JP6361358B2 - Electrophoresis device - Google Patents

Description

  The present invention relates to a capillary electrophoresis apparatus or an electrophoresis apparatus that performs electrophoresis on a plate (chip) whose flow path is finely processed.

  In general, a capillary electrophoresis apparatus fills a fused silica capillary having an inner diameter of 100 μm or less with a separation medium (an electrophoresis buffer solution containing an electrophoresis buffer solution or a sieving polymer solution), introduces a sample into one end of the capillary, A sample is electrophoresed and separated in a capillary by applying a voltage between both ends. In order to stabilize the separation performance in the capillary, the temperature around the capillary is kept constant. The analyte component separated by the capillary is detected at the other end of the capillary by fluorescence or other optical or electrochemical techniques. Some capillary electrophoresis apparatuses include one capillary or a plurality of capillaries.

  A plate used for electrophoresis includes, for example, a substrate provided with an electrophoresis channel therein, and is disposed at both ends of the electrophoresis channel to store the electrophoresis buffer solution, and the end of the electrophoresis channel passes through the electrophoresis buffer solution. And a pair of reservoirs that are electrically connected to the portion (see Patent Document 1). Some electrophoretic flow paths are provided on one substrate, and some electrophoretic flow paths are provided.

  As one method for introducing a sample into a channel formed in a capillary or a plate, there is an electric injection method (electrokinetic injection method: EKI method) (for example, see Non-Patent Document 1). In the electroinjection method, one end (injection end) of an electrophoresis channel filled with a separation medium is brought into contact with a sample, the other end of the electrophoresis channel is brought into contact with a buffer solution, and an electrode is attached to each of the sample and the buffer solution. A predetermined amount of sample is introduced into the flow path by applying a voltage for a certain period of time between both electrodes in the contact state.

  After introducing the sample into the electrophoresis channel, the sample injection end is brought into contact with the buffer solution, the electrode is immersed in the buffer solution in the respective reservoirs at both ends of the electrophoresis channel, and a voltage is applied between both electrodes. Perform sample electrophoresis.

JP2008-89440

Protein nucleic acid enzyme, 38 (3), 2243 (1993) Anal. Chem., 1998, 70, 2248-2253

  In the electric injection method, the quantitative relationship between the sample components introduced due to the difference in mobility of the sample components differs from the original composition of the sample, so that it is difficult to determine in principle.

  It is possible to correct the effective injection volume to some extent if the relative mobility of any component with respect to the internal standard is known, but this is limited because the injection rate and the temperature at the time of injection are constant. It must be a correction. Furthermore, when the ionic strength of the sample is different from that of the separation buffer solution or when the mobility at the time of injection changes, such as when the sample is injected into a separation medium containing an intercalator dye, correction becomes more difficult. .

  Attempts to reduce the difference in mobility of sample components by dividing a part of a capillary and using electroosmotic flow like a pseudo pump have been reported at various research levels (for example, non-patent literature). 12)), but there is no example of practical use because the convenience of the capillary is impaired and the cost is high.

  For certain types of electrophoresis plates, a cross flow channel is placed on the separation flow channel, the sample introduction process is continued until the sample composition in the cross portion reaches a steady state, and then the entire amount of sample components in the cross portion is injected. There are things that are supposed to do. In that case, a difference in injection amount due to a difference in mobility inherent in the sample component can be avoided. However, there are electrophoretic plates that do not have such a cross portion, and in the case of a capillary electrophoresis apparatus, it is difficult to form a cross flow path.

  The present invention is capable of quantitative analysis in an electrophoresis method and apparatus for injecting a sample by an electroinjection method for a capillary electrophoresis apparatus or an apparatus having an electrophoresis plate having no cross section. The purpose is to make it.

  Since there is a difference in the injection amount for each sample component in the sample injected by the electrical injection method, in the present invention, the detected peak area is corrected based on the injection speed for each sample component. Since the peak area corrected for speed represents the injection amount for each sample component, quantitative analysis becomes possible by correcting the peak area.

  In other words, the electrophoresis method of the present invention is a method for quantitatively analyzing sample components separated by electrophoresis, including the following steps. (A) A step of electrically injecting a sample from one end of the electrophoresis channel, and (B) a step of separating the injected sample by applying a voltage between both ends of the electrophoresis channel and performing electrophoresis. , (C) detecting the separated sample component at the detection position of the electrophoresis channel, (D) determining the peak area of the detected sample component, and (E) calculating the calculated peak area as the sample component. Correcting based on each injection rate.

