Electrophoresis device

Abstract

Provided is an electrophoresis device capable of acquiring an output signal that has a linear response to an input fluorescence signal. In an electrophoresis device (1), three pixels are defined as one bin, the amount of charge accumulated in a first pixel having the greatest light-reception signal strength among the three pixels is defined as a first charge amount, the amounts of charge accumulated in the remaining second pixel and third pixel are defined as second charge amounts, and the sum of the charge amounts of the three pixels when the first charge amount is in a saturated state and the charge amounts follow a Gaussian distribution with respect to each other is defined as an effective maximum charge amount, and in this case: the value of an output signal of the electrophoresis device (1) corresponding to the effective maximum charge amount is equal to the maximum value of the output signal of the electrophoresis device (1); the value of the output signal of the electrophoresis device (1) corresponding to charge amounts smaller than the effective maximum charge amount are smaller than the maximum value of the output signal of the electrophoresis device (1); and the value of the output signal of the electrophoresis device (1) corresponding to charge amounts larger than the effective maximum charge amount are equal to the maximum value of the output signal of the electrophoresis device (1).

Description

Electrophoresis equipment

[0001] The present invention relates to an electrophoresis device.

[0002] In the analysis of biological samples, a multi-capillary electrophoresis apparatus is widely used, in which multiple capillaries are filled with an electrophoretic separation medium such as an electrolyte solution or an electrolyte solution containing a polymer gel or polymer, and electrophoretic analysis is performed in parallel. The targets of electrophoresis range from small molecules to macromolecules such as proteins and nucleic acids.

[0003] In particular, in the detection of nucleic acids, excitation light is irradiated onto a sample containing fluorescently labeled nucleic acids, and the base sequence and length of the nucleic acids can be analyzed based on the fluorescent signals emitted by the fluorescent labels. The electrophoresis apparatus detects the fluorescent signals using an image sensor such as a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) image sensor. The electrophoresis apparatus then generates signal intensities for each wavelength based on the detected fluorescent signals, and analyzes the base sequence and length of the nucleic acids.

[0004] In an electrophoresis device, the higher the concentration of the sample being electrophoresed, the stronger the signal generated. However, when the sample concentration exceeds a certain level, the amount of charge generated in the image sensor by the fluorescent signal emitted by the fluorescent label exceeds the saturation charge of the image sensor. When this happens, the response between the sample concentration and the generated signal intensity no longer responds linearly. This is called saturation or being outside the detection limit. Saturation is determined by the saturation charge of the image sensor and the analog-to-digital converter.

[0005] Patent Document 1 states that "an analog-to-digital converter (ADC) providing 14 bits of precision, at a conversion gain that was software controllable." It also states that "if the capillary array is illuminated by a Gaussian-shaped light profile, the fluorescence from the center of the capillary array may saturate the middle pixels before the pixels on the sides have accumulated sufficient charge."

[0006] In electrophoresis devices, signal strength varies depending on the concentration of the sample. Therefore, improving the detection sensitivity of the image sensor is important to detect weak signals. Also, expanding the dynamic range is important to detect strong signal intensities.

[0007] Binning, which virtually combines multiple light-receiving surfaces (corresponding to pixels) of an image sensor and treats them as a single pixel, is known as a means of expanding the dynamic range of an image sensor. There are two types of binning: hardware binning and software binning. Software binning can handle higher fluorescence signal intensities than hardware binning.

[0008] Patent Document 2 states that "an optical detection unit that, if the fluorescence signal intensity acquired by executing the hardware binning exceeds a first threshold, switches to the software binning to acquire the fluorescence signal intensity, and, if the fluorescence signal intensity acquired by executing the software binning is equal to or less than the first threshold, switches to the hardware binning to acquire the fluorescence signal intensity."

[0009] Furthermore, Patent Document 3 discloses a binning process in which the signal intensity of each fluorescent label is calculated for bins generated by binning, and the degree of variation in signal intensity is evaluated as a value; if the value of the degree of variation is equal to or less than a predetermined value, the size of the bin corresponding to the peak value of the signal intensity for the fluorescent label with the largest peak value is reduced, and the size of the bin corresponding to the peak value of the signal intensity for the fluorescent label with the smallest peak value is increased.

[0010] Furthermore, Patent Document 4 discloses that in order to realize highly sensitive and wide dynamic range fluorescence measurement, the hard binning number, soft binning number, and noise of the image sensor are controlled so as to satisfy a predetermined relationship.

[0011] U.S. Patent No. 5,741,411 (Example III, Example IV) JP 2015-049179 A (Claim 2) WO 2023 / 058105 (Abstract) WO 2023 / 007567 (Claim 19)

[0012] In Patent Document 1, the electrophoresis apparatus allows the fluorescent signal received by the image sensor to be software-controlled by an ADC. Patent Document 1 also describes that when fluorescent labels flowing through a capillary array are irradiated with a laser and emit light according to a Gaussian distribution, the pixel corresponding to the center of the capillary array may saturate the central pixel before the pixels on either side have accumulated sufficient charge. However, Patent Document 1 does not describe at all how to process the fluorescent signal after the central pixel has saturated.

[0013] Patent Document 2 describes a threshold for switching between hardware binning and software binning. However, Patent Document 2 does not describe anything about the upper limit of detectable values ​​in software binning. Patent Documents 3 and 4 describe binning processing methods for achieving high sensitivity and a wide dynamic range. However, Patent Documents 3 and 4 do not describe anything about processing methods when pixels in a bin become saturated.

[0014] To achieve high sensitivity and a wide dynamic range, it is important to control the binning number, accumulate charge to the upper limit in each pixel of the image sensor, and ensure that the signal intensity output by the pixel is linear with respect to the intensity of the fluorescent signal. However, if the image sensor pixel corresponding to the center of the capillary array receives a fluorescent signal that exceeds its saturation charge, a blooming phenomenon occurs before adjacent pixels become saturated, i.e., before the saturation charge of the entire bin is exceeded, and the output signal intensity of the bin loses linearity. Note that blooming refers to the overflow of charge into adjacent pixels when the accumulated charge of a pixel exceeds a certain value. This phenomenon will be explained later.

