Δθ < 0. By setting Δθ < 0, the multifocal lens functions, allowing the laser beam to simultaneously irradiate multiple capillary tubes. Conversely, if Δθ > 0, the multifocal lens does not function, and the laser beam diverges from the plane of arrangement, thus preventing simultaneous irradiation of multiple capillary tubes. Typically, the capillary tubes are made of quartz glass, with a fixed n2 = 1.46. According to equation (1), to enhance the convex lens effect of each capillary (weaken the concave lens effect), a smaller n1 and a larger n3 are better. Conversely, a larger n1 and a smaller n3 result in a stronger concave lens effect of each capillary tube.

[0006] Even when multifocal functions, the intensity of the laser beam decreases as it travels through the capillary array due to reflection losses at the interfaces between the capillary and the external medium, and between the capillary and the internal medium. Consequently, the fluorescence intensity also decreases. If the fluorescence intensity varies significantly between capillaries, it becomes unsuitable to analyze multiple samples under identical conditions. (It should be noted that in the embodiments described later, fluorescence intensity is used as a representative of 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, the laser beam oscillating from a single laser source is split into two beams, which are then incident from opposite sides of the array plane, enabling multifocal functions. This homogenizes the combined intensity of the laser beam incident from one side of the array plane and the laser beam incident from the other side. A configuration where the laser beam enters from only one side of the array plane is called single-sided illumination, and a configuration where the 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 number n at the end of the laser beam entering the array is set to n=1, and the capillary number n at the end of the laser beam exiting the array is set to n=N. In the case of double-sided illumination, the capillary number n at one end of the array is set to n=1, and the capillary number n at the opposite end of the array is set to n=N.

[0007] Among the individual irradiation intensities and fluorescence intensities of multiple capillaries within the same capillary array, the lower the irradiation intensity and the higher the fluorescence intensity, the better. Empirically, when the desired fluorescence intensity is set to 1 when the full intensity of the laser beam oscillating from the laser source is irradiated into the interior of a single capillary, a practical sensitivity can be obtained if the minimum fluorescence intensity MIN is ≥ 0.2. Furthermore, the smaller the deviation in irradiation intensity and fluorescence intensity among multiple capillaries within the same capillary array, the better. Empirically, if the coefficient of variation (CV) of fluorescence intensity is ≤ 20%, or ≤ 15% under certain conditions, different samples can be analyzed under the same conditions. These are referred to as the practical performance of the capillary array electrophoresis device. In this disclosure, it is assumed that the phosphor concentration in the laser irradiation section of each capillary is constant; therefore, fluorescence intensity and laser irradiation intensity have the same meaning.

[0008] In DNA sequence or fragment analysis using the 3500 series and 3730 series gene analyzers, to enable electrophoretic separation of DNA fragments in the sample 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 commercially available separation media for the 3500 series and 3730 series gene analyzers, namely POP-4, POP-6, and POP-7, all contain 8M urea. Water has a refractive index of 1.33, while 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 separation.

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

[0010] Therefore, the 3730 series gene analyzer utilizes the configuration shown in Patent Document 2 to increase the number of capillaries that can be irradiated simultaneously. In the 3730 series gene analyzer, the laser irradiation section of multiple capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm is arranged in a fluorine solution with a refractive index 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, 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, thus increasing the number of capillaries that can be irradiated simultaneously. Therefore, it is possible to irradiate 48 or 96 capillaries simultaneously using a laser beam.

[0011] The configuration shown in Non-Patent Document 1 further increases the number of capillaries that can be simultaneously irradiated. In this configuration, the laser irradiation section of multiple capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm is arranged in a matching solution with a refractive index n1 = 1.46. Furthermore, among the multiple capillaries arranged, the odd-numbered capillaries from one end are used for analysis (hereinafter referred to as the analysis capillaries), and the even-numbered capillaries are used as rod-shaped lenses (hereinafter referred to as lens capillaries). That is, the analysis capillaries and lens capillaries are arranged alternately. The refractive index of the medium (separation medium) inside the analysis 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 of the capillaries is quartz glass with a refractive index n2 = 1.46. In addition, the reflection loss of the laser beam at the interface between the matching solution layer outside the capillaries and the capillaries (quartz glass) is zero, thus further increasing the number of capillaries that can be simultaneously irradiated. Furthermore, in Non-Patent Document 1, pages 2874-2875 describe the definition of the maximum number of capillaries that can be simultaneously irradiated by a laser beam. When irradiating from one side with the incident intensity set to 100%, the maximum number of capillaries that can be simultaneously irradiated is defined as twice the number of capillaries whose intensity is attenuated to 50%. This is because, when irradiating a capillary array with this number of capillaries from both sides, it is expected that the irradiation intensity of each capillary will be uniform. According to this definition, the maximum number of capillaries in Patent Document 2 is 150, and the maximum number of capillaries in Non-Patent Document 1 is 550.

[0012] In Patent Document 3, similarly to Non-Patent Document 1, analytical capillaries and lens capillaries are arranged alternately in the capillary array. Only the analytical capillaries are labeled with capillary numbers n = 1, 2, ..., N from the end of the capillary array. That is, the total number of capillaries combining analytical and lens capillaries is 2 × N.

[0013] Existing technical documents

[0014] Patent documents

[0015] Patent Document 1: Japanese Patent No. 3654290

[0016] Patent Document 2: Japanese Patent No. 5039156

[0017] Patent Document 3: Japanese Patent No. 3559648

[0018] Non-patent literature

[0019] Non-patent literature 1: Electrophoresis 2006, 27, 2869-2879 Summary of the Invention

[0020] The problem that the invention aims to solve

[0021] Here, we evaluate the number of capillaries capable of simultaneous irradiation considering reflection loss and the practicality of the aforementioned known technologies. This evaluation is the first of its kind in this disclosure. In order to approximate the transmittance of a laser beam considering reflection loss, we assume that the incident angle of a laser beam incident on the interface of two media with different refractive indices is 0°. 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, the reflectance is ref={(n1-n2) / (n1+n2)}^2, and the transmittance is tra=1-ref. Therefore, the transmittance T of the laser beam passing through one capillary is approximately obtained by the following equation (2).

[0022] [Number 2]

[0023]

[0024] Based on the 3500 series gene analyzer of the aforementioned Patent Document 1, T = 93% was calculated. In reality, the transmittance of components containing laser beams with incident angles not of 0° is slightly smaller than that of Equation (2). Therefore, Equation (2) represents the ideal transmittance. In the case of unilateral irradiation, if the laser irradiation intensity incident on the capillary with capillary number n = 1 is set to 1, then the laser irradiation intensity of the capillary with capillary number n is represented by the following Equation (3).

[0025] [Number 3]

[0026] L(n) = T n-1 …(3)

[0027] That is, in the above-mentioned 3500 series gene analyzer, when the number of capillaries N=24, the laser irradiation intensity decreases to 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, in the case of irradiation from both sides, if the laser irradiation intensity incident on the capillary with capillary number n=1 and n=N is set to 0.5 respectively, then the laser irradiation intensity of the capillary with capillary number n is expressed by the following formula (4).

[0028] [Number 4]

[0029] L(n) = 0.5·(T) n-1 +T N-n (4)

[0030] Unlike the case of unilateral illumination, the intensity attenuation of laser beams incident from both sides of the array plane is canceled out, thus improving the uniformity of laser illumination intensity in each capillary. Simultaneously, the lowest laser illumination intensity increases. Specifically, the capillaries located at the ends of the capillary array (n=1 and n=N) have the highest laser illumination 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 illumination intensity. That is, if plotted on the horizontal axis n and the vertical axis L(n), the distribution is convex downwards. Under the conditions of the aforementioned 3500 series gene analyzer, when the number of capillaries N=24, the laser irradiation intensity of the capillaries located at both ends of the capillary array (n=1 and n=24) is 0.60, and the laser irradiation intensity of the capillaries located in the center of the capillary array (n=12 and n=13) is 0.44, satisfying the practical performance requirement of MIN≥0.2. Furthermore, the coefficient of variation of the laser irradiation intensity of the 24 capillaries is 11%, satisfying the practical performance requirements of CV≤20% and CV≤15%.

[0031] However, under the conditions of the aforementioned 3500 series gene analyzer, if the number of capillaries is set to N=48, the laser irradiation intensity of the capillaries located at both ends of the capillary array (n=1 and n=48) is 0.52, and the laser irradiation intensity of the capillaries located in the center of the capillary array (n=24 and n=25) is 0.19, which does not meet the practical performance requirement of MIN≥0.2. Furthermore, the coefficient of variation of the laser irradiation intensity is 35%, and the practical performance requirements of CV≤20% and CV≤15% are not met. That is, even with irradiation from both sides, the uniformity of laser irradiation intensity of each capillary will decrease, and the lowest laser irradiation intensity will also decrease. Therefore, it is difficult to simultaneously irradiate 48 capillaries under these conditions.

[0032] Under the conditions of the 3730 series gene analyzer based on Patent Document 2, according to Equation (2), T = 99%, the transmittance is significantly improved compared to the conditions of the 3500 series gene analyzer. Furthermore, if the number of capillaries in Equation (4) is N = 48, the laser irradiation intensity of the capillaries (n = 1 and n = 48) located at both ends of the capillary array is 0.78, and the laser irradiation intensity of the capillaries (n = 24 and n = 25) located in the center of the capillary array is 0.74, satisfying MIN ≥ 0.2. In addition, the coefficient of variation of the laser irradiation intensity is 1%, satisfying the practical performance requirements of CV ≤ 20% and CV ≤ 15%. Furthermore, if the number of capillaries is N = 96 in equation (4), the laser irradiation intensity of the capillaries (n = 1 and n = 96) located at both ends of the capillary array is 0.65, and the laser irradiation intensity of the capillaries (n = 48 and n = 49) located in the center of the capillary array is 0.55, satisfying MIN ≥ 0.2. In addition, the coefficient of variation of the laser irradiation intensity is 5%, satisfying the practical performance requirements of CV ≤ 20% and CV ≤ 15%. In this way, the uniformity of the laser irradiation intensity of 48 and 96 capillaries is improved, and at the same time, the minimum laser irradiation intensity is increased, enabling simultaneous irradiation of 48 and 96 capillaries.

