JP3501090B2 - Microchip electrophoresis device - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microchip electrophoresis used for analyzing a very small amount of protein, nucleic acid, etc. at high speed and with high resolution using a microchip. More particularly, the present invention relates to a microchip electrophoresis apparatus in which an analyst can easily set a voltage value to be applied to an electrophoresis channel when performing analysis by electrophoresis. [0002] Capillary electrophoresis apparatuses are generally used for analyzing very small amounts of proteins, nucleic acids and the like. In recent years, capillary electrophoresis chips (called microchips) formed by joining two substrates have been developed.
A microchip electrophoresis apparatus using this has been devised. Examples of microchips used in this microchip electrophoresis apparatus are shown in FIGS. The microchip 10 shown in the figure is composed of a pair of transparent glass substrates 1 and 2, grooves 4 and 5 serving as migration channels intersecting each other are formed on the surface of one substrate 2 by an etching technique or the like, and the other substrate 1 is formed. In this, the port 3 is formed by a through hole at a position corresponding to each end of the grooves 4 and 5. In addition, even if the groove intersects the cross as shown in FIG.
It may be an intersection having small steps as shown in (A) to (C) (in some cases, more grooves may intersect). Microchip is substrate 1, 2
As shown in FIG. 1C, the buffer solution (electrophoretic solution) is injected into the grooves 4 and 5 from one of the ports 3.
Thereafter, a sample is introduced from the port 3 (S) at one end of the shorter groove 4, and the ports 3 (S), 3 at both ends of the groove 4 are introduced.
A high voltage is applied for a predetermined time between (W). Application of high voltage to the buffer solution may be performed by a method of piercing each port 3 with a needle electrode, or by previously patterning electrodes around the inner surface of each port and the extraction electrode portion electrically connected to this inner surface portion. Alternatively, a high voltage may be applied to the extraction electrode. When a high voltage is applied so that a potential difference is generated between the ports at both ends of the groove 4, a potential gradient is formed in the buffer solution injected into the groove 4, and an electrophoretic current flows. As a result, the sample introduced into the buffer solution Moves in the groove 4. Subsequently, the high voltage applied to the ports at both ends of the groove 4 is stopped, and a high voltage is applied between the ports 3 (B) and 3 (D) at both ends of the longer groove 5. As a result, the migration direction of the sample existing at the intersection 6 of the grooves 4 and 5 changes, and the sample moves in the groove 5. And groove 5
By placing an array-type detector over almost the entire area of the flow path of the groove 5 in a suitable location above the detector (UV-visible spectrophotometer, fluorometer, electrochemical detector, etc.) The sample separated in step (b) is detected. FIG. 2 is a schematic configuration diagram of a microchip electrophoresis apparatus used for performing electrophoresis analysis. An XY stage 12 for moving the microchip 10 is placed on the optical bench 14, and the microchip 10 is fixed on the XY stage 12, and can be moved manually or automatically in the horizontal planes in the X and Y directions. . In order to optically detect a sample (a fluorescent sample is used in this example) separated in the electrophoresis channel (grooves 4 and 5 in FIG. 1), the laser device 1
The laser beam generated in 6 is irradiated onto the sample by the irradiation / condensing unit 18 and the fluorescence from the sample is collected. The fluorescence condensed by the irradiation / condensing unit 18 is detected by the photomultiplier tube 20. Note that binoculars 22 used for aligning the optical axis of the irradiation / condensing unit 18 are provided, and the XY stage is moved as necessary to adjust the optical axis. The optical signal detected by the photomultiplier tube 20 is amplified by an amplifier 28, converted into a digital signal by an A / D converter 30, and taken into a personal computer 39 having a CPU 32. FIG. 3 shows a microchip electrophoresis apparatus (in this case, an ultraviolet-visible detector) having a measurement optical system different from the above. In this case, the light beam 42 from the light source 41 is condensed linearly by the cylindrical lens 43 so that the sample can be detected over almost the entire separation-side flow path (groove 5 in FIG. 1). The arrayed photocell 44 detects the transmitted light. The signal from each detected array is shown in FIG.