[0015] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an electrophoresis apparatus that can obtain an output signal that has a linear response to an input fluorescent signal.

[0016] The electrophoresis apparatus according to the present invention, which has solved the above-mentioned problems, comprises a capillary, an optical detection unit that irradiates the capillary with light and receives fluorescence from a sample flowing through the capillary, and an analysis unit that performs analysis of the sample based on the fluorescence received by the optical detection unit, wherein the optical detection unit includes a light receiving element that outputs a first voltage signal corresponding to the intensity of the received fluorescence, and has a configuration in which binning is performed, with three pixels allocated in the thickness direction of the capillary corresponding to one pixel of a plurality of pixels allocated in the longitudinal direction of the capillary being one bin, and an amount of charge accumulated in a first pixel of the three pixels having the greatest light receiving signal intensity is defined as a first charge amount, and a pixel of the three pixels having a light receiving signal intensity lower than the first pixel is defined as a second charge amount. When the amount of charge accumulated in one of the second pixel and the third pixel having the smaller signal strength is defined as a second charge amount, and the sum of the amounts of charge for the three pixels when the first charge amount is saturated and the first charge amount and the second charge amount mutually follow a Gaussian distribution is defined as an effective maximum charge amount, the value of the output signal of the electrophoretic device corresponding to the effective maximum charge amount is equal to the maximum value of the output signal of the electrophoretic device, the value of the output signal of the electrophoretic device corresponding to an amount of charge smaller than the effective maximum charge amount is smaller than the maximum value of the output signal of the electrophoretic device, and the value of the output signal of the electrophoretic device corresponding to an amount of charge larger than the effective maximum charge amount is equal to the maximum value of the output signal of the electrophoretic device.

[0017] According to the present invention, an electrophoresis device capable of obtaining an output signal that has a linear response to an input fluorescent signal can be provided. Problems, configurations, and advantages other than those described above will become apparent from the following description of the embodiments. Further features related to the present invention will become apparent from the description of this specification and the accompanying drawings.

[0018] 1 is a schematic diagram of an example of the configuration of an electrophoresis device according to the present embodiment. FIG. 1 is a schematic diagram of an example of the configuration of an optical detection unit according to the present embodiment. FIG. 2 is a schematic diagram showing an overview of an example of the configuration of a CCD image sensor according to the present embodiment. FIG. 3 is an explanatory diagram illustrating the operation of converting a spectrally dispersed fluorescent signal into a digital signal according to the present embodiment. FIG. 4 is an explanatory diagram illustrating the operation of converting a spectrally dispersed fluorescent signal into a digital signal according to the present embodiment. FIG. 5 is an explanatory diagram illustrating the operation of converting a spectrally dispersed fluorescent signal into a digital signal according to the present embodiment. FIG. 6 is an explanatory diagram illustrating the operation of converting a spectrally dispersed fluorescent signal into a digital signal according to the present embodiment. FIG. 7 is an explanatory diagram illustrating the operation of converting a spectrally dispersed fluorescent signal into a digital signal according to the present embodiment. FIG. 8 is an explanatory diagram illustrating an example of the configuration of a bin according to the present embodiment. FIG. 9 is an explanatory diagram illustrating the distribution of fluorescent signals in the bins according to the present embodiment. FIG. 10 is a graph illustrating an example of the relationship between fluorescent signal intensity before the application of this embodiment (i.e., conventional) and output digital signal intensity. FIG. 11 is a flowchart illustrating the procedure for generating an output digital signal for one bin in the optical detection unit of an electrophoresis device according to an example related to Aspect 1. FIG. 12 is a graph illustrating an example of the relationship between fluorescent signal intensity and output digital signal intensity obtained by an electrophoresis device according to an example related to Aspect 1. FIG. 13 is a flowchart illustrating the processing content of the analysis unit according to Aspect 3.

[0019] An electrophoresis device according to one embodiment of the present invention will be described below with reference to the accompanying drawings. Note that common components in the following description and drawings may be designated by the same reference numerals, and redundant description may be omitted. Furthermore, the present invention is not limited to the following embodiments. Furthermore, the descriptions in this specification are merely typical examples, and do not limit the scope of the claims or application examples in any sense.

[0020] 1 is a schematic diagram of an example of the configuration of an electrophoresis apparatus 1 according to this embodiment. As shown in Fig. 1, the electrophoresis apparatus 1 includes a sample tray 10, a transporter 20, a capillary array 30, a thermostatic bath 40, a pump unit 50, a high-voltage power supply 60, an excitation light source 70, a detection position 80, an optical detection unit 100, and an analysis unit 200 (Fig. 2).

[0021] The sample tray 10 accommodates a plurality of sample containers 11 containing samples (hereinafter referred to as samples) in which fluorescently labeled measurement targets such as DNA and proteins (hereinafter referred to as DNA) have been added. Each sample container 11 contains a different sample. The transporter 20 is movable in three directions, up and down, left and right, and depth, and transports the sample tray 10 so that each sample container 11 is positioned at the tip position of each capillary 31 in the capillary array 30.

[0022] The capillary array 30 is composed of a plurality of capillaries 31. Each capillary 31 is hollow. Each capillary 31 is inserted into a sample container 11 contained in a sample tray 10. The thermostatic bath 40 is covered with a heat insulating material, and maintains the temperature of the capillary array 30 uniformly and constantly by a heating / cooling mechanism and a fan (not shown). The pump unit 50 injects an electrophoretic medium 51 (e.g., a polymer) into each capillary 31. As a result, the interior of each capillary 31 is filled with the electrophoretic medium 51.

[0023] The high-voltage power supply 60 applies a high voltage to both ends of each capillary 31 filled with an electrophoresis medium 51. When the high voltage is applied by the high-voltage power supply 60, each sample stored in the sample container 11 electrophoreses within each capillary 31 toward a detection position 80. The detection position 80 is provided on a path along which the sample is electrophoresed. The excitation light source 70 continuously emits excitation light 71 so that all capillaries 31 passing through the detection position 80 are irradiated with light.