[0033] To compare the configuration of Patent Document 3 with that of Patent Document 2 and Non-Patent Document 1, the conditions are generalized to the extent possible. In the configuration of Patent Document 3, the laser irradiation section of multiple capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm is arranged in water with a refractive index n1 = 1.33. The refractive index of the medium (separation medium) inside the analytical capillary is set to n3 = 1.41, and the refractive index of the medium inside the lens capillary is set to n4 = 1.46. The raw material of the capillaries is quartz glass, n2 = 1.46. At this time, according to Equation (1), the refraction angle of one analytical capillary is Δθ. A = +0.03°, on the other hand, the refraction angle of a single lens capillary is Δθ. B = -2.1°. At this time, Δθ A +Δθ B = -2.07°, therefore, this group of one analytical capillary and one lens capillary acts as a convex lens, enabling multifocal focusing. Thus, this disclosure discovers that through Δθ A +Δθ B This is a method to evaluate the presence or absence of multifocal functionality. Furthermore, under these conditions, according to equation (2), the transmittance of one analytical capillary is T. A =99.5%, the transmittance of one lens capillary is T B =99.6%. Therefore, the transmittance of this group of one analytical capillary and one lens capillary is T = T A ×T B=99.1%. At this point, if the number of analytical capillaries in equation (4) is N = 96, the laser irradiation intensity of the analytical capillaries (n = 1 and n = 96) located at both ends of the capillary array is 0.71, and the laser irradiation intensity of the capillaries (n = 48 and n = 49) located in the center of the capillary array is 0.64, satisfying MIN ≥ 0.2. Furthermore, the coefficient of variation of the laser irradiation intensity is 3%, satisfying the practical performance requirements of CV ≤ 20% and CV ≤ 15%.

[0034] Furthermore, in the aforementioned known techniques, all separation media contain a high concentration of urea, n3 = 1.41. On the other hand, capillary electrophoresis apparatus using a single capillary tube is not limited to using a high concentration of urea in the separation medium; various 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. That is, typically, the refractive index of the separation medium used in capillary electrophoresis can be various values ​​ranging 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 the use of such various separation media in capillary array electrophoresis apparatuses.

[0035] However, in any of the aforementioned known techniques, if n3 = 1.33, the convex lens effect of each capillary disappears, the concave lens effect becomes stronger, and the multifocal structure fails to function. That is, parallel electrophoretic analysis using multiple capillary tubes cannot be performed. Specifically, as described below.

[0036] In the 3500 series gene analyzer based on Patent Document 1, when n3 = 1.33, according to Equation (1), Δθ = +1.3°, it can be seen that each capillary acts as a concave lens. Therefore, the multifocal function is not effective, and the laser beam cannot be used to simultaneously irradiate 8 or 24 capillaries.

[0037] In the 3730 series gene analyzer based on patent document 2, if n3 = 1.33, then according to equation (1), Δθ = +2.9°, it can be seen that each capillary acts as a concave lens. Therefore, the multifocal function is not effective, and the laser beam cannot be used to simultaneously irradiate 48 or 96 capillaries.

[0038] In the configuration 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 single lens capillary is Δθ. B = -3.0°. At this time, due to Δθ A +Δθ B= +3.6°, therefore, this group of one analytical capillary and one lens capillary acts as a concave lens, and the multifocal lens does not function. It should be noted that in the configuration of Non-Patent Document 1, when n3 = 1.41, according to Equation (1), the refraction angle of one analytical capillary is Δθ. A = +2.4°, on the other hand, the refraction angle of a single lens capillary is Δθ. B = -3.0°. At this time, Δθ A +Δθ B = -0.6°, therefore, this combination of one analytical capillary and one lens capillary acts as a convex lens, enabling multifocal operation. On page 2875 of Non-Patent Document 1, it is described that the configuration of Non-Patent Document 1 also functions advantageously when n3 = 1.33. However, according to the definition of the maximum number of capillaries in Non-Patent Document 1, based on Non-Patent Document 1... Figure 11 When n3 = 1.33, the maximum number of capillaries is only about 8. Therefore, when n3 = 1.33, the configuration in Non-Patent Document 1 does not function.

[0039] In the configuration based on Patent Document 3, if n3 = 1.33, then according to Equation (1), the refraction angle of one analytical capillary is Δθ. A = +3.7°, on the other hand, the refraction angle of a single lens capillary is Δθ. B = -2.1°. At this time, Δθ A +Δθ B = +1.6°, therefore, this group of one analytical capillary and one lens capillary acts as a concave lens, and the multifocal method does not function. Therefore, it is not possible to use a laser beam to simultaneously irradiate multiple capillaries.

[0040] In view of this situation, this disclosure proposes a technique that enables electrophoretic analysis even when using various separation media with arbitrary refractive indices in the range of 1.33≤n3≤1.41 in a capillary array electrophoresis apparatus (and of course, separation media with refractive indices outside the range of 1.33≤n3≤1.41).

[0041] Methods for solving problems

[0042] To address the aforementioned issues, this disclosure proposes a capillary array, for example, where N is set to an integer greater than or equal to 2. The array is configured such that the laser irradiation sections of N analytical capillaries used for electrophoretic analysis, which are irradiated by the same laser beam, and the laser irradiation sections of N±1 lens capillaries not used for electrophoretic analysis, which are also irradiated by the same laser beam, are alternately arranged approximately on the same plane. Specifically, when the outer radius of each of the N analytical capillaries in the laser irradiation section and the N±1 lens capillaries in the laser irradiation section is set to R, the inner radius to r, the refractive index of the external medium to n1, and the refractive index of the raw material to n2, and the refractive index of the medium inside the N analytical capillaries in the laser irradiation section to n3, and the refractive index of the medium inside the N±1 lens capillaries in the laser irradiation section to n4, assuming n3 < 1.36, R, r, n1, n2, and n4 satisfy a predetermined relationship.

[0043] Further features relating to this disclosure will become apparent from the description and accompanying drawings. Furthermore, this disclosure is achieved and implemented through elements and combinations of elements, as well as through the detailed description below and the appended claims.

[0044] It should be understood that the description in this specification is merely a typical example and is not, in any sense, a limitation on the claims or applications of this disclosure.

[0045] Invention Effects

[0046] According to the technology disclosed herein, capillary array electrophoresis apparatuses can perform electrophoretic analyses using various separation media with arbitrary refractive indices in the range of 1.33 ≤ n3 ≤ 1.41. In particular, capillary electrophoretic analyses using separation media with a refractive index similar to or close to that of water (1.33) can be performed. This significantly expands the application range of capillary array electrophoresis apparatuses, which can increase analytical throughput and reduce the analytical cost per sample.

[0047] Other issues, structures, and effects not mentioned above can be more clearly understood through the following description of the implementation methods. Attached Figure Description

[0048] Figure 1 This is a diagram illustrating an example of the configuration of a capillary array electrophoresis apparatus.

[0049] Figure 2 This is a diagram illustrating an example of the optical system configuration of a capillary array electrophoresis apparatus.

[0050] Figure 3 This is a diagram illustrating an example of the collaboration between a display sensor and a computer.

[0051] Figure 4 This is a diagram showing the structure of the capillary array based on Patent Document 1 and the results of laser beam ray tracing.

[0052] Figure 5 This is a graph showing the relative fluorescence intensity distribution of the capillary array based on Patent Document 1.

[0053] Figure 6 This is a diagram illustrating the structure of the capillary array and the results of laser beam ray tracing according to the present disclosure.

[0054] Figure 7 This is a graph showing the relative fluorescence intensity distribution of the capillary array of the present disclosure.

[0055] Figure 8 This is a diagram showing the structure of the capillary array based on Patent Document 2 and the results of laser beam ray tracing.

[0056] Figure 9 This is a graph showing the relative fluorescence intensity distribution of the capillary array based on Patent Document 2.

[0057] Figure 10 This is a graph showing the relative fluorescence intensity distribution of the capillary array based on Non-Patent Document 1.

[0058] Figure 11 This is a graph showing the relative fluorescence intensity distribution of the capillary array based on Non-Patent Document 1.

[0059] Figure 12 This is a diagram showing the structure of the capillary array based on Patent Document 3 and the results of laser beam ray tracing.

[0060] Figure 13 This is a graph showing the relative fluorescence intensity distribution of the capillary array based on Patent Document 3.

[0061] Figure 14 This is a diagram illustrating the structure of the capillary array and the results of laser beam ray tracing according to the present disclosure.

[0062] Figure 15 This is a graph showing the relative fluorescence intensity distribution of the capillary array of the present disclosure.

[0063] Figure 16 This is a diagram illustrating the structure of the capillary array and the results of laser beam ray tracing according to the present disclosure.

[0064] Figure 17 This is a graph showing the relative fluorescence intensity distribution of the capillary array of the present disclosure.

[0065] Figure 18 This is a diagram illustrating the structure of the capillary array and the results of laser beam ray tracing according to the present disclosure.

[0066] Figure 19 This is a graph showing the relative fluorescence intensity distribution of the capillary array of the present disclosure.

[0067] Figure 20 This is a graph showing the relative fluorescence intensity distribution of the capillary array of the present disclosure.

[0068] Figure 21 This is a graph used to illustrate the effect of the external refractive index of the capillary on the relative fluorescence intensity and its coefficient of variation.

[0069] Figure 22 This is a graph used to illustrate the effect of capillary outer diameter on relative fluorescence intensity and its coefficient of variation.

[0070] Figure 23 This diagram illustrates an example of a capillary array consisting of alternating analytical capillary and lens capillary structures. Detailed Implementation

[0071] The technology disclosed herein relates to a capillary array electrophoresis apparatus, which simultaneously irradiates multiple capillaries with laser beams during electrophoresis and detects the fluorescence emitted from each capillary, thereby simultaneously analyzing multiple samples.

[0072] (A) Technical Summary of this Disclosure

[0073] This disclosure primarily proposes a technique for using a separation medium with a refractive index equivalent to water (1.33) or with a low refractive index less than 1.36. When using such a low refractive index separation medium, regardless of which of the known examples (Patent Documents 1 to 3 and Non-Patent Document 1) is used, the multifocal method does not function, making it difficult to simultaneously irradiate multiple capillaries with a laser beam.