In the same manner as above, it is amplified by the amplifier 28, converted into a digital signal by the A / D converter 30, and taken into the personal computer 39 having the CPU 32. In this example, the distribution of the separated sample in the groove 5 can be detected and displayed. Next, the application of a high voltage to the microchip 10 will be described again with reference to FIG. Microchip 1
In order for the sample injected into 0 to be separated by electrophoretic action, it is necessary to apply a high voltage to both ends of the electrophoresis flow path, and high voltage power supply devices 34, 35, 36, and 37 for that purpose are provided. A voltage can be applied independently from the port at the end of the flow path. These high voltage power supplies are CPU32
A predetermined high voltage is generated by the control from. In order to set the applied voltage, an input device such as a keyboard 39 and a display device 40 such as a liquid crystal screen are connected to the personal computer 38, and application is performed using the keyboard 39 while viewing the screen of the display device 40. By inputting the voltage value, the personal computer 3
The data is transmitted from 9 to each of the high voltage power supply devices 34 to 37, and a desired voltage is applied at a desired timing. The microchip electrophoresis apparatus has an excellent feature that it can analyze a very small amount of sample at high speed and with high resolution. However, in order to perform an optimal analysis with this apparatus, it is necessary to adjust the high voltage applied to each flow path to an optimal value. For example, in the case where voltages are independently applied to the four flow channel ends of the electrophoresis flow channel crossing the cross as described above using four power supplies, how much high voltage is applied to each flow channel end. It is necessary to determine whether it is good, but this operation is difficult and cumbersome and requires skill. Therefore, it is desired that a simpler setting can be made. In addition, in the microchip in which the grooves crossing the cross are formed, the migration channel needs to be switched from the short migration channel on the sample introduction side to the long migration channel on the sample separation side. Since the optimal setting conditions differ between the electrophoresis state in the short flow path at the time of sample introduction and the migration state in the long flow path after switching the flow path, the migration program from the time of sample introduction to the end of analysis is simple and easy. It is desired that it can be set optimally. Accordingly, an object of the present invention is to provide an electrophoresis apparatus in which an analyst can easily set optimum electrophoresis conditions when setting a voltage value to be applied to an electrophoresis channel. And It is another object of the present invention to easily perform high-resolution and rapid migration analysis by performing electrophoresis under suitable setting conditions. In particular, it is an object of the present invention to make it possible to easily find different optimum conditions in each process when performing complicated parameter condition setting as in the case of switching channels. [0008] In order to solve the above problems, a microchip electrophoresis apparatus of the present invention has an electrophoresis channel formed on a microchip and an opening provided on the electrophoresis channel. In the microchip electrophoresis apparatus in which the sample is introduced from the electrophoresis and the electrophoresis is performed by applying a voltage to the electrophoresis channel, the microchip electrophoresis apparatus sets the applied voltage value and / or the parameter value for grasping the electrophoresis state. When a necessary electrical characteristic parameter value is input from the control unit having a screen for input and the applied voltage and the parameter for grasping the migration state from this screen, application is performed based on the input electrical characteristic parameter value. An electrical characteristic calculating means for calculating other electrical characteristic parameter values that are undecided among the parameters for grasping the voltage and the electrophoretic state; And a screen composition means for simultaneously displaying the calculated electrical characteristic parameter value and the inputted electrical characteristic parameter value on the screen. The parameters input by the analyst directly from the keyboard and set are the voltage values of the high-voltage power supply applied to each flow path. The information necessary for the analyst to grasp the migration state is the voltage value itself. Rather, it is a parameter that determines an electrophoretic state other than a voltage value such as a potential gradient or electrophoretic current generated in each channel. This potential gradient or the like is determined by the relative relationship between the voltage values applied to the ends of each flow path. Therefore, parameters can be set while displaying such information on the screen to the analyst. That is, when a parameter (for example, voltage value) necessary for performing electrophoresis on the screen is input, a parameter (for example, potential gradient of each channel) useful for grasping the electrophoresis state is calculated according to the input data. The calculation result is displayed on the screen together with the input parameters. The analyst looks at and confirms these pieces of information and changes the settings as necessary to obtain the optimum parameter settings. Electrophoresis is performed under the optimal parameter conditions finally set in this manner, and analysis is performed under the optimal conditions. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to embodiments. The measurement optical system of the microchip electrophoresis apparatus main body and the hardware configuration of the high-voltage power supply shown in the following examples are basically the same as those shown in FIGS. 2 and 3 and are used. In addition, since there are various variations of the type of microchip used (type of shape of the electrophoresis channel engraved on the microchip) depending on the purpose of electrophoresis, the screen shape described later can be changed appropriately according to the shape. It goes without saying. For convenience of explanation, the microchip used in the following explanation is shown in FIG.
It shall be shown in (D). That is, the port 3 at both ends of the groove 4
S, 3W, ports 3B, 3D, groove 4 and groove 5 at both ends of groove 5
Intersects the cross to form intersection 6, port 3
Sample is introduced from B, and each port 3B, 3W, 3B, 3
Needle electrodes are respectively inserted into D, and voltages HV1, HV2, HV3, and HV4 are independently applied from independent high-voltage power supply devices 34, 35, 36, and 37 as shown in FIG. FIG. 4 is a view showing an example of a screen displayed on the display device 40 of the microchip electrophoresis apparatus according to the embodiment of the present invention. This embodiment is useful when the analyst wants to set the voltage applied to the electrodes at the four port portions provided at the end of the electrophoresis channel to an optimum value by trial and error. As shown in FIG. 4, a display mode input unit 101 for inputting a display mode (potential gradient display, current display, etc.) is displayed in a window frame shape at the lower right of the screen. A graphic screen corresponding to the shape of the migration channel of the microchip 10 is displayed at the center of the screen. On the graphic, each port 3S, 3W, 3B, 3 of the microchip 10 is displayed.