[0024] FIG. 2 is a schematic diagram of an exemplary configuration of the optical detection unit 100 in this embodiment. The optical detection unit 100 receives fluorescence from a sample (specimen) flowing through the capillary 31. As shown in FIG. 2, the optical detection unit 100 includes a detection unit 110 and a conversion unit 120. The detection unit 110 includes a spectroscope 111 that separates the sample's emitted light generated at the detection position 80 into individual wavelengths, a CCD image sensor 300 (hereinafter referred to as CCD 300) that detects the separated light, and a CCD control unit 301. The fluorescent labels attached to the DNA electrophoresing in each capillary 31 and passing through the detection position 80 are excited by irradiation with excitation light 71, emitting a fluorescent signal 1001 ( FIG. 12 ). The generated fluorescent signal 1001 is separated into individual wavelengths by the spectroscope 111 and irradiated onto the light-receiving surface 310 ( FIG. 3 ) of the CCD 300.

[0025] Signal charges 1002 ( FIG. 12 ) are generated on the light-receiving surface 310 of the CCD 300 when the fluorescence signals 1001 separated into wavelengths are irradiated. The configuration and behavior of the CCD 300 will be described later. The CCD 300 may be of any of the following types: frame transfer type, full frame transfer type, interline transfer type, and frame inter-transfer type. Furthermore, a CMOS image sensor (Complementary Metal Oxide Semiconductor) may be used instead of the CCD 300. When a CMOS image sensor is used, it becomes possible to directly acquire digital signals from each light-receiving surface 310.

[0026] The conversion unit 120 has an amplifier 121 that amplifies the analog signal output from the CCD 300, and an ADC 122 (Analog Digital Converter) that converts the analog signal output from the amplifier 121 into a digital signal. The digital signal output from the ADC 122 is output to the analysis unit 200. The analysis unit 200 analyzes the sample based on the fluorescence received by the optical detection unit 100.

[0027] Next, the flow of sample measurement when the sample is DNA will be described with reference to FIGS. 1 and 2 . First, fluorescently labeled DNA is placed in the sample container 11. Then, a high voltage is applied to both ends of each capillary 31 by the high-voltage power supply 60, causing the DNA to move from the sample container 11 into the capillary 31. As a result, the DNA passes through the detection position 80 and undergoes electrophoresis inside the capillary 31 toward the discharge container 12. As the DNA electrophoreses, the migration speed varies depending on the base length, so DNA with shorter base lengths arrive at the detection position 80 first. Excitation light 71 emitted by the excitation light source 70 is irradiated onto the DNA that has reached the detection position 80. The excitation light source 70 irradiates all capillaries 31 passing through the detection position 80 with the excitation light 71. The irradiation with the excitation light 71 excites the fluorescent labels attached to the DNA, causing them to emit fluorescent signals 1001. The fluorescent signals 1001 are then separated into wavelengths by the spectrometer 111. The dispersed fluorescent signal 1001 is received by the CCD 300. The light receiving surface 310 of the CCD 300 is positioned so that images of all the capillaries 31 (the number of capillaries 31 in the capillary array 30) are projected onto it. The fluorescent signal 1001 received by the CCD 300 is converted from an analog signal to a digital signal by the conversion unit 120. The analysis unit 200 analyzes the sample using intensity information for each capillary 31 and for each wavelength of the digital signal output from the conversion unit 120. In this embodiment, fluorescently labeled DNA fragments are assumed as the sample passing through the inside of the capillaries 31, but samples other than DNA fragments may also be used.

[0028] (CCD) Fig. 3 is a schematic diagram showing an example of the configuration of a CCD 300 in this embodiment. As shown in Fig. 3, the CCD 300 has light-receiving surfaces 310 arranged in a grid pattern, a horizontal register 311, and a summing gate 312. Each light-receiving surface 310 receives a fluorescence signal 1001 obtained by dispersing the emitted light, which is excited when the excitation light 71 is irradiated onto the sample passing through the inside of the capillary 31, into wavelengths by the spectroscope 111. When the light-receiving surface 310 receives the fluorescence signal 1001, it outputs a signal charge 1002 corresponding to the intensity of the fluorescence signal 1001.

[0029] The horizontal register 311 vertically integrates the signal charges 1002 accumulated on the light-receiving surface 310. In this embodiment, the direction from the light-receiving surface 310 toward the horizontal register 311 is defined as the vertical direction, and the direction from the horizontal register 311 toward the summing gate 312 is defined as the horizontal direction. The summing gate 312 integrates the charges integrated by the horizontal register 311 in the horizontal direction.

[0030] 4 to 8 are explanatory diagrams illustrating the operation of converting a spectrally dispersed fluorescent signal 1001 into a digital signal in this embodiment. The CCD control unit 301 applies a pulse to a pulse line 401 to vertically transfer the signal charge accumulated in the light-receiving surface 310. When a pulse is applied to the pulse line 401, the charge accumulated in each light-receiving surface 310 is transferred vertically, pixel by pixel. As a result, the charge is transferred vertically from the position shown in FIG. 4 to the position shown in FIG. 5. Furthermore, as shown in FIG. 5, the charge transferred to the light-receiving surface 310 at the very end in the vertical direction is transferred to a horizontal register 311.

[0031] Next, the CCD control unit 301 again applies a pulse to the pulse line 401 to vertically transfer the signal charges 1002 accumulated in each light-receiving surface 310. When a pulse is applied to the pulse line 401, the signal charges 1002 accumulated in each light-receiving surface 310 are vertically transferred pixel by pixel. As a result, each signal charge 1002 is vertically transferred from the position shown in FIG. 5 to the position shown in FIG. 6. Furthermore, as shown in FIG. 6, the signal charge 1002 transferred to the light-receiving surface 310 at the very end in the vertical direction is transferred to the horizontal register 311. Furthermore, each horizontal register 311 accumulates the charge transferred previously and the signal charge 1002 transferred this time.