[0074] Furthermore, this disclosure also proposes the following technique: capillary electrophoresis analysis can be performed not only with low-refractive-index separation media as described above, but also with high-refractive-index separation media, typically those with a refractive index of 1.36 or higher and 1.42 or lower. The maximum number of capillaries that can be simultaneously irradiated is better, and can be set to 24 or more, or even 48 or more depending on the situation. As mentioned above, among the irradiation intensities and fluorescence intensities of multiple capillaries within the same capillary array, the lower the irradiation intensity and fluorescence intensity, the better. Empirically, when the desired fluorescence intensity is set to 1 when the full intensity of the laser beam oscillating from the laser source is irradiated into the interior of a single capillary, a practical sensitivity can be 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. Experience shows that if the coefficient of variation (CV) of irradiation intensity and fluorescence intensity is ≤20% and, depending on the condition, ≤15%, different samples can be analyzed under the same conditions. This disclosure aims to achieve the practical performance of such a capillary array electrophoresis device.

[0075] In-depth research was conducted on the above-mentioned topic, and the results showed that for each capillary in the capillary array, when the capillary outer diameter 2R = 126 μm, the capillary inner diameter 2r = 50 μm, the outside of the capillary is air and n1 = 1.00, the capillary material 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), Δθ = -3.2°. Therefore, each capillary exhibits a convex lens effect, and the multifocal function is realized. In the 3500 series gene analyzer based on the above-mentioned patent document 1, the difference from n3 = 1.33 is that the outer diameter 2R of the capillary is reduced from 323 μm to 126 μm. As a result, the concave lens effect of each capillary is converted into a convex lens effect.

[0076] If the number of capillaries is set to N=24, and this condition is substituted into equations (2) and (4), the laser irradiation intensity of the capillaries (n=1 and n=24) at both ends of the capillary array is 0.59, and the laser irradiation intensity of the capillaries (n=12 and n=13) in the center of the capillary array is 0.42, satisfying the practical performance requirement of MIN≥0.2. In addition, the coefficient of variation of the laser irradiation intensity of the 24 capillaries is 12%, satisfying the practical performance requirements of CV≤20% and CV≤15%. Further research shows that if the outer diameter 2R of the capillary is less than 220μm, then Δθ<0, and the convex lens plays a role. If the case of the inner diameter 2r=50μm of the capillary is not limited to and is generalized, then when R / r≤4.4, Δθ<0, and it can be seen that the convex lens plays a role. Patent document 1 does not study the separation medium with a low refractive index of n3=1.33. That is, the above-mentioned conditions are first discovered in the technology disclosed herein.

[0077] Furthermore, when the capillary outer diameter 2R = 126 μm and the capillary inner diameter 2r = 50 μm, and the refractive index of the separation medium inside the capillary is n3 = 1.34, 1.35, and 1.36, according to equation (1), Δθ = -3.5°, -3.8°, and -4.2°, indicating that each capillary still exhibits the function of a convex lens, and the multifocal structure functions. Further research shows that if the capillary outer diameter 2R is below 240 μm, below 264 μm, and below 293 μm, respectively, then Δθ < 0, and the convex lens functions. In a conventional case, when R / r ≤ 4.8, R / r ≤ 5.3, and R / r ≤ 5.9, respectively, Δθ < 0, indicating that the convex lens functions. That is, it can be seen that when using a separation medium with a low refractive index of less than 1.36, it is sufficient to set R / r < 5.9. Such a low refractive index separation medium was not studied in Patent Document 1. That is, the above conditions were first discovered in the technology disclosed herein.

[0078] On the other hand, under the conditions of capillary outer diameter 2R = 126 μm, capillary inner diameter 2r = 50 μm, air outside the capillary and n1 = 1.00, quartz glass as the capillary material and n2 = 1.46, and separation medium inside the capillary and n3 = 1.33, if the number of capillaries is set to N = 48, then according to equations (2) and (4), the laser irradiation intensity of the capillaries (n = 1 and n = 48) at both ends of the capillary array is 0.51, and the laser irradiation intensity of the capillaries (n = 24 and n = 25) in the center of the capillary array is 0.17, which does not meet the practical performance requirement of MIN ≥ 0.2. In addition, the coefficient of variation of the laser irradiation intensity of 48 capillaries is 38%, and neither the practical performance requirements of CV ≤ 20% nor CV ≤ 15% are met. Therefore, this condition is not suitable for the simultaneous irradiation of more than 48 capillaries.

[0079] Therefore, a new configuration was designed that allows simultaneous irradiation of 48 or more capillaries using a laser beam while employing a low-refractive-index separation medium. Similar to the configurations shown in Non-Patent Document 1 and Patent Document 3, an array of analytical capillaries and lens capillaries is formed by alternating arrangements. Here, the number of analytical capillaries and lens capillaries does not necessarily need to be the same. When the two ends of the capillary array are set as analytical capillaries, N analytical capillaries and N-1 lens capillaries can be alternated, as long as N is an integer greater than or equal to 2. When the two ends of the capillary array are set as lens capillaries, N analytical capillaries and N+1 lens capillaries can be alternated. The capillary numbers n = 1, 2, ..., N are used only to indicate analytical capillaries. The laser irradiation section of the multiple capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm is placed in a fluorine solution with a refractive index n1 = 1.25. The capillary material is quartz glass, n2 = 1.46; the analytical capillary contains a separation medium, n3 = 1.33; and the lens capillary contains a matching solution, n4 = 1.46. Therefore, according to equation (1), the refraction angle of one analytical capillary is Δθ. A = +2.0°, on the other hand, the refraction angle of a single lens capillary is Δθ. B = -3.3°. At this time, Δθ A +Δθ B = -1.3°, therefore, this group of one analytical capillary and one lens capillary acts as a convex lens, and the multifocal function is utilized. Furthermore, under these conditions, according to equation (2), the transmittance of one analytical capillary is T. A =98.4%, the transmittance of one lens capillary is T B =98.8%. Therefore, the transmittance of this group of one analytical capillary and one lens capillary is T = T A ×T B =97.2%. At this time, if the number of capillaries analyzed is set to N=48 in equation (4), then according to equations (2) and (4), the laser irradiation intensity of the capillaries (n=1 and n=48) at both ends of the capillary array is 0.63, and the laser irradiation intensity of the capillaries (n=24 and n=25) in the center of the capillary array is 0.51, which satisfies the practical performance requirement of MIN≥0.2. In addition, the coefficient of variation of the laser irradiation intensity of the 48 capillaries is 7%, which simultaneously satisfies the practical performance requirements of CV≤20% and CV≤15%.

[0080] Next, if the number of capillaries is set to N = 72, then according to equations (2) and (4), the laser irradiation intensity of the capillaries (n = 1 and n = 72) at both ends of the capillary array is 0.57, and the laser irradiation intensity of the capillaries (n = 36 and n = 37) at the center of the capillary array is 0.36, satisfying the practical performance requirement of MIN ≥ 0.2. In addition, the coefficient of variation of the laser irradiation intensity of the 72 capillaries is 14%, simultaneously satisfying the practical performance requirements of CV ≤ 20% and CV ≤ 15%. Further, if the number of capillaries is set to N = 96, then according to equations (2) and (4), the laser irradiation intensity of the capillaries (n = 1 and n = 96) at both ends of the capillary array is 0.53, and the laser irradiation intensity of the capillaries (n = 48 and n = 49) at the center of the capillary array is 0.26, satisfying the practical performance requirement of MIN ≥ 0.2. Furthermore, the coefficient of variation of the laser irradiation intensity of 96 capillaries is 24%, which does not meet the practical performance requirements of CV ≤ 20% and CV ≤ 15%. Under the above conditions, if n3 = 1.33 is changed back to n3 = 1.41, the performance of all the above indicators will naturally improve. This configuration is significantly different from the configurations of Patent Document 2, Non-Patent Document 1, and Patent Document 3, except for changing n3 = 1.41 to n3 = 1.33. In Patent Document 2, a lens capillary is not used, and the refractive index of the medium outside the capillary is n1 = 1.29, both of which differ from the configuration of this disclosure. Furthermore, in Non-Patent Document 1, the refractive index of the medium outside the capillary is n1 = 1.46, and the interior of the lens capillary is a high-refractive-index solution, n4 = 1.53, both of which differ from the configuration of this disclosure. Moreover, in Patent Document 3, the refractive index of the medium outside the capillary is n1 = 1.33, which differs from the configuration of this disclosure. These differences, as described above, represent different functions resulting from fundamentally different constructions, and are not simply design changes.

[0081] The embodiments of this disclosure will now be described in detail. Furthermore, while each embodiment will be described separately, the techniques shown in these embodiments are not exclusive and can be appropriately combined with each other.

[0082] (B) First Embodiment

[0083] <Example of the configuration of a capillary array electrophoresis apparatus>

[0084] Figure 1 This diagram illustrates an example of the configuration of a capillary array electrophoresis apparatus. In this apparatus, in addition to DNA sequence and single-stranded DNA fragment analysis performed in conventional capillary array electrophoresis apparatuses, double-stranded DNA fragment analysis is also performed. In this embodiment, 24 capillaries are used (wherein, Figure 1(Only four capillaries are shown in the diagram). First, DNA sequencing of different samples is performed in each capillary. Then, double-stranded DNA fragment analysis of different samples is performed in each capillary. The DNA sequencing samples contain single-stranded DNA fragments of various lengths labeled with four fluorescent agents corresponding to four bases. The electrophoresis separation medium filling 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 samples for double-stranded DNA fragment analysis contain double-stranded DNA fragments of various lengths labeled with two fluorescent agents. One fluorescently labeled double-stranded DNA fragment is a PCR product, and the other fluorescently labeled double-stranded DNA fragment is a molecular weight marker. The electrophoresis separation medium filling 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 using the following steps (i) to (vi).

[0085] (i) First, the sample injection end 2 of the 24 capillaries 1 is immersed in the cathode-side buffer 6, and the sample elution end 3 is connected to the anode-side buffer 7 via the polymer block 9.