  In addition to the correction (E1) based on the relative mobility of the sample component, the correction based on the injection speed in the step (E) includes (E2) correction based on the linear velocity at the time of sample injection for each sample component, and ( E3) At least one of correction based on the current integration value at the time of sample injection is included.

  The electrophoresis apparatus of the present invention that realizes the electrophoresis method of the present invention includes an electrophoresis channel, a voltage for sample injection into one end of the electrophoresis channel, and a voltage for electrophoresis in the electrophoresis channel. A power supply device applied between both ends of the electrophoresis channel, an electrophoresis unit having a detector for detecting the sample component separated by the electrophoresis channel, and an electrophoresis signal from the detector for each sample component And a control unit for performing quantitative analysis.

  The control unit includes a peak area calculation unit that obtains a peak area from the electrophoresis signal, and a correction unit that corrects the obtained peak area based on an injection rate for each sample component. The correction unit includes a peak area correction unit based on relative mobility that performs correction based on the injection rate based on the relative mobility of the sample component, and the correction unit further performs correction based on the injection rate for each sample component. At least one of a peak area correction unit based on the linear velocity during injection performed based on the linear velocity during injection and a peak area correction unit based on the current integral value performing correction based on the current integration value is provided. .

  According to the present invention, in the electrophoresis method and apparatus for electrically injecting a sample, the obtained peak area is corrected based on the injection speed for each sample component, so that quantitative determination that is difficult in principle is possible. become. In addition, since the peak area can be corrected without processing the capillary or adding a cross flow path, it can be quantified at low cost without impairing convenience.

It is a block diagram which shows schematically the capillary electrophoresis apparatus of a 1st Example. It is a figure which shows the electrophoresis apparatus of 2nd Example, (A) is a figure which shows the plate for electrophoresis in a top view, and shows a control apparatus etc. with a block diagram, (B) is the sample injection of the plate for electrophoresis It is an enlarged view of an end. (C) is a schematic perspective view which shows the sample injection | pouring end side. It is a block diagram which shows the control part in a 1st Example and a 2nd Example with a detector and a high voltage power supply. It is a flowchart which shows the capillary electrophoresis method of one Example. It is a graph which shows the relationship between the mobility of a sample component, and injection | pouring volume. − It is a figure of the electrophoresis waveform which shows the relationship between the distance from a capillary injection end, and a separation peak waveform. It is a graph which shows the relationship between the distance from the injection | pouring end for every DNA sample component from which size differs based on the data of FIG. 6, and a linear velocity. It is a graph which shows the relationship between the DNA sample component size based on the data, and a linear velocity in the initial state (upper graph) of injection | pouring, and a steady state (lower graph). It is a graph which shows the time change of the electric current value at the time of sample injection. It is a figure which shows the electrophoresis waveform in one Example. It is a graph which shows each sample component density | concentration and peak area of the Example on the time-axis reference | standard as it is measured. It is a graph which converts the time axis of the data of the graph of Drawing 11 into a mobility axis, and shows it on a mobility axis basis. It is a graph which shows the relationship between each sample component density | concentration after correcting by the mobility to the data of the graph of FIG. 12, and a peak area. It is a graph which shows the relationship between each sample component density | concentration after performing correction | amendment by the linear velocity by an intercalator further to the data of the graph of FIG. 13, and a peak area.

  In one embodiment, the difference in the amount of sample component injected in the electrical injection method is corrected by the difference in relative mobility of each ion component, and the obtained peak area is corrected based thereon. The difference in relative mobility is caused by, for example, the DNA chain length. Even if the charge amount is constant, if the DNA chain length is different, the relative mobility is different due to the different molecular weight.

For example, the difference in relative mobility of each ion component is that the mobility is larger than any sample component (the shortest chain length in the case of DNA) and the mobility is smaller than any sample component (in the case of DNA) Using two types of internal standard substances with known concentrations consisting of the components having the longest chain length, the relative mobility for any component is calculated. That is, in the step (A), two types of internal standard substance A having a mobility lower than any sample component and an internal standard substance B having a mobility higher than any sample component are injected into the capillary together with the sample. In step (E), an interpolation coefficient F _i for each sample component is used as the relative mobility. The interpolation coefficient F is a coefficient normalized so that the mobility μ _A of the internal standard substance _A is 0 and the mobility μ _B of the internal standard substance _B is 1. In this way, by performing relative mobility correction using two kinds of internal standards, the obtained peak area can be corrected even when the ionic strength of the sample is different from that of the separation buffer solution as the separation medium.