[0032] Next, the CCD control unit 301 again applies a pulse to the pulse line 401 to vertically transfer the signal charges 1002 accumulated in each light-receiving surface 310. When a pulse is applied to the pulse line 401, the signal charges 1002 accumulated in each light-receiving surface 310 are vertically transferred pixel by pixel. As a result, each signal charge 1002 is vertically transferred from the position shown in FIG. 6 to the position shown in FIG. 7. Furthermore, as shown in FIG. 7, the signal charge 1002 transferred to the light-receiving surface 310 at the very back in the vertical direction is transferred to the horizontal register 311. Furthermore, each horizontal register 311 accumulates the charges transferred two times previously and the signal charge 1002 transferred last time, as well as the signal charge 1002 transferred this time.

[0033] Next, the CCD control unit 301 applies a pulse to the pulse line 402 to transfer the signal charges 1002 accumulated in each horizontal register 311 in the horizontal direction. When a pulse is applied to the pulse line 402, the signal charges 1002 accumulated in each horizontal register 311 are transferred horizontally one pixel at a time. As a result, each charge is transferred horizontally from the position shown in Fig. 7 to the position shown in Fig. 8. Furthermore, as shown in Fig. 8, the charge transferred to the horizontal register 311 at the end in the horizontal direction is transferred to the summing gate 312.

[0034] All of the signal charges 1002 accumulated in the summing gate 312 are converted into a voltage all at once. Then, the CCD 300 outputs the converted voltage as an analog signal to the conversion unit 120. The analog signal input to the conversion unit 120 is amplified by the amplifier 121 and converted into a digital signal by the ADC 122. The ADC 122 outputs the converted digital signal (output digital signal 1006) ( FIG. 12 ) to the analysis unit 200.

[0035] The signal charges 1002 corresponding to the multiple light-receiving surfaces 310 are accumulated in the summing gate 312 by the operation described with reference to FIGS. 4 to 8 , allowing the multiple light-receiving surfaces 310 to be treated as a single light-receiving surface 310 in a pseudo-manner. Treating the multiple light-receiving surfaces 310 as a single light-receiving surface 310 in this manner is referred to as hardware binning, and the pseudo-combined light-receiving surfaces 310 are referred to as bins 320. In other words, in this embodiment, the optical detection unit 100 includes light-receiving surfaces 310 (light-receiving elements) that output a first voltage signal corresponding to the intensity of the received fluorescence. At the same time, the optical detection unit 100 performs binning, with one bin consisting of three pixels allocated in the thickness direction of the capillary 31, which corresponds to one pixel out of the multiple pixels allocated in the longitudinal direction of the capillary 31.

[0036] In the examples shown in FIGS. 4 to 8 , a total of three photosensitive surfaces 310 (three in the vertical direction and one in the horizontal direction) are hardware binned into one bin 320. For each photosensitive surface 310 corresponding to a bin 320, signal charges 1002 are accumulated in the horizontal register 311 and summing gate 312. That is, as in the examples shown in FIGS. 4 to 8 , a pulse is applied three times in succession to the photosensitive surface 310, and then a pulse is applied once to the horizontal register 311. Then, a pulse is applied once to the summing gate 312. Note that the area subjected to hardware binning does not have to be limited to the examples shown in FIGS. 4 to 8 . By changing (making variable) the area subjected to hardware binning, the sensitivity of the CCD 300 can be changed. In other words, the size of the bin 320 is variable.

[0037] (Overall Processing) The upper limit of the detectable signal intensity of fluorescence passing through the capillaries 31 will be described with reference to Figs. 9 to 11. Fig. 9 is an explanatory diagram illustrating an example of the configuration of the bins 320 in this embodiment. Note that in this embodiment, it is assumed that the fluorescence signal 1001 of the fluorescence passing through the capillaries 31 in the capillary thickness direction follows a Gaussian distribution. Also, as shown in Fig. 9, it is assumed that hard binning is performed on the image of each capillary 31 (each capillary 31), with one bin consisting of three pixels in the capillary thickness direction and one pixel in the capillary longitudinal direction.

[0038] The distribution of the accumulated charge amount within one bin will now be described with reference to FIG. 10 . FIG. 10 is an explanatory diagram illustrating the distribution of the fluorescent signal 1001 within the bin 320 in this embodiment. As shown in FIG. 10 , the fluorescent signal 1001 of the pixel 501 corresponding to the center of the capillary in the capillary thickness direction (hereinafter referred to as the central pixel 501) is the strongest, and the fluorescent signals 1001 of the two pixels 502 adjacent to the central pixel 501 (hereinafter referred to as the adjacent pixels 502) are weaker than the fluorescent signal 1001 of the central pixel 501. The relationship between the signal intensities of the central pixel 501, the adjacent pixel 502, and the two pixels can be derived using the following equations (1) and (2). Here, the Gaussian function f(x), the pixel position x in the capillary thickness direction, and the Gaussian function coefficient a １ ~a ４ , the signal intensity ratio s between the central pixel 501 and the adjacent pixel 502, and p the pixel position of the peak (central pixel 501) of the fluorescent signal 1001 in the capillary thickness direction. The integral range of the numerator of equation (2) may be from p+0.5 to p+1.5.

[0039]

[0040]

[0041] In equation (2), the denominator calculates the area of ​​the peak shape of the Gaussian distribution. Furthermore, in equation (2), the numerator calculates the area in the range of -0.5 to -1.5, which is the area in the range of ±0.5 when the pixel position in the width direction of the capillary 31 is p. The area in the range of ±0.5 is the area of ​​the fluorescent signal 1001 in the central pixel 501. The area in the range of -0.5 to -1.5 is the area of ​​the fluorescent signal 1001 in one of the adjacent pixels 502. In equation (2), the ratio is s (signal intensity ratio).