[0086] (ii) Next, close the valve 10 of the polymer block 9 and press the piston of the syringe 11 connected to the polymer block 9 to pressurize the internal polymer solution and fill the polymer solution from the sample elution end 3 toward the sample injection end 2 into the interior of each capillary 1.

[0087] (iii) Next, valve 10 is opened, and different samples are injected into the electric field of each capillary 1 from the sample injection end 2. Then, a high voltage is applied between the cathode 4 and the anode 5 using power supply 8, thereby starting capillary electrophoresis. DNA fragments labeled with various fluorescent agents are electrophoresed from the sample injection end 2 toward the sample elution end 3.

[0088] (iv) In parallel, the position of each capillary 1 at which it has been electrophoretically extended a certain distance from the sample injection end 2 is designated as a laser irradiation section 14. A laser beam 13 oscillating from the laser source 12 is simultaneously irradiated onto the laser irradiation section 14 using a multifocal method. Here, the coating on each capillary 1 near the laser irradiation section 14 is removed beforehand, and the capillary 1s near the laser irradiation section 14 are arranged on an arrangement plane. The laser beam 13 is focused and then incident from the side of the arrangement plane along the arrangement plane. Figure 1 For simplicity, it is described as irradiating one side of the laser beam 13, but in reality, the laser beam 13 is divided into two parts and irradiated from both sides.

[0089] (v) Then, DNA fragments labeled with various fluorophores are electrophoresed 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. That is, various fluorophores emit fluorescence from 24 luminescent points (laser irradiation sections), and the fluorescence intensity of each fluorophore changes constantly during electrophoresis.

[0090] (vi) Finally, the fluorescence emitted from each luminescent point is detected by multicolor detection, and the obtained time series data is analyzed to analyze the samples injected into each capillary.

[0091] The steps (i) to (vi) above are common to both DNA sequencing and double-stranded DNA fragment analysis, but the polymer solution and buffer solution can be appropriately changed. That is, the capillary array electrophoresis apparatus of this embodiment can, for example, switch between multiple analysis modes with different conditions, including a first analysis mode for double-stranded DNA fragment analysis and a second analysis mode for DNA sequencing. In each analysis mode, it is effective to appropriately change the electrophoresis analysis conditions according to their respective purposes. The electrophoresis analysis conditions that can be changed 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, it is effective to change the capillary control temperature in each analysis mode, such as adjusting the capillary temperature to 30°C in the first analysis mode and adjusting it to 60°C in the second analysis mode. Furthermore, the designations "first" and "second" are merely for convenience in distinguishing the analysis modes and do not indicate the order in which the analysis modes are performed. In the above example, the refractive index of the electrophoretic separation medium in DNA fragment analysis is n3 = 1.33, and the refractive index of the electrophoretic separation medium in DNA sequencing is n3 = 1.41. Therefore, the refractive index of the electrophoretic separation medium in the first analysis mode is n3 < 1.36, and the refractive index of the electrophoretic separation medium in the second analysis mode is n3 ≥ 1.36. Depending on the situation, the refractive index of the electrophoretic separation medium in the first analysis mode can be set to 1.33 ≤ n3 < 1.36, and the refractive index of the electrophoretic separation medium in the second analysis mode can be set to 1.36 ≤ n3 ≤ 1.42. Furthermore, the analysis session consisting of steps (i) to (vi) can be repeated multiple times. For example, by analyzing samples 1 to 24 in the first analysis session, and samples 25 to 48, ... in the second analysis session, multiple different samples can be analyzed. At this time, DNA sequencing can be repeatedly performed using the same polymer solution and buffer solution, or the process can be switched to double-stranded DNA fragment analysis midway. Any application can be selected in any analysis session.

[0092] <Example of the configuration of an optical system for fluorescence detection>

[0093] Figure 2 This is a cross-sectional view illustrating 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 Similarly, in Figure 2 The image depicts single-sided illumination of four capillary arrays, but in reality, it's irradiation from both sides of a 24-capillary array. The capillary 1, arranged in the array plane, is simultaneously irradiated by a multi-focal laser beam 13. 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, and the laser is blocked by a laser cutoff filter 16, allowing it to pass through a transmission-type diffraction grating 17. This disperses the wavelength along the central axis of each capillary, and the imaging lens 18 images the imaging points 22 onto the sensor 19. The sensor 19 can be any sensor capable of simultaneously measuring multiple imaging points 22, such as a CCD, CMOS, or photodiode array. Each imaging point 22 is actually located in... Figure 2 Wavelength dispersion was achieved along the depth direction, but... Figure 2 The single wavelength portion of each imaging point 22 is schematically depicted in the figure.

[0094] In such an optical system, the focusing efficiency decreases as the light moves away from the optical axis 23 of the optical system. This is because, as... Figure 2 As shown, the focusing angle of the emitted fluorescence 21 from the emission point 20, which is far 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 extent of the vignetting effect, i.e., the optical system correction coefficient based on the vignetting effect, is determined by the optical system and can be investigated through calculation or experiment. Using the optical system correction coefficient based on the vignetting effect, the fluorescence intensity at each imaging point 22 can be calculated based on the fluorescence intensity at each emission point 20.

[0095] <Example of system configuration for data analysis and device control>

[0096] Figure 3This diagram 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 units. The raw time-series data of the signals output from the sensor is sequentially stored in memory. Additionally, analysis parameter information stored in a database located inside the HDD is stored in memory. The CPU uses the analysis parameter information stored in memory to analyze the raw time-series data stored in memory and derives time-series analysis data, which is sequentially stored in memory and simultaneously displayed 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).

[0097] <Example of the configuration of a capillary array in the past (Patent Document 1)>

[0098] Figure 4 (a) is a cross-sectional view showing the configuration of the capillary array in 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 370 μm intervals. The outside of the capillaries is air, n1 = 1.00, and the capillary material is quartz glass, n2 = 1.46.

[0099] Figure 4 (b) represents the situation where, under the above conditions, the inside of the capillary is a separation medium with a high refractive index and n3 = 1.41, irradiation is performed from the left side only. The image shows the laser beam ray tracing results. The multifocal design clearly demonstrates its functionality, efficiently illuminating the interior of all 24 capillaries. According to equation (1), Δθ = -1.3°, which corresponds to the convex lens effect of each capillary.

[0100] In contrast, Figure 4 (c) is a graph showing the same laser beam tracing results under the above conditions, where the capillary interior is a low-refractive-index separation medium and n3 = 1.33. Clearly, the multifocal configuration fails to function, and the laser beam diverges from the capillary array, failing to efficiently illuminate the entire array. According to equation (1), Δθ = +1.3°, which corresponds to the concave lens effect of each capillary.

[0101] <Relative fluorescence intensity distribution based on capillary arrays as described in previous patent document 1>

[0102] Figure 5 (a) indicates that... Figure 4The results of unilateral irradiation shown in (b) and (c) are plots of the relative fluorescence intensities of each capillary under bilateral irradiation. The capillary numbering is based on... Figure 4 The leftmost capillary is designated as 1, and the numbers are sequentially labeled towards the right. The relative fluorescence intensity is calculated based on the irradiation intensity of each capillary, assuming a certain concentration of phosphor in the laser-irradiated portion of each capillary, taking into account the laser beam reflection loss. The expected fluorescence intensity is set to 1 when the full intensity of the laser beam oscillating from the laser source is irradiated into the interior of one capillary. In the calculation of irradiation from both sides, half of the full intensity of the laser beam is irradiated from both sides of the capillary array. This graph corresponds to the laser irradiation intensity of each capillary as expressed in equations (2) and (4). Equations (2) and (4) are approximate formulas assuming the incident angle of the laser beam at the interface between different media is 0°. In contrast, this graph is calculated based on the actual incident angle obtained through laser beam tracing, and is therefore more accurate. The following... Figure 7 (a) Figure 9 , Figure 11 , Figure 13 , Figure 15 , Figure 17 , Figure 19 as well as Figure 20 The same chart shown is also a more accurate result calculated based on the actual angle of incidence. Figure 5 (a) With n3 = 1.41, for 24 capillaries, the minimum relative fluorescence intensity is MIN = 0.42, and the coefficient of variation (CV = standard deviation of relative fluorescence intensity / average of relative fluorescence intensity) is CV = 11%. This indicates that the practical performance requirements of MIN ≥ 0.2, CV ≤ 20%, and CV ≤ 15% are met. The downward convex distribution of relative fluorescence intensity relative to capillary number is because, although the multifocal system functions, the intensity of the laser beam attenuates due to reflection losses as it travels within the capillary array. In contrast, with n3 = 1.33, MIN = 0.068 and CV = 74% are obtained, indicating that neither of these practical performance requirements is met.

[0103] Figure 5 (b) indicates that through the Figure 5(a) shows the results plus the vignetting effect of the 3500 series gene analyzer's optical system, i.e., multiplied by the vignetting effect-based optical system correction coefficient, resulting in a graph of the optical system-corrected relative fluorescence intensity for each capillary. With n3 = 1.41, the downward convex distribution of relative fluorescence intensity relative to capillary number cancels out the distribution of the optical system correction, resulting in a flat distribution of the optical system-corrected relative fluorescence intensity. As a result, the minimum fluorescence intensity MIN = 0.42 remains unchanged, but the coefficient of variation is significantly reduced to CV = 0.76%. Of course, all practical performance requirements are met. In contrast, with n3 = 1.33, MIN = 0.066 and CV = 61% remain largely unchanged, and the practical performance requirements remain unmet.

[0104] <Example of the configuration of the capillary array in the first embodiment>

[0105] Figure 6 (a) is a cross-sectional view showing a configuration example of the capillary array based on the first 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 outside of the capillaries is air, n1 = 1.00, and the capillary material is quartz glass, n2 = 1.46.

[0106] Figure 6 (b) represents the situation where, under the above conditions, the inside of the capillary is a separation medium with a high refractive index and n3 = 1.41, irradiation is performed from the left side only. The image shows the laser beam ray tracing results. The multifocal design clearly demonstrates its functionality, efficiently illuminating the interior of all 24 capillaries. According to equation (1), Δθ = -5.8°, which corresponds to the convex lens effect of each capillary.