  Other embodiments correct for mobility changes during sample injection. For example, when the capillary is filled with a separation medium containing an intercalator dye, the mobility changes due to a change in charge amount due to the formation of a complex of the sample component and the intercalator dye. Therefore, this embodiment includes correction based on the linear velocity at the time of sample injection for each sample component as correction based on the injection velocity. As correction based on the linear velocity at the time of sample injection, a linear velocity conversion table obtained in advance under specific conditions can be used. Specific conditions include the type, concentration, sample injection voltage / time, etc. of the intercalator dye. As described above, the correction accuracy is further improved by correcting even a system including an intercalator dye.

  In yet another embodiment, the sample injection amount due to a current change in the sample injection process is corrected. The current change in the sample injection process is caused by a temperature change or the like. Therefore, this embodiment further includes a step of calculating a current integrated value at the time of sample injection, and includes a correction based on the current integrated value calculated as a correction based on the injection speed. The linear velocity at the time of injection changes linearly with changes in the current value during the sample injection process (however, the coefficient determined by pH, dielectric constant, ionic strength, viscosity, DNA chain length, capillary / sample zeta potential, etc.) Constant).

  In one embodiment, when the peak area of each component obtained at the end of the analysis is corrected based on the injection speed for each sample component, the injection amount for each sample component is corrected based on the relative mobility of the sample component. It is basic. Specifically, the injection amount of each component is obtained as a peak area. When the peak area is obtained, the time axis is corrected by converting it to a moving speed (= proportional to the mobility) at the detection point. That is, the peak height is integrated by the moving speed at that time (= [distance between injection end-detection point] / [electrophoresis time]) and is displayed in units of (mV · μm). Further, it is preferable to further include one or both of correction due to mobility change during sample injection and correction of sample injection amount due to current change during the sample injection process. The peak area of each component obtained at the end of the analysis or a value obtained from the peak area (the unit is arbitrary) is preferably quantified from the known concentration value of the internal standard substance.

  In this way, quantitativeness can be greatly improved even with an electrical sample injection method. Preferably, it is possible to approach a quantitative property equivalent to that of microchip electrophoresis using a cross flow channel.

  FIG. 1 schematically shows a capillary electrophoresis apparatus according to a first embodiment. In the fused silica capillary 2, both ends of the capillary 2 are immersed in the migration buffer solutions 4 and 6, as indicated by solid lines during migration, and migrate between the ends of the capillary 2 from the high-voltage power supply 8 through the migration buffer solutions 4 and 6. A voltage is applied. The high-voltage power supply 8 includes an ammeter that detects a current flowing through the capillary 2.

  An autosampler 10 is provided to inject a sample into one end of the capillary 2 prior to electrophoresis. The autosampler 10 holds a plurality of sample containers 12 on a sample plate, and moves the sample container 12 containing a sample to be analyzed to a sample injection position. At the time of sample injection, one end of the capillary 2 is immersed in the sample in the sample container 12 as indicated by a broken line, and an electrode is also immersed in the sample 12, so that a voltage for sample injection is inserted between the sample 12 and the migration buffer solution 6. Is applied. In this way, the sample is injected into one end of the capillary 2.

  A detector 14 is arranged at a detection position on the other end side of the capillary 2. The detector 14 is, for example, a UV detector (ultraviolet visible absorptiometer).

  The capillary 2 is accommodated in the thermostat 16 and is kept at a constant temperature.

  The operation of the autosampler 10, the application of the sample injection voltage by the high voltage power supply 8, the application of the electrophoresis voltage by the high voltage power supply 8, etc. are controlled, and the electrophoresis signal of the detector 14 is taken in, processed, and separated by electrophoresis. A control unit 20 is provided to perform sample component identification and quantification operations. The controller 20 is connected to a dedicated computer 22 for this capillary electrophoresis apparatus.

  2A and 2B show examples of plates used in the electrophoresis apparatus of the second embodiment. A plurality of electrophoresis channels 24 are arranged in the glass substrate 26 so as not to cross each other, and a reservoir 28 is attached to the cathode side end of the electrophoresis channel 24 on the surface of the glass substrate 26, and an end on the anode side The reservoir 3 is attached to the part. The electrophoresis channel 24 has a width of, for example, 90 μm and a depth of 40 μm, extends in the longitudinal direction of the glass substrate 26, and has a plurality of, for example, 384 so as not to cross each other in a radial region that spreads from the anode side to the cathode side. Books are arranged.