[0042] On the light receiving surface 310 of the CCD 300, the fluorescent signal 1001 emitted from the inside of the capillary 31 saturates earlier at the central pixel 501 than at the adjacent pixels 502. Here, saturation means that the charge amount obtained by photoelectric conversion of the fluorescent signal 1001 reaches the saturated charge amount in one pixel on the light receiving surface 310 of the CCD 300. In this embodiment, as an example, the saturated charge amount on the light receiving surface 310 of the CCD 300 is set to e １, the saturated charge amount of the horizontal register 311 and the summing gate 312 is e ２ When this is the case, e １ and e ２ The relationship is 3 x e １ <e ２ The ADC 122 converts a 5V analog signal into a 16-bit (maximum 65535 ADU) digital signal. １ 11 shows an example of the relationship between the fluorescent signal 1001 from the fluorescent label and the digital signal output by the ADC 122 when the amplifier 121 is set so that a charge amount three times the saturation charge amount of one bin is 65,535 ADU, which is the maximum output of the output digital signal 1006 from the ADC 122. Fig. 11 is a graph illustrating an example of the relationship between the fluorescent signal intensity and the output digital signal intensity before the application of this embodiment (i.e., the conventional relationship). Note that Fig. 11 was obtained when the gain of the amplifier 121 was set to, for example, 2.37.

[0043] As shown in FIG. 11 , there is a linear relationship between the intensity of the fluorescent signal and the intensity of the output digital signal up to the threshold value 601 at which the central pixel 501 saturates. However, at the threshold value 601 in FIG. 11 , the central pixel 501 saturates. When the CCD 300 receives a fluorescent signal 1001 that exceeds the threshold value 601, a blooming phenomenon occurs on the light-receiving surface 310. The blooming phenomenon occurs when the accumulated charge of a pixel exceeds a certain level and the charge overflows into adjacent pixels. Therefore, before all three pixels constituting one bin saturate, i.e., before two adjacent pixels 502 saturate, the fluorescent signal 1001 and the output digital signal 1006 no longer have a linear relationship. Specifically, as shown in FIG. 11 , the relationship between the threshold value 601 and 65,535 ADU is not linear but rather curved. Therefore, there is a range (between the threshold value 601 and 65,535 ADU) in which the output digital signal 1006 is inaccurate relative to the actual intensity of the fluorescent signal 1001. Because there is such a range of inaccurate values, conventional electrophoretic devices have room for improvement, particularly in terms of high sensitivity, in order to achieve high sensitivity and a wide dynamic range.Here, the amount of charge in one bin (corresponding to three pixels) at the time when the central pixel 501 is saturated (hereinafter referred to as the effective maximum charge amount) is defined as E, and the effective maximum charge amount E is calculated using equations (2) and (3).

[0044]

[0045] Therefore, in this embodiment, in order to realize high sensitivity, the upper detection limit of the fluorescent signal 1001 at which the output signal intensity of the bin 320 maintains linearity is defined, as will be explained in the following aspects 1 to 3, and processing is performed.

[0046] <Aspect 1> In aspect 1, the amount of charge accumulated in the central pixel 501 (first pixel), which has the greatest received light signal strength among the three pixels, is defined as the first amount of charge. Furthermore, in aspect 1, the amount of charge accumulated in either one of the second pixel (adjacent pixel 502) and the third pixel (adjacent pixel 502), which have a weaker received light signal strength than the first pixel, is defined as the second amount of charge. In aspect 1, when the sum of the amounts of charge for the three pixels when the first amount of charge is saturated and the first amount of charge and the second amount of charge mutually follow a Gaussian distribution is defined as the effective maximum amount of charge E, processing is performed by defining the effective maximum amount of charge E so as to satisfy all of the following (a1) to (a3):

[0047] (a1) The value of the output signal of the electrophoresis device 1 corresponding to the effective maximum charge amount E is set to be equal to the maximum value of the output signal of the electrophoresis device 1. This can be realized by determining the gain so that these values ​​are equal. In this way, the maximum value that maintains linearity is defined and set. (a2) The value of the output signal of the electrophoresis device 1 corresponding to a charge amount smaller than the effective maximum charge amount E is set to be smaller than the maximum value of the output signal of the electrophoresis device 1. At this time, linearity is maintained between a charge amount smaller than the effective maximum charge amount E and the value of the output signal of the electrophoresis device 1. In this way, linearity is maintained up to the maximum value. (a3) ​​The value of the output signal of the electrophoresis device 1 corresponding to a charge amount larger than the effective maximum charge amount E is set to be equal to the maximum value of the output signal of the electrophoresis device 1. In this way, charge amounts larger than the effective maximum charge amount E are uniformly treated as the maximum value, thereby eliminating the range of inaccurate values ​​described above.

[0048] In the first aspect, the maximum value at which linearity can be maintained is set by the definitions (a1) to (a3), and if a signal exceeds the maximum value, it is output as the maximum value, so that linearity is maintained regardless of the value of the signal input. The definitions (a1) to (a3) ​​may be set in advance as defaults for the electrophoresis device 1, or may be set by the saturated charge amount e １The gain may be set appropriately depending on the device configuration, etc. In the latter case, the setting can be performed by the user determining the gain, or the electrophoresis device 1 may determine the gain autonomously through calculation.

[0049] 12 is a flowchart showing a procedure for generating an output digital signal 1006 for one bin in the optical detection unit 100 of the electrophoresis device 1 according to an embodiment of the aspect 1. In this embodiment, the saturation charge amount e １ The signal intensity ratio s is calculated using the reference value of the saturated charge amount e １ For example, the value can be a value written in the specifications of the CCD 300, but it can also be determined by conducting a test or the like in advance.

[0050] In explaining this embodiment, the saturated charge amount e １ is 320ke - , the saturated charge amount e of the horizontal register 311 and the summing gate 312 ２ is 1000ke - Assume also that the fluorescence signal 1001 is measured in advance, Gaussian fitting is performed using equation (1), and the signal intensity ratio s is calculated to be 0.8 using equation (2). This value may be different. In this case, the effective maximum charge amount E is (1 + 2 × 0.8) × 320 keV. - =832ke - is.

[0051] As shown in FIG. 12, the effective maximum charge E (in this embodiment, 832 keV) - ) enters the optical detection unit 100. The fluorescence signal 1001 entering the optical detection unit 100 is dispersed by the spectroscope 111 of the detection unit 110. The dispersed fluorescence signal 1001 is accumulated as signal charges 1002 on the light receiving surface 310 of the CCD 300. The accumulated signal charges 1002 are integrated into signal charges 1003 of one bin (corresponding to three pixels) in the horizontal register 311. The signal charges 1003 are transferred to the summing gate 312 and output as an output analog signal 1004 of one bin.