[0107] In contrast, Figure 6 (c) is a graph showing the same laser beam tracing results under the above conditions, where the separation medium inside the capillary is of low refractive index and n3 = 1.33. In this case, the multifocal focal spot also functions significantly, efficiently illuminating the interior of all 24 capillaries. According to equation (1), Δθ = -3.2°, which corresponds to the convex lens effect of each capillary. Thus, whether it is a separation medium of high refractive index (n3 ≥ 1.36) or a separation medium of low refractive index (n3 < 1.36), each capillary exhibits a convex lens effect, and the multifocal focal spot functions, a situation that cannot be achieved in any known example, but is achieved for the first time by the technology of this disclosure. That is, the multifocal focal spot of the capillary array electrophoresis apparatus of this embodiment functions in either the first analysis mode where n3 < 1.36 or the second analysis mode where n3 ≥ 1.36.

[0108] <Based on the first implementation method ( Figure 6 The relative fluorescence intensity distribution formed by the capillary array of ) >

[0109] Figure 7 (a) indicates that... Figure 6 The results of unilateral irradiation shown in (b) and (c) are plots of the relative fluorescence intensities of each capillary under bilateral irradiation. Figure 7 (a) With n3 = 1.41, for 24 capillaries, the minimum relative fluorescence intensity is MIN = 0.42 and the coefficient of variation is CV = 11%, which satisfies the practical performance requirements of MIN ≥ 0.2, CV ≤ 20%, and CV ≤ 15%. On the other hand, with n3 = 1.33, MIN = 0.40 and CV = 12%, which also satisfies the practical performance requirements.

[0110] Figure 7 (b) indicates that through the Figure 7 (a) is a graph showing the relative fluorescence intensity of each capillary after adding the vignetting effect of the optical system of the 3500 series gene analyzer, i.e., multiplying by the optical system correction coefficient based on the vignetting effect. The results of the optical system correction are MIN=0.42 and CV=9.0% when n3=1.41, and MIN=0.40 and CV=10% when n3=1.33, which also meet the practical performance requirements.

[0111] according to Figure 7 ,and Figure 5 In different cases, the relative fluorescence intensity does not change much depending on whether or not an optical system is used for correction. This is because... Figure 4 The entire width of the capillary array is 370μm × (24 tubes - 1 tube) = 8.5mm. In contrast, Figure 6 The entire width of the capillary array is 155μm × (24 capillaries - 1 capillary) = 3.6mm, which is relatively narrow. That is, the distance between each capillary and the optical axis is relatively short, so the vignetting effect of the optical system is smaller.

[0112] As a result, in Figure 4 With n3 = 1.41 in the capillary array of the 3500 series gene analyzer shown, the CV = 11% was significantly reduced to CV = 0.76% through optical system correction based on the vignetting effect of the optical system. In contrast, in Figure 6 In the capillary array of this embodiment, when n3 = 1.41, the CV = 11% is reduced to CV = 9% only through optical system correction based on the vignetting effect of the optical system. Similarly, when n3 = 1.33 in the capillary array of this embodiment, the CV = 12% is also reduced to CV = 10% only through optical system correction based on the vignetting effect of the optical system.

[0113] <Summary of the First Implementation>

[0114] As can be seen from the above, in the configuration of the first embodiment, when using a separation medium with an arbitrary refractive index of n3 ≥ 1.33, including n3 = 1.41, each capillary acts as a convex lens, and the multifocal function is achieved. Furthermore, as a variation of this configuration, when using any capillary with R / r ≤ 4.4, for example, a capillary with an inner diameter of 2r = 50 μm and an outer diameter of 2R ≤ 220 μm, since each capillary acts as a convex lens under the condition that n3 ≥ 1.33, the multifocal function can also be achieved.

[0115] (C) Second Embodiment

[0116] In the first embodiment, the case where the outside of the capillary is air (refractive index n1 = 1.00) was described. In the second embodiment, the case where the outside of the capillary is not air (refractive index n1 ≠ 1.00) was described. In this case, according to the technology of this disclosure, when using a separation medium having any refractive index including n3 ≥ 1.33 (n3 = 1.41), each capillary also exhibits the function of a convex lens, and the multifocal function is realized.

[0117] <Example of the configuration of a capillary array in the past (Patent Document 2)>

[0118] Figure 8 (a) is a cross-sectional view showing the configuration of the capillary array in the 3730 series gene analyzer based on Patent Document 2. The laser irradiation section of 96 capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm is arranged on the same plane at intervals of 155 tubes. The outer surface of the capillaries is a fluorine solution, n1 = 1.29, and the raw material for the capillaries is quartz glass, n2 = 1.46.

[0119] Figure 8 (b) represents the situation where, under the above conditions, the inside of the capillary is a separation medium with a high refractive index and n3 = 1.41, irradiation is performed from the left side only. The image shows the laser beam ray tracing results. The multifocal system functions effectively, efficiently illuminating the interior of all 96 capillaries. According to equation (1), Δθ = -0.69°, which corresponds to the convex lens effect of each capillary.

[0120] In contrast, Figure 8(c) is a graph showing the same laser beam tracing results under the above conditions, where the capillary interior is a low-refractive-index separation medium and n3 = 1.33. The multifocal path does not function properly; the laser beam diverges from the capillary array and cannot efficiently illuminate the entire array. According to equation (1), Δθ = +2.9°, which corresponds to the concave lens effect of each capillary.

[0121] <Relative fluorescence intensity distribution based on capillary arrays as described in previous patent document 2>

[0122] Figure 9 It means to Figure 8 The results of unilateral irradiation shown in (b) and (c) are rewritten as graphs of the relative fluorescence intensity of each capillary under bilateral irradiation. With n3 = 1.41, for 96 capillaries, the minimum relative fluorescence intensity is MIN = 0.63, and the coefficient of variation is CV = 3.2%, which satisfies the practical performance requirements of MIN ≥ 0.2, CV ≤ 20%, and CV ≤ 15%. In contrast, with n3 = 1.33, MIN = 0.00067 and CV = 192%, indicating that the practical performance requirements are not met.

[0123] <Examples of the configuration of capillary arrays in the past (non-patent document 1)>

[0124] Figure 10 (a) is a cross-sectional view showing the configuration of the capillary array based on Non-Patent Document 1. The laser irradiation sections of 96 capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm are arranged on the same plane at 155 μm intervals. In the capillary array, 48 analytical capillaries and 48 lens capillaries are arranged alternately. The outer layer of the capillaries is a matching solution, n1 = 1.46, and the capillary material is quartz glass, n2 = 1.46. The refractive index of the medium inside the lens capillaries is set to n4 = 1.53.

[0125] Figure 10 (b) represents the analysis under the above conditions, where the separation medium inside the capillary is of high refractive index and n3 = 1.41, with irradiation from the left side only. The image shows the laser beam ray tracing results. The multifocal setup effectively illuminates the interior of all 96 capillaries. This is because, according to equation (1), the refraction angle of one analyzed capillary is Δθ. A = +2.4°, on the other hand, the refraction angle of a single lens capillary is Δθ. B = -3.0°, Δθ A +Δθ B = -0.6°, therefore, this group of one analytical capillary and one lens capillary acts as a convex lens, and the multifocal function is realized.

[0126] In contrast, Figure 10 (c) is a graph showing the laser beam tracing results under the above conditions, where the separation medium inside the capillary is of low refractive index and n3 = 1.33. The multifocal path does not function properly; the laser beam diverges from the capillary array and cannot efficiently illuminate the entire array. This is because, 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 single lens capillary is Δθ. B = -3.0°. At this time, because Δθ A +Δθ B = +3.6°, so this group of one analytical capillary and one lens capillary shows the function of a concave lens, and the multifocal lens does not function.

[0127] <Relative fluorescence intensity distribution based on capillary arrays (non-patent document 1)>

[0128] Figure 11 It means to Figure 10 The results of unilateral irradiation shown in (b) and (c) are rewritten as graphs of the relative fluorescence intensity of each capillary under bilateral irradiation. With n3 = 1.41, for 48 analytical capillaries, the minimum relative fluorescence intensity was MIN = 0.58, and the coefficient of variation was CV = 7%, which satisfies the practical performance requirements of MIN ≥ 0.2, CV ≤ 20%, and CV ≤ 15%. In contrast, with n3 = 1.33, MIN = 0.00247 and CV = 193%, indicating that the practical performance requirements are not met.

[0129] <Example of the configuration of a capillary array in the past (Patent Document 3)>

[0130] Figure 12 (a) is a cross-sectional view showing a configuration example of the capillary array based on Patent Document 3. The laser irradiation section of 96 capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm are arranged on the same plane at 155 μm intervals. In the capillary array, 48 analytical capillaries and 48 lens capillaries are arranged alternately. The outer surface of the capillaries is water, n1 = 1.33, and the capillary material is quartz glass, n2 = 1.46. The refractive index of the medium inside the lens capillaries is set to n4 = 1.53.

[0131] Figure 12 (b) represents the analysis under the above conditions, where the separation medium inside the capillary is of high refractive index and n3 = 1.41, with irradiation from the left side only. The image shows the laser beam ray tracing results. The multifocal setup effectively illuminates the interior of all 96 capillaries. This is because, according to equation (1), the refraction angle of one analyzed capillary is Δθ. A = +0.03°, on the other hand, the refraction angle of a single lens capillary is Δθ. B = -2.1°, Δθ A +Δθ B = -2.07°, therefore, this group of one analytical capillary and one lens capillary acts as a convex lens, and the multifocal function is realized.

[0132] In contrast, Figure 12 (c) is a graph showing the laser beam tracing results under the above conditions, where the separation medium inside the capillary is of low refractive index and n3 = 1.33. The multifocal path does not function properly; the laser beam diverges from the capillary array and cannot efficiently illuminate the entire array. This is because, according to equation (1), the refraction angle of one analytical capillary is Δθ. A = +3.7°, on the other hand, the refraction angle of a single lens capillary is Δθ. B = -2.1°, Δθ A +Δθ B = +1.6°, therefore, this group of one analytical capillary and one lens capillary acts as a concave lens, and the multifocal system does not function. Therefore, when using a separation medium with a low refractive index, it is not possible to simultaneously irradiate multiple capillaries with a laser beam.