  Each of the reservoirs 28 and 3 constitutes a container having an opening at the top, accommodates the electrophoresis buffer solution, and is electrically connected to the end of the electrophoresis channel 24 through the electrophoresis buffer solution at the bottom. ing. The reservoir 28 on the cathode side has a plurality of 384 openings 24a connected to one end of the electrophoresis channel 24 at the bottom thereof, and in this example, 384 openings 24a are disposed, and these openings 24a serve as sample dispensing positions. The reservoir 28 is a single container surrounding a region where all the openings 24a are arranged. The anode-side reservoir 3 is also connected to the other end of the electrophoresis channel 24 at the bottom.

  As the material of the glass substrate 26, quartz glass or borosilicate glass can be used, and another material such as a resin can be used instead of the glass substrate 26. Here, since the components separated by electrophoresis are optically detected, the glass substrate 26 made of a transparent material is selected. However, when a detection means other than the optical detector is used, the material of the glass substrate 26 is limited to a transparent one. Is not to be done.

  The glass substrate 26 can be a laminate of two glass plates. The electrophoresis channel 24 can be formed on the bonding surface of one glass plate by lithography and etching (wet etching or dry etching). The openings 24a and the like can be formed as through holes in the other glass plate or the same glass plate at positions on both ends of the electrophoresis channel 24 by a method such as sandblasting or laser drilling.

  Prior to the electrophoresis, in order to inject the sample into one end of the electrophoresis channel 24, the sample is injected into the sample dispensing opening 24a by the pipetter mechanism. Thereafter, the tip of each electrode is immersed in the sample injected into the sample dispensing opening 24a, a predetermined voltage is applied between the electrode immersed in the migration buffer solution of the anode reservoir 3, and the sample migrates. It is introduced into the flow path 24.

  Thereafter, the sample remaining in the sample dispensing opening 24 a is removed by suction with a suction mechanism such as an aspirator, and then the electrophoresis buffer solution is supplied into the cathode reservoir 3. In the electrophoretic separation, both ends of the electrophoresis channel 24 are electrically connected to the power supply device 8 via electrodes in the electrophoresis buffer solution, and electrophoresis is started.

  A detector 14 a such as a laser-excited fluorescence detector is disposed at a detection position on the cathode reservoir 3 side of the electrophoresis channel 24.

  As in the embodiment of FIG. 1, the sample injection operation to the sample dispensing opening 24a, the application of the sample injection voltage by the high-voltage power supply 8, the application of the migration voltage by the high-voltage power supply 8 and the like are controlled. A control unit 20a is provided for taking in the electrophoresis signal, processing the data, and performing identification and quantification operations of the sample components separated by electrophoresis. A dedicated computer 22a for the electrophoresis plate electrophoresis apparatus is connected to the controller 20a.

  Since the control units 20 and 20a and the computers 22 and 22a perform the same function regarding data processing for correction in an embodiment, they will be described together below.

  As illustrated in FIG. 3, the control units 20 and 20 a include a peak area calculation unit 30 and a correction unit 32. The peak area calculation unit 30 is configured to obtain the peak area from the electrophoresis signals detected by the detectors 14 and 14a. The peak area corresponds to the injection amount of the sample component. The correction unit 32 is configured to correct the peak area calculated by the peak area calculation unit 30 based on the injection rate for each sample component.

  The correction unit 32 includes a peak area correction unit 36 based on relative mobility, and the peak area correction unit 36 based on relative mobility uses the peak area of each sample component calculated by the peak area calculation unit 30 as the relative movement of each sample component. Correct based on degree.

  The correction unit 32 includes a peak area correction unit 40 based on the linear velocity at the time of injection so that the electrophoresis channels 2 and 24 can be applied even when they are filled with a separation medium containing an intercalator dye. preferable. The peak area correction unit 40 based on the linear velocity at the time of injection corrects the peak area based on the linear velocity at the time of sample injection for each sample component.

  When the peak area correction unit 40 based on the linear velocity during injection is provided, the control unit 20 preferably further includes a linear velocity conversion table holding unit 38 that holds a linear velocity conversion table. In that case, the peak area correction unit 40 based on the linear velocity during injection corrects the peak area using the linear velocity conversion table held in the linear velocity conversion table holding unit 42.

  The control units 20 and 20a are provided with a current integration value calculation unit 42, and the correction unit 32 is provided with a peak area correction unit 44 based on the current integration value so that it can be applied even when the current value at the time of sample injection changes. Is preferred. The current integration value calculation unit 42 takes in a current value at the time of sample injection from the high-voltage power supply 8 and obtains the current integration value. The peak area correction unit 44 based on the current integration value corrects the peak area based on the current integration value obtained by the current integration value calculation unit 42.