[0052] The output analog signal 1004 corresponding to the effective maximum charge amount E is amplified by the amplifier 121 of the conversion unit 120 to 5 V, for example, when the reference voltage of the ADC 122 is 5 V, to produce an amplified analog signal 1005. Note that the above-mentioned 5 V is an example of the maximum output of the CCD 300. The amplification by the amplifier 121 can be set appropriately according to the maximum output of the CCD 300 using the definitions (a1) to (a3) ​​above. The illustrated amplification to 5 V by the amplifier 121 can be achieved by determining and adopting a predetermined gain. The predetermined gain can be, for example, 2.73, but is not limited to this.

[0053] The amplified analog signal 1005 corresponding to the effective maximum charge amount E is output by the ADC 122 to the analysis unit 200 as an output digital signal 1006 (digital fluorescence signal) of 65535 ADU, which is the maximum value of 16 bits.

[0054] (Effect) Because the device is configured so that the effective maximum charge amount E is output as the maximum value (65,535 ADU) of the output digital signal 1006 output by the ADC 122, the relationship between the fluorescent signal 1001 and the output digital signal 1006 output by the ADC 122 is as shown in Fig. 13. Fig. 13 is a graph illustrating an example of the relationship between the fluorescent signal intensity and the output digital signal intensity obtained by an electrophoresis device 1 according to an example related to aspect 1. Fig. 13 was obtained when the gain of the amplifier 121 was set to, for example, 2.73.

[0055] Comparing the graphs shown in FIG. 13 and FIG. 11 reveals that in Aspect 1, there is no range in which the output signal (output digital signal 1006) from CCD 300 has an inaccurate value relative to the intensity of the actual fluorescent signal 1001. Therefore, as shown in FIG. 13, it can be confirmed that in Aspect 1, linear data can be acquired up to the maximum output digital signal intensity (65,535 ADU). In this case, the threshold value 602 of the fluorescent signal intensity at which linearity is lost in FIG. 11 is equal to the threshold value 603 of the fluorescent signal intensity at which the output digital signal intensity due to the fluorescent signal 1001 saturates at 65,535 ADU in FIG. 13. In other words, the electrophoresis apparatus 1 according to Aspect 1 can acquire an output signal (output digital signal 1006) that responds linearly to the input fluorescent signal 1001 up to the maximum value (65,535 ADU).

[0056] <Aspect 2> In aspect 2, the electrophoresis apparatus 1 includes a conversion unit 120 that converts the fluorescence received by the optical detection unit 100 into a signal. The conversion unit 120 includes an amplifier 121 that amplifies the first voltage signal with a predetermined gain and outputs the amplified signal as a second voltage signal. The conversion unit 120 also includes an ADC 122 (A-D converter) that performs A-D conversion on the second voltage signal and outputs a digital signal corresponding to the second voltage signal.

[0057] In this aspect 2, the predetermined gain is determined to satisfy all of the following conditions (b1) to (b3): ​​(b1) The value of the second voltage signal corresponding to the effective maximum charge amount E is equal to the value of the input signal to ADC 122 corresponding to the maximum value of the digital signal that is the output signal of ADC 122. This defines and sets a maximum value that maintains linearity. (b2) The value of the second voltage signal corresponding to a charge amount smaller than the effective maximum charge amount E is smaller than the value of the input signal to ADC 122 corresponding to the maximum value of the digital signal that is the output signal of ADC 122. At this time, linearity is maintained between a charge amount smaller than the effective maximum charge amount E and the value of the input signal to ADC 122. This maintains linearity up to the maximum value. (b3) The value of the second voltage signal corresponding to a charge amount larger than the effective maximum charge amount E is equal to the value of the input signal to ADC 122 corresponding to the maximum value of the digital signal that is the output signal of ADC 122. This uniformly treats charge amounts larger than the effective maximum charge amount E as the maximum value, thereby eliminating the range of inaccurate values ​​described above.

[0058] In the second aspect, the definitions (b1) to (b3) (gain determination) set the maximum value at which linearity can be maintained, and any value exceeding the maximum value is output as the maximum value, so that linearity is maintained regardless of the value of the signal input. The definitions (b1) to (b3) can be determined as appropriate for each individual electrophoresis device 1.

[0059] In the above-mentioned aspect 1, preferably, the saturated charge amount e １ The electrophoresis device 1 is assumed to have a saturated charge amount e of the light receiving surface 310 of the CCD 300. １ The signal intensity ratio s may differ when Gaussian fitting is performed on the capillary 31 depending on the device configuration, etc. In this embodiment, even in such a case, the effective maximum charge amount E1 is calculated taking these factors into account, so that the performance of each individual device can be reflected in the detection upper limit more accurately than in embodiment 1.

[0060] <Example of Aspect 2> In Aspect 2, the electrophoresis device 1 has the same configuration as that of Aspect 1, but the saturated charge amount e １ In the first embodiment, the saturated charge amount e of the light receiving surface 310 of the CCD 300 is measured. １ and the saturated charge amount e of the horizontal register 311 and the summing gate 312 ２ The values ​​used were the default values ​​given in advance.

[0061] In the second aspect, the saturated charge amount e of the light receiving surface 310 of the CCD 300 is １ and the saturated charge amount e of the horizontal register 311 and the summing gate 312 ２ Therefore, in the second embodiment, the saturated charge amounts e of the light receiving surface 310, the horizontal register 311, and the summing gate 312 for the CCD 300 are different. １ , e ２ The value of the saturated charge amount e on the light receiving surface 310 of the CCD 300 is measured, and the value of the saturated charge amount obtained by the measurement is used. １ The measurement may be performed before or after the assembly to the optical detection unit 100 .

[0062] In addition, in embodiment 1, the signal intensity ratio s is calculated by measuring the fluorescent signal 1001 in advance and performing Gaussian fitting using equation (1). In embodiment 2, the signal intensity ratio s is calculated by measuring the fluorescence for each electrophoresis apparatus 1 and performing Gaussian fitting using equation (1). As a result, the signal intensity ratio s for each electrophoresis apparatus 1 is calculated using equation (2).