[0133] Based on past ( Figure 12 The relative fluorescence intensity distribution formed by the capillary array of ) >

[0134] Figure 13 It means to Figure 12 The results of unilateral irradiation shown in (b) and (c) are rewritten as graphs of the relative fluorescence intensity of each capillary under bilateral irradiation. With n3 = 1.41, for 48 analytical capillaries, the minimum relative fluorescence intensity was MIN = 0.79, and the coefficient of variation was CV = 1%, which satisfies the practical performance requirements of MIN ≥ 0.2, CV ≤ 20%, and CV ≤ 15%. In contrast, with n3 = 1.33, MIN = 0.00975 and CV = 119%, indicating that the practical performance requirements are not met.

[0135] <Example of the configuration of the capillary array in the second embodiment>

[0136] Figure 14(a) is a cross-sectional view showing a configuration example of the capillary array according to the second embodiment. The laser irradiation sections of 96 capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm are arranged on the same plane at 155 μm intervals. In the capillary array, 48 analytical capillaries and 48 lens capillaries are arranged alternately. The outer surface of the capillaries is a fluorine solution, n1 = 1.29, and the capillary material is quartz glass, n2 = 1.46. The refractive index of the medium inside the lens capillaries is set to n4 = 1.46. Since n2 = n4 = 1.46, a rod-shaped lens with an outer diameter 2R = 126 μm and a refractive index of 1.46 can be used instead of the lens capillaries. However, it is generally difficult to make the outer diameter of the capillaries match the outer diameter of the rod-shaped lens. Therefore, it is easier to make the outer diameters of both the analytical capillaries and the lens capillaries match when using capillaries of the same specifications and from the same batch.

[0137] Figure 14 (b) represents the analysis under the above conditions, where the separation medium inside the capillary is of high refractive index and n3 = 1.41, with irradiation from the left side only. The image shows the laser beam ray tracing results. The multifocal setup effectively illuminates the interior of all 96 capillaries. This is because, according to equation (1), the refraction angle of one analyzed capillary is Δθ. A = -0.7°, on the other hand, the refraction angle of a single lens capillary is Δθ. B = -2.7°, Δθ A +Δθ B = -3.4°, therefore, this group of one analytical capillary and one lens capillary acts as a convex lens, and the multifocal function is realized.

[0138] In contrast, Figure 14 (c) is a graph showing the laser beam tracing results under the above conditions, where the separation medium inside the capillary is of low refractive index and n3 = 1.33. Although the multifocal method works, the laser beam diverges slightly from the capillary array, failing to efficiently illuminate the entire array. This is because, according to equation (1), the refraction angle of one analytical capillary is Δθ. A = +2.9°, on the other hand, the refraction angle of a single lens capillary is Δθ. B = -2.7°, Δθ A +Δθ B = +0.2°, therefore, this combination of one analytical capillary and one lens capillary slightly exhibits the effect of a concave lens, reducing the multifocal capability. However, compared with Figure 10 (c) and Figure 12 Compared to the results of the existing method shown in (c), it can efficiently irradiate each capillary simultaneously.

[0139] <Based on the second implementation method ( Figure 14 The relative fluorescence intensity distribution formed by the capillary array of ) >

[0140] Figure 15 It means to Figure 14 The results of unilateral irradiation shown in (b) and (c) are rewritten as graphs of the relative fluorescence intensity of each capillary under bilateral irradiation. With n3 = 1.41, for 48 analytical capillaries, the minimum relative fluorescence intensity is MIN = 0.68, and the coefficient of variation is CV = 2%, satisfying the practical performance requirements of MIN ≥ 0.2, CV ≤ 20%, and CV ≤ 15%. In contrast, with n3 = 1.33, MIN = 0.42 and CV = 11%, satisfying MIN ≥ 0.2 and CV ≤ 20%, but not CV ≤ 15%. Therefore, a method for more effectively irradiating more than 48 analytical capillaries simultaneously with a laser beam is proposed.

[0141] <Other configuration examples of the capillary array in the second embodiment>

[0142] Figure 16 (a) is a cross-sectional view showing a configuration example of the capillary array according to the second embodiment. The laser irradiation sections of 96 capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm are arranged on the same plane at 155 μm intervals. In the capillary array, 48 analytical capillaries and 48 lens capillaries are arranged alternately. The outer surface of the capillaries is a fluorine solution, n1 = 1.25, and the capillary material is quartz glass, n2 = 1.46. For the fluorine solution with n1 = 1.25, Figure 8 and Figure 14 The fluorine solution used, with n1 = 1.29, is of a different specification and has not been used within the scope of known technology. Reducing the refractive index outside the capillary enhances the convex lens effect of each capillary, but simultaneously increases reflection loss; therefore, it is unclear whether it is effective for simultaneous irradiation of more than 48 analytical capillaries. It should be noted that the fluorine solution with n1 = 1.25 could, for example, be Fluorinert (a registered trademark) sold by 3M. The refractive index of the medium inside the lens capillary is set to n4 = 1.46.

[0143] Figure 16 (b) represents the analysis under the above conditions, where the separation medium inside the capillary is of high refractive index and n3 = 1.41, with irradiation from the left side only. The image shows the laser beam ray tracing results. The multifocal setup effectively illuminates the interior of all 96 capillaries. This is because, according to equation (1), the refraction angle of one analyzed capillary is Δθ. A = -1.4°, on the other hand, the refraction angle of a single lens capillary is Δθ.B = -3.3°, Δθ A +Δθ B = -4.7°, therefore, this group of one analytical capillary and one lens capillary acts as a convex lens, and the multifocal function is realized.

[0144] In contrast, Figure 16 (c) is a graph showing the laser beam tracing results under the above conditions, where the interior of the capillary is a low-refractive-index separation medium and n3 = 1.33. The multifocal setup functions effectively, efficiently illuminating the interior of all 96 capillaries. This is because, according to equation (1), the refraction angle of one analytical capillary is Δθ. A = +2.0°, on the other hand, the refraction angle of a single lens capillary is Δθ. B = -3.3°, Δθ A +Δθ B = -1.3°, therefore, this group of one analytical capillary and one lens capillary acts as a convex lens, and the multifocal function is realized.

[0145] <Based on the second implementation method ( Figure 16 The relative fluorescence intensity distribution formed by the capillary array of ) >

[0146] Figure 17 It means to Figure 16 The results of unilateral irradiation shown in (b) and (c) are rewritten as graphs of the relative fluorescence intensity of each capillary under bilateral irradiation. With n3 = 1.41, for 48 analytical capillaries, the minimum relative fluorescence intensity is MIN = 0.54, and the coefficient of variation is CV = 6%, satisfying the practical performance requirements of MIN ≥ 0.2, CV ≤ 20%, and CV ≤ 15%. In contrast, with n3 = 1.33, MIN = 0.47 and CV = 8%, satisfying MIN ≥ 0.2, CV ≤ 20%, and CV ≤ 15%. Therefore, this configuration is suitable for simultaneously irradiating 48 or more analytical capillaries with a laser beam.

[0147] <Other configuration examples of the capillary array in the second embodiment>

[0148] Figure 18 (a) is a cross-sectional view of the capillary array configuration of this embodiment. 192 capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm, whose laser irradiation sections are arranged on the same plane at 155 μm intervals. In the capillary array, 96 analytical capillaries and 96 lens capillaries are arranged alternately. The outer layer of the capillaries is a fluorine solution, n1 = 1.25, and the capillary material is quartz glass, n2 = 1.46. The refractive index of the medium inside the lens capillaries is set to n4 = 1.46.

[0149] Figure 18 (b) represents the analysis under the above conditions, where the separation medium inside the capillary is of high refractive index and n3 = 1.41, with irradiation from the left side only. The image shows the laser beam tracing results. The multifocal setup effectively illuminates the interior of all 192 capillaries. The refraction angle of each capillary is shown in the figure. Figure 16 The situation is the same as in (b).

[0150] In contrast, Figure 18 (c) is a graph showing the laser beam tracing results under the above conditions, where the interior of the capillary is a low-refractive-index separating medium and n3 = 1.33. The multifocal setup functioned effectively, efficiently illuminating the interior of all 192 capillaries. The refraction angle of each capillary and... Figure 16

[0151] The situation is the same as in (c).

[0152] <Based on the second implementation method ( Figure 18 The relative fluorescence intensity distribution formed by the capillary array of ) >

[0153] Figure 19 It means to Figure 18 The results of unilateral irradiation shown in (b) and (c) are rewritten as graphs of the relative fluorescence intensity of each capillary under bilateral irradiation. With n3 = 1.41, for 96 analytical capillaries, the minimum relative fluorescence intensity was MIN = 0.30, and the coefficient of variation was CV = 20%. This indicates that while MIN ≥ 0.2 and CV ≤ 20% are met as practical performance criteria, CV ≤ 15% is not satisfied. Conversely, with n3 = 1.33, MIN = 0.23 and CV = 27%, indicating that MIN ≥ 0.2 is met, but CV ≤ 20% and CV ≤ 15% are not satisfied. However, this configuration and... Figure 9 Compared to the case of n3=1.33 in (Patent Document 2), this method is clearly more suitable for simultaneously irradiating more than 96 analytical capillaries with a laser beam.

[0154] <Changes in the number of capillaries>

[0155] Figure 20 and Figures 16-19Similarly, this graph shows the relative fluorescence intensity of each analytical capillary when irradiated from both sides by a laser beam, with 24, 48, 72, and 96 analytical capillaries (outer diameter 2R = 126 μm, inner diameter 2r = 50 μm) arranged at 155 μm intervals on the same plane. The external capillary is a fluorine solution (n1 = 1.25), the capillary material is quartz glass (n2 = 1.46), the internal analytical capillary is a low-refractive-index separation medium (n3 = 1.33), and the refractive index of the medium inside the lens capillary is set to n4 = 1.46. That is, Figure 20 The chart showing the 48 analytical capillaries plotted with black diamonds and Figure 17 The chart for n3 = 1.33 is the same, and the chart for 96 analytical capillaries plotted with black squares is the same. Figure 19 The graph for n3 = 1.33 is the same. Thus, it can be seen that the configuration of this embodiment is suitable for simultaneous irradiation of analytical capillaries with various numbers of capillaries, from 24 to 96, using a laser beam. Of course, it is also effective for analytical capillaries with fewer than 24 capillaries or more than 96 capillaries.