  The correction unit 32 includes the peak area correction unit 36 based on relative mobility as an essential element. The peak area correction unit 40 based on the linear velocity during injection and the peak area correction unit 44 based on the current integration value include one or both of them. As a result, as the corrected peak area output value, in addition to the correction by the peak area correction unit 36 based on the relative mobility, the correction by the peak area correction unit 40 by the linear velocity during injection and the correction by the peak area correction unit 44 by the current integration value It is possible to obtain a peak area output value to which one or both of the above are applied. This is indicated by a solid line, a broken line, and an alternate long and short dash line leading to the output in FIG. The path indicated by the two-dot chain line is a comparative example, and shows a case where the peak area correction unit 40 based on the linear velocity at the time of injection and the peak area correction unit 44 based on the current integration value are not provided, and the corrected peak As the area output value, a peak area output value that has been corrected only by the peak area correction unit 36 based on relative mobility is obtained.

  FIG. 4 is a flowchart showing an electrophoresis method according to an embodiment. Although the case where the electrophoresis channel is the capillary 2 will be described here, the operation relating to the correction is the same when the electrophoresis channel is the electrophoresis channel 24 of the plate. Although there is a difference between the sample injection operation to the capillary 2 or the electrophoresis channel 24 and the electrophoresis operation for separation, the difference has already been described above.

  First, analysis parameters (sample injection voltage, injection time, electrophoresis voltage) are set. Two types of internal standard substances A and B are mixed in the sample. The internal standard substance A is an internal standard substance having a smaller mobility than any sample component, and the internal standard substance B is an internal standard substance having a greater mobility than any sample component.

  The injection end of the capillary 2 is immersed in the sample in the sample container 12, the other end of the capillary 2 is immersed in the buffer solution 6, and a voltage is applied between both ends of the capillary 2 to electrically inject the sample into the capillary 2. The amount of the sample stored in the sample container 12 is set to be larger than the amount injected into the capillary 2 during the injection time.

  After the sample injection, the capillary 2 injection end is taken out of the sample, immersed in the buffer solution 4, a voltage is applied between both ends of the capillary 2, and the sample is electrophoresed by the capillary 2 to be separated.

  The separated sample component is detected by the detector 14, and the peak area for each sample component is obtained.

  The mobility interpolation coefficient Fi of each sample component is calculated from the peak detection times of the internal standard substances A and B and the peak detection time of each sample component. The mobility interpolation coefficient Fi will be described later with reference to FIG.

  When the linear velocity of sample component injection is constant at the time of injection, that is, when no intercalator dye is mixed in the separation medium, the peak area of each sample component, that is, the injection amount is corrected using the mobility interpolation coefficient Fi as the relative mobility. To do. A correction method will also be described later.

  If the linear velocity of sample component injection is not constant at the time of injection, that is, if an intercalator dye is mixed in the separation medium, refer to the linear velocity conversion table prepared in advance to determine the linear velocity at the time of sample injection for each sample component. Ask.

  Next, when the current value at the time of injection is constant, for example, when the change of the current value is 5% or less during the sample injection time, in addition to correcting the peak area by relative mobility, the peak area by the linear velocity at the time of injection is further added. That is, the injection amount is corrected.

  If the current value at the time of injection is not constant, the current value obtained by the ammeter of the high-voltage power supply 8 is integrated over the injection time. In this case, in addition to the correction of the peak area based on the relative mobility, or the correction of the peak area based on the relative mobility and the correction of the peak area based on the linear velocity during injection, the peak area is corrected based on the current integration value.

  Next, the correction method will be described in detail.

(1) Correction using two types of internal standard substances

In general, in an electrical sample injection method into a capillary, the effective injection volume (V _i ) of each ion component in the sample is:
V _i = t _inj ν _i (πr ² )
Can be shown. here,
t _inj : injection time,
ν _i : Linear velocity when each ion is injected from the capillary end,
r: Capillary radius.

Assuming that there is no hydrodynamic movement velocity component caused by the water head difference (pressure difference) between both ends of the capillary, the zeta potential between the capillary inner wall and the solution is negligible, and there is no velocity component generated by electroosmotic flow.
ν _i = μ _i E _inj
Since E _inj is the electric field strength at the time of implantation and μ _i is the mobility of each ion component, the implantation volume V _i is V _i = t _inj E _inj (πr ² ) · μ _i
And is proportional to μ _i . This is illustrated in FIG. 5, and its slope (proportional constant) is πr ² t _inj E _inj .