[0063] By the above measurement, the saturated charge amount e of the light receiving surface 310 of the CCD 300 is calculated for each individual electrophoresis device 1. １, and the signal intensity ratio s is calculated. Here, the effective maximum charge amount calculated by equation (3) for each individual electrophoresis device 1 is defined as E1. When the effective maximum charge amount E1 calculated for each individual electrophoresis device 1 is incident on the optical detection unit 100, it passes through the detection unit 110 and outputs an output analog signal 1004, as in embodiment 1. Because the effective maximum charge amount E1 differs for each individual electrophoresis device 1, the output analog signal 1004 also differs for each individual electrophoresis device 1. For example, when the reference voltage of the ADC 122 is 5 V, the conversion unit 120 sets a different amplifier 121 for each individual electrophoresis device 1 so that the amplified analog signal 1005 outputs 5 V. The setting (amplification) by the amplifier 121 can be appropriately set according to the maximum output of the CCD 300 using the definitions (b1) to (b3) above (gain determination).

[0064] The amplified analog signal 1005 corresponding to the effective maximum charge amount E1 is output by the ADC 122 to the analysis unit 200 as an output digital signal 1006 (digital fluorescence signal) of 65535 ADU, which is the maximum value of 16 bits.

[0065] (Effects) In addition to achieving the same effects as in aspect 1, aspect 2 calculates the effective maximum charge amount E1 for each individual electrophoresis device 1, and also incorporates the amplifier 121 into the electrophoresis device 1. As a result, in aspect 2, even if the upper detection limit differs depending on the individual electrophoresis device 1, the detection limit can more accurately reflect the capabilities of each individual electrophoresis device than in aspect 1.

[0066] <Aspect 3> In Aspects 1 and 2, the electrophoresis apparatus 1 is provided by controlling amplification using the amplifier 121. In Aspect 3, an electrophoresis apparatus 1 is provided that is capable of acquiring linear data up to the upper detection limit using the analysis unit 200 (i.e., software that operates the analysis unit 200) instead of the amplifier 121.

[0067] To realize this, the electrophoresis apparatus 1 according to Aspect 3 employs the following configuration and performs processing. The electrophoresis apparatus 1 according to Aspect 3 includes a conversion unit 120 that converts the fluorescence received by the optical detection unit 100 into a signal. The analysis unit 200 is configured to perform analysis of the sample based on the characteristics of the signal converted by the conversion unit 120.

[0068] Furthermore, the conversion unit 120 is configured to include an amplifier 121 that amplifies the first voltage signal with a predetermined gain and outputs the amplified signal as a second voltage signal. The predetermined gain in aspect 3 can be set to a value similar to that of the conventional technology. The conversion unit 120 is also configured to include an ADC 122 (A-D converter) that performs A-D conversion of the second voltage signal and outputs a digital signal corresponding to the second voltage signal.

[0069] In addition, the analysis unit 200 is configured to include a calculation unit (not shown) that performs calculations using the digital signals output from the conversion unit 120. The calculation unit corresponds to a central processing unit (CPU) or the like that is provided in the analysis unit 200.

[0070] In aspect 3, when the effective maximum charge amount E passes through the conversion unit 120 and the analysis unit 200 and the intensity of the digital signal output from the electrophoresis device 1 is defined as the effective maximum digital fluorescent signal intensity, processing is performed by defining it so as to satisfy both of the following (c1) and (c2).

[0071] (c1) The digital signal output from the electrophoresis apparatus 1 is output in a linear response up to the effective maximum digital fluorescent signal intensity. This maintains linearity up to the maximum value. (c2) For digital signals output from the electrophoresis apparatus 1 that are greater than the effective maximum digital fluorescent signal intensity, the maximum digital fluorescent signal intensity is output as the upper detection limit. This allows digital signals greater than the effective maximum digital fluorescent signal intensity to be uniformly treated as the maximum value, eliminating the range of inaccurate values ​​described above.

[0072] In Aspect 3, the hardware configuration of the electrophoresis apparatus 1 does not have to be the same as in Aspects 1 and 2. In Aspect 3, the hardware configuration of the conventional electrophoresis apparatus is retained, and the upper detection limit (effective maximum charge amount E) is set by software settings. In Aspect 3, even if a fluorescent signal 1001 equal to or greater than the effective maximum charge amount E is obtained, the analysis unit 200 determines that the signal is saturated (upper detection limit) and performs processing so that all linearity is maintained.

[0073] <Example of Aspect 3> The optical detection unit 100 and the analysis unit 200 of the electrophoresis apparatus 1 according to Aspect 3 will be described with reference to Fig. 12 and Fig. 14. Fig. 14 is a flowchart illustrating the processing details of the analysis unit 200 in Aspect 3. In Aspect 3, the configuration of the optical detection unit 100 in Fig. 12 differs from that of Aspects 1 and 2. For example, in Aspect 3, the amplifier 121 is not limited to amplification according to the effective maximum charge amount E and the ADC 122, and any amplification setting can be used.

[0074] In the third embodiment, similarly to the first embodiment, the saturated charge amount e １ The signal intensity ratio s is calculated using the reference value of . Also, it is assumed that the fluorescent signal 1001 is measured in advance, Gaussian fitting is performed using equation (1), and the signal intensity ratio s is calculated using equation (2). １ The effective maximum charge amount E is calculated from the signal intensity ratio s and equation (3).

[0075] When the fluorescent signal 1001 corresponding to the charge amount of this effective maximum charge amount E is converted into an output digital signal 1006 by the optical detection unit 100 and input to the analysis unit 200, this output digital signal 1006 is set as an effective maximum digital signal D. Then, in this analysis unit 200, the effective maximum digital signal D is set as the upper limit of detection.