[0156] (D) Third Implementation

[0157] In the third embodiment, Figure 16 In the capillary array configuration shown in the second embodiment, the influence of the refractive index of the medium outside the capillary is evaluated. The laser irradiation sections of 96 capillaries with an outer diameter 2R = 126 μm and an inner diameter 2r = 50 μm are arranged on the same plane at 155 μm intervals. In the capillary array, 48 analytical capillaries and 48 lens capillaries are arranged alternately. The capillary material is quartz glass, n2 = 1.46; the interior of the analytical capillary is a low-refractive-index separation medium, n3 = 1.33; and the refractive index of the medium inside the lens capillary is n4 = 1.46.

[0158] Figure 21 In (a), the maximum relative fluorescence intensity of 48 analyzed capillaries is plotted using black triangles when the refractive index n1 outside the capillary varies between 1.20 and 1.33 under the given conditions. The average value is plotted using black dots, and the minimum value is plotted using black squares. Based on this result, it can be seen that when n1 ≤ 1.31, the practical performance requirement MIN ≥ 0.2 is met. Furthermore, by making n1 ≤ 1.30 and 1.23 ≤ n1 ≤ 1.29, satisfying MIN ≥ 0.3 and MIN ≥ 0.4 respectively, even higher performance can be obtained.

[0159] Figure 21 (b) indicates that in relation to Figure 21(a) The coefficient of variation (CV) of the relative fluorescence intensity of 48 analytical capillaries when the refractive index n1 of the capillary exterior varies between 1.20 and 1.33 under the same conditions. Based on this result, it can be seen that when n1 ≤ 1.30, the CV for practical performance is ≤ 20%. Furthermore, it can be seen that when 1.21 ≤ n1 ≤ 1.29, the CV for practical performance is ≤ 15%. Moreover, it can be seen that when 1.24 ≤ n1 ≤ 1.28, a higher performance CV is ≤ 10%.

[0160] Figure 16 In this context, n1 = 1.25 becomes the condition for obtaining CV ≤ 10%. In practice, as shown... Figure 17 As shown, MIN = 0.47 and CV = 8%. Figure 14 The condition n1 = 1.29 becomes the condition that allows us to obtain CV ≤ 15% but not CV ≤ 10%. In fact, as... Figure 15 As shown, MIN = 0.42 and CV = 11%. That is, from Figure 21 It can be seen that n1 = 1.25 is the preferred condition compared to n1 = 1.29. Therefore, in summary, higher performance can be obtained when 1.24 ≤ n1 ≤ 1.28, and furthermore, the highest performance can be obtained when n1 = 1.26.

[0161] (E) Fourth Embodiment

[0162] In the fourth embodiment, Figure 16 In the capillary array configuration shown in the second embodiment, the influence of the capillary outer diameter and the ratio of outer diameter to inner diameter is evaluated. The laser irradiation sections of 96 capillaries with an inner diameter of 2r = 50 μm are arranged on the same plane. In the capillary array, 48 analytical capillaries and 48 lens capillaries are arranged alternately. The outer layer of the capillaries is a fluorine solution, n1 = 1.25; the capillary material is quartz glass, n2 = 1.46; the inner layer of the analytical capillaries is a low-refractive-index separation medium, n3 = 1.33; and the inner layer of the lens capillaries is a matching solution, n4 = 1.46.

[0163] Figure 22In (a), the maximum relative fluorescence intensity of 48 analytical capillaries under these conditions, with the outer diameter 2R varying between 75 μm and 250 μm, is plotted using black triangles, the average value using black dots, and the minimum value using black squares. The capillary spacing is set to the outer diameter 2R plus 29 μm. Based on this result, when the outer diameter 2R ≤ 190 μm, the practical performance requirement MIN ≥ 0.2 is met. Furthermore, by setting 2R ≤ 175 μm and 90 μm ≤ 2R ≤ 150 μm, MIN ≥ 0.3 and MIN ≥ 0.4 are satisfied, respectively, resulting in even higher performance. That is, when the outer diameter to inner diameter ratio R / r ≤ 3.8, the practical performance requirement MIN ≥ 0.2 is met. Furthermore, by setting R / r ≤ 3.5 and 1.8 ≤ R / r ≤ 3.0, MIN ≥ 0.3 and MIN ≥ 0.4 are satisfied, respectively, resulting in even higher performance.

[0164] Figure 22 (b) indicates that in relation to Figure 22 (a) The coefficient of variation (CV) of relative fluorescence intensity of 48 analytical capillaries when the outer diameter 2R of the capillaries varied between 75 μm and 250 μm under the same conditions. Based on this result, it can be seen that when the outer diameter 2R ≤ 175 μm, the CV for practical performance is ≤ 20%. Furthermore, it can be seen that when 85 μm ≤ 2R ≤ 160 μm, the CV for practical performance is ≤ 15%. Furthermore, it can be seen that when 100 μm ≤ 2R ≤ 140 μm, the CV for even higher performance is ≤ 10%. That is, it can be seen that when the outer diameter to inner diameter ratio R / r ≤ 3.5, the CV for practical performance is ≤ 20%. Furthermore, it can be seen that when 1.7 ≤ R / r ≤ 3.2, the CV for practical performance is ≤ 15%. Furthermore, it can be seen that when 2.0 ≤ R / r ≤ 2.8, the CV for even higher performance is ≤ 10%.

[0165] As can be seen from the above, in summary, the highest performance can be obtained when 100μm≤2R≤140μm, that is, when 2.0≤R / r≤2.8.

[0166] (F) Fifth Embodiment

[0167] <Standardization of the technology disclosed herein>

[0168] In the fifth embodiment, the structural features of the capillary array with alternating analytical capillaries and lens capillaries disclosed in this disclosure are standardized. Here, the number of analytical capillaries and lens capillaries does not necessarily need to be the same. When the two ends of the capillary array are set as analytical capillaries, N analytical capillaries and N-1 lens capillaries can be alternately arranged as long as N is an integer greater than or equal to 2. When the two ends of the capillary array are set as lens capillaries, N analytical capillaries and N+1 lens capillaries can be alternately arranged. Hereinafter, the term "capillary" refers to both "analytical capillaries" and "lens capillaries". In the laser irradiation section of the capillary, 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 medium outside the capillary is set to n1, the refractive index of the raw material of the capillary is set to n2, the refractive index of the medium inside the analytical capillary (separation medium) is set to n3, and the refractive index of the medium inside the lens capillary is set to n4. The refraction angle when the laser beam passes through one analytical capillary is set to θ. A Let θ be the angle of refraction of the laser beam as it passes through a lens capillary. B When the laser beam passes through a set of one analytical capillary and one lens capillary, the angle of refraction is expressed by the following equation (5) according to equation (1).

[0169] [Number 5]

[0170]

[0171] As described in the second to fourth embodiments above, in the technical configuration of this disclosure, the condition for enabling the multifocal system to function is Δθ. A +Δθ B ≤0. This condition is first discovered in the art of this disclosure. In particular, when the interior of the analytical capillary is filled with a separation medium of low refractive index, specifically, under the conditions that n3 < 1.36, ideally n3 ≤ 1.35, further ideally n3 ≤ 1.34, and ultimately n3 = 1.33, Δθ is required. A +Δθ B The condition ≤0 holds true. This has not been achieved in any known example. In addition, in order to reduce the reflection loss of the laser beam caused by the lens capillary, it is preferable to assume the condition n2 = n4. In this case, equation (5) is transformed into the following equation (6).

[0172] [Number 6]

[0173]

[0174] When a rod-shaped lens with a refractive index of n2 is used instead of a lens capillary, it is also represented by equation (6).

[0175] <Regarding the analysis of the ratio of capillary to lens capillary>

[0176] The above describes an alternating arrangement of analytical capillaries and lens capillaries in a 1:1 ratio, but this ratio is not strictly necessary. For example, when using one lens capillary for two analytical capillaries, as long as the overall structure exhibits a convex lens effect and the multifocal function is achieved, it is sufficient. In this case, the capillaries can be arranged in a repeating manner, with the group of two analytical capillaries and one lens capillary as the unit. It is not necessary for the units to repeat throughout the entire capillary array; the units can be repeated only in a portion of the capillary array. Typically, when m is set to a positive integer and one lens capillary is used for m analytical capillaries, that is, in order to exhibit a convex lens effect and achieve the multifocal function with the group of m analytical capillaries and one lens capillary as the unit, as shown in the following equation (7), it is necessary to make mΔθ A +Δθ B ≤0 is true.

[0177] [Number 7]

[0178]

[0179] When m = 1, equation (7) is the same as equation (5).

[0180] On the other hand, as shown in the above embodiments, even when the multifocal configuration is functional, if the reflection loss of the laser beam due to the capillary is large, it becomes difficult to simultaneously irradiate all analytical capillaries with the laser beam. Let T be the transmittance of the laser beam passing through one analytical capillary. A Let T be the transmittance of the laser beam passing through a lens capillary. B According to equation (2), the transmittance of the laser beam when passing through a set of one analytical capillary and one lens capillary is expressed by the following equation (8).

[0181] [Number 8]

[0182]

[0183] In addition, to reduce the reflection loss of the laser beam caused by the lens capillary, it is preferable to set the condition n2 = n4. In this case, equation (8) is transformed into the following equation (9).

[0184] [Number 9]

[0185]

[0186] When a rod-shaped lens with a refractive index of n2 is used instead of a lens capillary, it is also represented by equation (9).

[0187] When a laser beam is applied to both sides of a capillary array consisting of N analytical capillaries and N lens capillaries arranged alternately, the analytical capillary located in the center of the capillary array experiences the lowest laser irradiation intensity. The capillary numbering of the analytical capillary located in the center of the capillary array is n = (N+1) / 2 when N is odd, and n = N / 2 or n = N / 2+1 when N is even. Therefore, according to equation (4), the minimum laser irradiation intensity when N is odd is expressed by equation (10).