Two types of internal standard substances, internal standard substance A (injection volume V _A ) having a lower mobility than any sample component, and internal standard substance B (injection volume V _B ) having a higher mobility than any sample component are sampled. 5, the relative mobility with respect to an arbitrary component i can be obtained as the interpolation coefficient F as shown in FIG. The interpolation coefficient F is standardized so that the mobility μ _A of the internal standard substance _A is 0 and the mobility μ _B of the internal standard substance _B is 1. Interpolation coefficients F _i is the following equation for any component i (1), obtained by (2).
V _i = F _i V _B + (1−F _i ) V _A (1)
F _i = (V _i −V _A ) / (V _B −V _A )
= (Ν _i −ν _A ) / (ν _B −ν _A )
= (Μ _i −μ _A ) / (μ _B −μ _A ) (2)

It is approximated that ions move to the detection unit while maintaining the linear velocity ν when ions are injected from the sample injection end of the capillary. When the separation length from the sample injection end of the capillary to the detection unit is L, and the peak detection time at the detection unit is t, the linear velocity ν is ν = L / t. The mobility μ is μ = ν / E _inj = L / E _inj t
Can be obtained as In the examples described later, the mobility μ (μ _i , μ _A , μ _B ) was tested by _setting the electric field strength E _inj at the time of injection to 200 V / cm, the separation length from the sample injection end to the detection part to 8.5 cm. It was determined in units of [cm ² / V seconds] from the peak detection time t obtained automatically. The mobility μ (μ _i , μ _A , μ _B ) thus obtained can be applied to the equation (2) to calculate the interpolation coefficient F _i for an arbitrary component i.

Correction of the peak area of the arbitrary component i is preferably performed by the following procedure.
1) A peak area is obtained for a migration pattern whose horizontal axis is the time axis.
2) The peak area is obtained after converting the horizontal axis of the migration pattern from the time axis to the mobility axis.
(Peak area) = Σ _n h _n × Δμ _n
= Σ _n h _n × (μ _{n + 1} −μ _n )
It becomes. Here, h _n is the peak height and μ _n is the mobility.
3) The peak area of an arbitrary component obtained for the mobility axis is corrected using the interpolation coefficient F _i . Assuming that the peak area and concentration of the internal standard substance B are A _B and C _B respectively, the concentration C _i before correction of any component is C _i = (A _i / A _B ) × C _B , and the concentration after correction C _i c is
C _i c = F _i × C _i .

(2) Correction in systems containing intercalator dyes

  In DNA fragment analysis, there is a capillary electrophoresis analysis method in which a separation buffer solution containing an intercalator dye is preliminarily filled into a capillary as a separation medium, and then a sample is injected from the capillary injection end by an electric injection method. In this method, it is not necessary to label the sample with a fluorescent dye in advance, and the sample DNA injected from the capillary injection end forms a complex with the intercalator dye, so that the detection unit can detect fluorescence.

  In this electrophoretic analysis method using an intercalator dye, the charge changes in the process of forming a complex between the sample DNA and the intercalator dye, so the injection rate of the sample components is not constant.

  For example, the distance from the injection end and the electrophoretic waveform are measured using an apparatus capable of detecting the fluorescence of the complex at an arbitrary position in the capillary. 6A-6E show the electrophoretic waveforms at the distances of 1 mm, 2 mm, 5 mm, 10 mm, and 23 mm from the injection end, respectively. FIG. 7 shows the relationship between the distance from the injection end and the moving speed based on the measurement results.

  However, in the measurement where the measurement results of FIGS. 6 and 7 were obtained, in order to eliminate the influence of the mobility change due to EKI itself, unlike the example, a full volume injection method using a cross flow path was adopted. The sample is a HaeIII digest of φX174 DNA (product of Promega Corporation). The separation medium is a separation buffer solution (DNA-1000: a product of Shimadzu Corporation) containing Gelstar (registered trademark) as an intercalator dye at a 20,000-fold dilution.

  As is apparent from FIG. 7, the speed of migration in the capillary gradually decreases according to the distance from the injection end. This is because the net charge changes when the negatively charged DNA migrates while forming a complex with the positively charged intercalator dye. In the vicinity of the injection end, the difference in the injection amount due to the sample component seems to be slight, but the mobility changes according to the charge of the DNA-dye complex with the injection time, and the introduction amount changes accordingly.

  Therefore, as one embodiment, a linear velocity conversion table for each DNA size corresponding to the injection time under a predetermined condition is prepared, and the injection amount for a component having a known size is corrected.