[0076] Next, the processing performed by the analysis unit 200 to linearly acquire data up to the upper detection limit will be described. FIG. 14 is a flowchart illustrating the processing performed by the analysis unit 200 in aspect 3. As shown in FIG. 14, the analysis unit 200 acquires the output digital signal 1006 from the conversion unit 120 of the optical detection unit 100 (step S701). If the output digital signal 1006 exceeds the effective maximum digital signal D, which is the threshold value (step S702 → Yes), the process proceeds to step S703. This means that the acquired output digital signal 1006 exceeds the upper detection limit. On the other hand, if the output digital signal 1006 is equal to or less than the effective maximum digital signal D, which is the threshold value (step S702 → No), the process proceeds to step S704. This means that the acquired output digital signal 1006 is within the upper detection limit, which is the linear range of the data.

[0077] In the third embodiment, as in the first embodiment, the saturated charge amount e １ The signal intensity ratio s is not limited to a value prepared in advance. In the third embodiment, as in the second embodiment, the effective maximum digital signal D may be calculated for each individual electrophoresis apparatus 1 and set as a value specific to that apparatus. Furthermore, when the electrophoresis apparatus 1 is used, the effective maximum digital signal D may be measured before detecting nucleic acids and set in the analysis unit 200 each time.

[0078] (Effects) In Aspect 3, as in Aspects 1 and 2, an electrophoresis apparatus 1 can be provided that can linearly obtain data up to the maximum output (maximum value / upper limit of detection). That is, the electrophoresis apparatus 1 according to Aspect 3 can obtain an output signal (output digital signal 1006) that responds linearly to the input fluorescent signal 1001. In addition, in Aspect 3, the electrophoresis apparatus 1 can be provided using the analysis unit 200 instead of the amplifier 121, eliminating the need to modify the hardware components of the apparatus. In Aspect 3, the upper limit of detection can be measured by the analysis unit 200 even when the electrophoresis apparatus 1 is in use, and data can be linearly obtained up to the maximum output.

[0079] The electrophoresis device 1 according to the present invention has been described in detail above through embodiments. As described above, the electrophoresis device 1 ensures linearity of the output of the electrophoresis device 1 up to the effective maximum charge amounts E and E1 (effective maximum digital fluorescent signal intensity), thereby enabling both an expansion of the dynamic range of the electrophoresis device 1 and an improvement in the reliability of the output data.

[0080] The present invention is not limited to the above-described embodiments, but includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, or to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with other configurations.

[0081] REFERENCE SIGNS LIST 1 Electrophoresis apparatus 10 Sample tray 11 Sample container 12 Discharge container 20 Conveyor 30 Capillary array 31 Capillary 40 Thermostatic bath 50 Pump unit 51 Electrophoresis medium 60 High voltage power supply 70 Excitation light source 71 Excitation light 80 Detection position 100 Optical detection unit 120 Conversion unit 121 Amplifier 122 ADC (Analog-to-digital converter) 200 Analysis unit

Claims

1. A device comprising: a capillary; an optical detection unit that irradiates the capillary with light and receives fluorescence from a sample flowing through the capillary; and an analysis unit that performs analysis of the sample based on the fluorescence received by the optical detection unit, wherein the optical detection unit includes a light receiving element that outputs a first voltage signal corresponding to the intensity of the received fluorescence, and has a configuration that performs binning, with three pixels allocated in the thickness direction of the capillary corresponding to one pixel of a plurality of pixels allocated in the longitudinal direction of the capillary being set as one bin; wherein the amount of charge accumulated in a first pixel of the three pixels having the greatest received light signal strength is set as a first amount of charge, and the amount of charge accumulated in one of a second pixel and a third pixel of the three pixels having a light receiving signal strength smaller than that of the first pixel is set as a second amount of charge, and the sum of the amounts of charge for the three pixels when the first amount of charge is saturated and the first amount of charge and the second amount of charge mutually follow a Gaussian distribution is set as an effective maximum amount of charge. an electrophoresis device, characterized in that the value of the output signal of the electrophoresis device corresponding to the effective maximum charge amount is equal to the maximum value of the output signal of the electrophoresis device, the value of the output signal of the electrophoresis device corresponding to a charge amount smaller than the effective maximum charge amount is smaller than the maximum value of the output signal of the electrophoresis device, and the value of the output signal of the electrophoresis device corresponding to a charge amount larger than the effective maximum charge amount is equal to the maximum value of the output signal of the electrophoresis device.

2. An electrophoresis apparatus according to claim 1, further comprising a conversion unit that converts the fluorescence received by the optical detection unit into a signal, the conversion unit comprising an amplifier that amplifies the first voltage signal by a predetermined gain and outputs it as a second voltage signal, and an AD converter that performs AD conversion on the second voltage signal and outputs a digital signal corresponding to the second voltage signal, the predetermined gain being such that: the value of the second voltage signal corresponding to the effective maximum amount of charge is equal to the value of the input signal of the AD converter that corresponds to the maximum value of the digital signal that is the output signal of the AD converter; the value of the second voltage signal corresponding to an amount of charge smaller than the effective maximum amount of charge is smaller than the value of the input signal of the AD converter that corresponds to the maximum value of the digital signal that is the output signal of the AD converter; and the value of the second voltage signal corresponding to an amount of charge larger than the effective maximum amount of charge is equal to the value of the input signal of the AD converter that corresponds to the maximum value of the digital signal that is the output signal of the AD converter.

3. An electrophoresis apparatus according to claim 1, wherein the electrophoresis apparatus comprises a conversion section that converts the fluorescence received by the optical detection section into a signal; the analysis section is configured to perform analysis of the sample from the characteristics of the signal converted by the conversion section; the conversion section comprises an amplifier that amplifies the first voltage signal with a predetermined gain and outputs it as a second voltage signal, and an AD converter that performs AD conversion of the second voltage signal and outputs a digital signal corresponding to the second voltage signal; the analysis section comprises a calculation section that performs calculations using the digital signal output from the conversion section; and when the intensity of the digital signal output from the electrophoresis apparatus after the effective maximum amount of charge has passed through the conversion section and the analysis section is defined as an effective maximum digital fluorescent signal intensity, the digital signal output from the electrophoresis apparatus is a signal that is output in a linear response up to the effective maximum digital fluorescent signal intensity, and a digital signal output from the electrophoresis apparatus that is greater than the effective maximum digital fluorescent signal intensity is output as the maximum digital fluorescent signal intensity as an upper detection limit.

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