[0188] [Number 10]

[0189]

[0190] When N is even, it is represented by the following formula (11).

[0191] [Number 11]

[0192]

[0193] To meet practical performance requirements, equation (10) or (11) is needed to ensure that MIN ≥ 0.2. Specifically, to achieve simultaneous irradiation of 24 or more analytical capillaries using a laser beam, MIN ≥ 0.2 is required under the conditions of N = 24 and N = 48. In equation (11), when N = 48, to satisfy MIN ≥ 0.2, T... A ×T B ≥93% is sufficient. These conditions were discovered for the first time in this disclosure. In particular, when the interior of the analytical capillary is filled with a separation medium of low refractive index, specifically, under the conditions that n3 < 1.36, ideally n3 ≤ 1.35, further ideally n3 ≤ 1.34, and ultimately n3 = 1.33, MIN ≥ 0.2 is required.

[0194] The coefficient of variation (CV) of the laser irradiation intensity of N analytical capillaries can be approximately calculated using equation (4). The maximum value (MAX) of the laser irradiation intensity is obtained using capillaries positioned at both ends of the capillary array (n = 1 and n = N), and the minimum value (MIN) of the laser irradiation intensity is obtained using capillaries positioned in the center of the capillary array (n = (N+1) / 2 when N is odd, n = N / 2 or n = N / 2+1 when N is even). According to equation (4), regardless of whether N is odd or even, MAX is represented by the following equation (12).

[0195] [Number 12]

[0196] MAX = 0.5 · {(T A ×T B ) N-1 +1}…(12)

[0197] At this point, CV can be approximated by dividing MAX-MIN by the average of MAX and MIN and then dividing by 3. That is, it can be represented by the following equation (13).

[0198] [Number 13]

[0199]

[0200] To meet practical performance requirements, equations (10), (11), (12), and (13) need to be used to ensure that CV ≤ 20% and CV ≤ 15%.

[0201] As can be seen from the above, in order to obtain higher practical performance, (i)Δθ A +Δθ B ≤0 or mΔθ A +Δθ B The simultaneous fulfillment of the following three conditions is necessary for the efficient use of a laser beam to simultaneously irradiate multiple analytical capillaries: (ii) MIN ≥ 0.2, and (iii) CV ≤ 20% or CV ≤ 15%. In particular, when the interior of the analytical capillary is filled with a low-refractive-index separation medium, specifically, these conditions must be met simultaneously under the following conditions: n3 < 1.36, ideally n3 ≤ 1.35, further ideally n3 ≤ 1.34, and ultimately n3 = 1.33.

[0202] (G) Sixth Implementation Method

[0203] In the sixth embodiment, a specific configuration of a capillary array in which analytical capillaries and lens capillaries are arranged alternately is shown. Hereinafter, the term "capillary" refers to both "analytical capillary" and "lens capillary".

[0204] Figure 23 This diagram shows the periphery of the laser irradiation section 14 of multiple capillaries. The laser irradiation sections 14 of multiple analytical capillaries 1 and multiple lens capillaries 24 are arranged alternately on an array plane, forming a capillary array. The analytical capillaries 1 are represented by dashed lines, and the lens capillaries 24 are represented by solid lines. The laser beam 13 enters from the side of the array plane, and the laser irradiation sections 14 of each capillary are simultaneously irradiated.

[0205] exist Figure 23In the diagram, only the area near the laser irradiation section 14 within the analytical capillary 1 is shown, while the entire lens capillary 24 is shown. That is, the total length of the lens capillary is shorter than that of the analytical capillary. This is because the lens capillary only needs to exist within the laser irradiation section 14. Therefore, the overall capillary array can be simplified while reducing the amount of capillary consumption. A high-refractive-index medium is filled inside the lens capillary. For example, if the capillary material is quartz glass with n2 = 1.46, a matching solution with n4 = 1.46 can be filled inside the lens capillary. Furthermore, sealing both ends of the lens capillary is effective in preventing the solution filling the lens capillary from evaporating or detaching. Furthermore, as... Figure 23 As shown, the sealing part 25 can be used to bundle and seal the two ends of multiple lens capillaries. This further simplifies the overall capillary array. The sealing part 25 and the lens capillaries can be easily bonded using an adhesive.

[0206] The above explanation describes capillaries made of quartz glass with a refractive index n² = 1.46 for both analytical capillaries and lens capillaries. However, a quartz glass rod can also be used instead of a lens capillary, with the outer diameter of the rod being equal to that of the analytical capillary. In this case, the total length of the quartz glass rod can also be shorter than the total length of the analytical capillary.

[0207] Symbol Explanation

[0208] 1. Capillary (Analytical Capillary)

[0209] 2. Sample injection end

[0210] 3 Sample elution end

[0211] 4 cathodes

[0212] 5 anodes

[0213] 6 Cathode-side buffer

[0214] 7 Anode-side buffer

[0215] 8 power supplies

[0216] 9 polymer blocks

[0217] 10 valves

[0218] 11 syringes

[0219] 12 laser sources

[0220] 13 laser beams

[0221] 14 Laser Irradiation Section

[0222] 15 Condensing Lens

[0223] 16 Laser Cut-off Filter

[0224] 17 Transmission-type diffraction gratings

[0225] 18 Imaging Lenses

[0226] 19 sensors

[0227] 20 light-emitting points

[0228] 21 fluorescence

[0229] 22 imaging points

[0230] 23 optical axes

[0231] 24-lens capillary

[0232] 25. Sealing section.

Claims

1. A capillary array, wherein N is set to an integer greater than or equal to 2, and the laser irradiation portions of N analytical capillaries used for electrophoretic analysis, which are irradiated by a laser beam, and the laser irradiation portions of N±1 lens capillaries not used for electrophoretic analysis, which are irradiated by the laser beam, are alternately arranged approximately on the same plane, wherein, When the outer radius of each of the N analytical capillaries and the N±1 lens capillaries in the laser irradiation section is set to R, the inner radius to r, the refractive index of the external medium to n1, and the refractive index of the raw material to n2, the refractive index of the medium inside the N analytical capillaries in the laser irradiation section is set to n3, and the refractive index of the medium inside the N±1 lens capillaries in the laser irradiation section is set to n4, n3＜1.36， R, r, n1, n2, n3, and n4 satisfy [Number 1] 。 2. The capillary array according to claim 1, wherein, n3≤1.34。 3. The capillary array according to claim 1, wherein, Let the transmittance of each of the N analytical capillaries be T. A Let the transmittance of each of the N±1 lens capillaries be T. B , set as [Number 2] hour, When N is odd, it satisfies [Number 3] ， When N is even, the following conditions are met: [Number 4] 。 4. The capillary array according to claim 1, wherein, Let the transmittance of each of the N analytical capillaries be T. A Let the transmittance of each of the N±1 lens capillaries be T. B , set as [Number 5] ， Set as [Number 6] ， When N is odd, let it be... [Number 7] , When N is even, let it be... [Number 8] hour, satisfy [Number 9] 。 5. The capillary array according to claim 4, wherein, satisfy [Number 10] 。 6. The capillary array according to claim 1, wherein, 1.23≤n1≤1.29。 7. The capillary array according to claim 6, wherein, 1.24≤n1≤1.28。 8. The capillary array according to claim 1, wherein, 1.8≤R / r≤3.0。 9. The capillary array according to claim 8, wherein, 2.0≤R / r≤2.8。 10. The capillary array according to claim 1, wherein, n2 = n4 = 1.46 ± 0.

01.

11. The capillary array according to claim 1, wherein, 1.33≤n3＜1.36。 12. The capillary array according to claim 1, wherein, It has multiple analysis modes, including a first analysis mode of 1.33≤n3<1.36 and a second analysis mode of 1.36≤n3≤1.

42.

13. The capillary array according to claim 1, wherein, N≥48。 14. A capillary array, wherein N is set to an integer greater than or equal to 2, and laser irradiation portions of N analytical capillaries used for electrophoretic analysis, which are irradiated by a laser beam, and laser irradiation portions of N±1 lens capillaries not used for electrophoretic analysis, which are irradiated by the laser beam, are alternately arranged approximately on the same plane, wherein... When the outer radius of the N analytical capillaries and the N±1 lens capillaries in the laser irradiation section are set to R, the inner radius to r, the refractive index of the external medium to n1, and the refractive index of the raw material to n2, the refractive index of the medium inside the N analytical capillaries in the laser irradiation section is set to n3, and the refractive index of the medium inside the N±1 lens capillaries in the laser irradiation section is set to n4, the following conditions are met: 1.23≤n1≤1.29、 n2=n4=1.46±0.01、 1.33≤n3＜1.36、 R / r≤3.5。 15. The capillary array according to claim 14, wherein, 1.24≤n1≤1.28。 16. The capillary array according to claim 14, wherein, 2.0≤R / r≤2.8。 17. The capillary array according to claim 14, wherein, N≥48。 18. A capillary array, wherein N is set to an integer greater than or equal to 2, wherein laser irradiation portions of N analytical capillaries used for electrophoretic analysis, which are irradiated by a laser beam, and laser irradiation portions of N±1 lens capillaries not used for electrophoretic analysis, which are irradiated by the laser beam, are alternately arranged approximately on the same plane, wherein, When the outer radius of the N analytical capillaries and the N±1 lens capillaries in the laser irradiation section are set to R, the inner radius to r, the refractive index of the external medium to n1, and the refractive index of the raw material to n2, the refractive index of the medium inside the N analytical capillaries in the laser irradiation section is set to n3, and the refractive index of the medium inside the N±1 lens capillaries in the laser irradiation section is set to n4, It has multiple analysis modes, including a first analysis mode of 1.33≤n3<1.36 and a second analysis mode of 1.36≤n3≤1.42 under the conditions of 1.23≤n1≤1.29, n2=n4=1.46±0.01, and R / r≤3.

5.

19. The capillary array according to claim 18, wherein, 1.24≤n1≤1.28。 20. The capillary array according to claim 18, wherein, 2.0≤R / r≤2.8。 21. The capillary array according to claim 18, wherein, N≥48。

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