  The conditions to be specified in preparing the linear velocity conversion table are the composition of the separation medium, the type and concentration of the intercalator dye, the injection parameters, and the like. FIG. 8 shows the relationship between the linear velocity and the DNA fragment size from the same experimental data shown in FIG. It can be seen that the “brake effect” at the time of sample injection by the intercalator is greater for short-chain DNA.

  The ratio of (initial state) / (steady state) in FIG. 8 is the “linear velocity conversion coefficient”. Table 1 shows an example of the linear velocity conversion table conversion table obtained here.

  The linear velocity conversion coefficient is multiplied by the injection amount for each sample component to obtain an injection amount correction value. The injection amount is preferably the injection amount after correction by mobility.

(3) Correction when monitor current changes during injection

  When a separation buffer solution is used as the separation medium, the current value is proportional to the voltage when the salt concentration of the sample is the same as the salt concentration of the separation buffer solution and there is no temperature change due to the application of voltage. Since the moving speed of the sample component is proportional to the current value, it is also proportional to the applied voltage. In this case, under the electrophoresis voltage that is constant over time, the current value at the time of injection is also constant over time, and the electrophoresis waveform of the sample component is rectangular.

  Whether the current value at the time of injection is constant or not can be determined to be constant if the fluctuation width is actually within 5% (corresponding to the temperature change) from the instruction of the ammeter.

  However, generally, when the salt concentration of the sample does not match that of the separation buffer solution or when a temperature change occurs, the current value is not constant over time even if the applied voltage is constant over time.

The moving speed ν _inj at the time of sample injection is given by the following equation.
ν _inj = K _a / πr ²
Here, K _a pH of separation buffer solution, dielectric constant, ionic strength, viscosity, DNA chain length, capillary internal wall - potential difference between the electrophoretic solution (zeta potential) is a constant determined by such.

Distance from the injection end x, injection volume n _a of the injection time t, the current I (t) and concentration c _a (x, t) can be defined by the following equation.
dn _a (x, t) = K _a c _a (x, t) × I (t) dt

Since the injection time t _inj is short and c _a (x, t) can be regarded as _a constant c _a , the injection amount when the injection time t _inj elapses is
It becomes.

  An example of a current waveform at the time of sample introduction is shown in FIG. This shows the current value at the rise from the start of sample injection to 20 seconds. Immediately after the start of sample injection, the current value increases and indicates that the steady state has been reached from around 10 seconds, and it can be said that the sample injection amount has changed with time for 10 seconds after the start of sample injection.

  Since the moving speed of the sample component is proportional to the current value, the sample injection amount is proportional to the current integrated value at the time of sample injection. Therefore, the current integration value at the time of sample injection is detected, and the sample injection amount is corrected as being proportional to the current integration value.

  Since the current at the time of sample injection represents the movement of all the components in the capillary, it is useful for monitoring the parallel accuracy between analyses.

(Example)
The results of quantitative analysis of the sample components separated by capillary electrophoresis by the method of one embodiment are shown below.

The analysis conditions are as follows.
Capillary: inner diameter 50 μm × effective length 85 mm, LPA (linear polyacrylamide) coated capillary separation buffer solution: DNA-1000 (product of Shimadzu Corporation),
Intercalator: GelStar (registered trademark) 20,000-fold diluted Sample: φX174-HaeIII digest (1 ng / μL) (product of Promega Corporation)
Injection conditions: 200 V / cm × 3 seconds Separation conditions: 200 V / cm

  The obtained electrophoretic pattern is as shown in FIG. A fluorescent dye (5-FAM) irrelevant to DNA is mixed with a sample for electrophoresis and used as a marker having a higher mobility than any sample. Also, a 2720 bp DNA fragment sample is used as an internal standard substance with the lowest mobility.

FIG. 11 shows the results of plotting the respective theoretical concentrations (ng / μL) and peak area values for 11 peaks of the DNA fragment sample excluding 2720 bp based on the electrophoresis waveform data of FIG. The determination coefficient R ² was 0.9011. The theoretical concentration is the concentration of each DNA fragment sample determined from the known concentration before enzyme digestion and the total DNA chain length.

Next, when the time axis of the electrophoresis pattern was converted into a mobility axis and the peak area based on the mobility axis was obtained, the determination coefficient R ² was improved to 0.9509 as shown in FIG.

Furthermore, as a result of correcting the peak areas of the respective components obtained for the mobility axis using the interpolation coefficient F _i , the determination coefficient R ² was improved to 0.989 as shown in FIG. Thus, it can be seen that the peak area detected by correcting the injection amount for each sample component based on the relative mobility of the sample component, that is, the quantitative accuracy of the amount corresponding to the detected peak area is improved.

Next, FIG. 14 shows the result of correcting the data shown in FIG. 13 by the linear velocity at the time of injection with the intercalator dye using the linear velocity conversion coefficient shown in Table 1. DNA size linear velocity conversion factors not shown in Table 1 were determined by linear interpolation based on the DNA size linear velocity conversion factors shown in Table 1. In the data shown in FIG. 14, the determination coefficient R ² was improved to 0.9933. In this way, it can be seen that the quantitative accuracy of the amount corresponding to the detected peak area is further improved by correcting the injection amount for each sample component based on the linear velocity at the time of sample injection for each sample component. .

2 Capillary 4, 6 Buffer solution 8 High-voltage power supply 10 Syringe pump 12 Sample container 14, 14a Detection unit 20, 20a Control unit 24 Electrophoretic flow path 30 Peak area calculation unit 32 Correction unit 36 Peak area correction unit based on relative mobility 38 lines Speed conversion table holding unit 40 Peak area correction unit based on linear velocity during injection 42 Area integrated value calculation unit 44 Peak area correction unit based on area integrated value

Claims (6)

(A) a step of electrically injecting a sample containing a plurality of sample components having different sizes from one end into an electrophoresis channel filled with a gel ; and (B) the injected sample being placed in the electrophoresis channel. step separated by their size the sample components by applying a voltage to the electrophoretic across, detecting the sample components are (C) separating the detection position of the electrophoresis flow channels, (D) detection are steps determine the peak area of the sample components, and (E) the peak area obtained comprises the step of correcting, based on the injection rate for each of the sample components,
In addition to the correction (E1) based on the relative mobility according to the size of each sample component , the correction based on the injection speed in the step (E)
(E2) Correction based on the braking effect on the linear velocity at the time of sample injection of each sample component by the intercalator in the electrophoresis flow path , and (E3) Correction based on the current integration value at the time of sample injection,
An electrophoresis method for quantitatively analyzing a sample component electrophoretically separated including at least one of the above. In the step (A), two types of internal standard substance A having a mobility lower than any sample component and an internal standard substance B having a mobility higher than any sample component are injected into the electrophoresis channel together with the sample,
The electrophoresis method according to claim 1, wherein in the correction (E1), an interpolation coefficient F _i for each sample component is used as the relative mobility.
Here, the interpolation coefficient F is a coefficient normalized so that the mobility μA of the internal standard substance _A is 0 and the mobility μB of the internal standard substance _B is 1. The linear velocity conversion table for converting the fragment size of the sample component obtained in advance under a specific condition into a coefficient indicating the magnitude of the brake effect is used as the correction (E2). Electrophoresis method. Electrophoresis channel filled with gel , injection of sample containing a plurality of sample components having different sizes into one end of the electrophoresis channel, and voltage for electrophoresis in the electrophoresis channel and the power supply, as well as electrophoresis unit equipped with a detector for detecting the sample components separated by size by the electrophoretic flow path applied across the flow path,
And a control unit that performs quantitative analysis of each of the sample components takes in electrophoresis signal from the detector,
The control unit, a peak area calculation unit for obtaining a peak area from the electrophoresis signal,
The peak area obtained by the peak area calculation unit, and a correction unit that corrects, based on the injection rate for each of the sample components,
The correction unit includes a peak area correction unit by relative mobility that performs correction based on the injection rate based on relative mobility according to the size of the sample component,
The correction unit further performs a correction based on the injection speed based on a braking effect on the linear speed at the time of sample injection of each sample component by the intercalator in the electrophoresis flow channel, and a peak area correction unit based on the linear speed at the time of injection, And an electrophoretic device comprising at least one of a peak area correction unit based on an integrated current value that performs correction based on an integrated current value based on an injection rate. The electrophoresis apparatus includes a linear velocity conversion table holding unit that holds a linear velocity conversion table that is obtained in advance under specific conditions and converts a fragment size of a sample component into a coefficient indicating the magnitude of the brake effect. In addition,
The electrophoresis apparatus according to claim 4, wherein the peak area correction unit based on the linear velocity at the time of injection is configured to use a linear velocity conversion table held in the linear velocity conversion table holding unit. The electrophoresis apparatus includes a current integral value calculation unit that takes in a current value at the time of sample injection from the power supply device and obtains a current integral value thereof,
The electrophoretic device according to claim 4, wherein the peak area correction unit is configured to perform correction based on an injection rate based on the current integration value obtained by the current integration value calculation unit.