D is a graphic port 103S, 103 respectively
Corresponding to W, 103B, 103D, groove 4 and groove 5 are groove 10
4 and 105, and the intersection 6 corresponds to the intersection 106. On this screen, further, high-voltage power supply devices 34, 35, 3
6 and 37 are voltages HV1, HV2, and HV applied to the respective electrodes.
3. Voltage input units 111, 1 for setting the value of HV4
12, 113, 114 are displayed in a window frame shape. When “potential gradient display” is input in the display mode input unit 101, between the port 103S and the intersection 106,
Between intersection 106 and port 103W, port 103B
And intersection 106, intersection 106 and port 103D
Numerical value display of the potential gradient generated between and the potential gradient display unit and direction display units 121, 121A, 122, 122A, 12 for displaying the gradient direction
3, 123A, 124, and 124A are formed (these actually indicate potential gradients at corresponding positions on the actual microchip). Intersection 1
Crossing voltage display 1 for displaying the voltage generated at the position 06
26 is also formed. Further, an electrophoretic display unit 130 is provided on the electrophoretic flow path to display that the electrophoretic state is being performed when voltage is applied and electrophoretic migration is performed. The system configuration of the computer for performing this screen display will be described with reference to the configuration block diagram shown in FIG. The system stores an input unit 141 that captures parameter data input from the keyboard 39, a calculation formula necessary for calculating migration characteristics, and fixed parameters required for the calculation formula (channel length, buffer liquid conductivity, etc.). The calculation formula / fixed parameter storage unit 142, the graphic screen file storage unit 143 for storing the graphic screen file corresponding to the microchip shape (there is a file corresponding to each microchip shape), and the input unit 141 An electric characteristic calculation unit 144 that calculates other electric characteristic parameters such as a potential gradient based on the calculated parameter data and the calculation formula stored in the calculation formula / fixed parameter storage unit 142, and a parameter that is input to the input unit 141 Other electrical characteristics parameters calculated by the data and electrical characteristics calculation unit 144 Having a screen combining unit 145 for combining the data on one screen, a display unit 146 for displaying the synthesized screen on the display device 40. Here, the screen synthesis unit 145 functions as an input screen before data of other electrical characteristic parameters is sent from the electrical characteristic calculation unit, and after that is sent, a screen for displaying the electrophoretic characteristics by adding the data. Function as. Next, a method for calculating electrical characteristic parameters will be described with reference to FIG. Each port 103 in FIG.
The electrical resistance of each flow path from S, 103W, 103B, 103D to the intersection 106 is R1, R2, R3, R
4 (actually, it means the electric resistance of each channel on the microchip shown in FIG. 1D corresponding to FIG. 4). Here, R1 to R4 represent the cross-sectional area of the flow path as S (m ² ), the length of the flow path as L1 to L4 (m), and the specific resistance of the buffer solution as U (Ω
m), R1 = L1 * U / S (Ω) can be obtained, and R2, R3, and R4 are obtained in the same manner. S, L1 to L4, and U are numerical values stored in advance in a computer memory as known fixed parameters. Assuming that the center voltage at the intersection 106 is V0 (V) and the voltages at the respective ports are HV1, HV2, HV3, and HV4, the following relationship is established according to Kirchhoff's current law. (HV1-V0) / R1 + (HV2-V0) / R2 + (HV3-V0) / R3 + (HV4-V0) / R4 = 0 (1) From equation (1), the voltage at the intersection 106 is It becomes like this. V0 = (R1 // R2 // R3 // R4) * (HV1 / R1 + HV2 / R2 + HV3 / R3 + HV4 / R4) (2) where (R1 // R2 // R3 // R4) is Resistance R1
It means the parallel connection resistance value of R4. The potential gradients G1, G2, G3, and G4 of the respective channels can be obtained by the following calculation formula using (2). G1 = (HV1-V0) / L1 (V / m) (3) G2 = (HV2-V0) / L2 (V / m) (4) G3 = (HV3-V0) / L3 ( V / m) (5) G4 = (HV4-V0) / L4 (V / m) (6) Next, the operation performed by the analyst in this embodiment is shown in FIG. This will be described with reference to a flow diagram. First,
The type of microchip to be used is input (201). When the analyst wishes to set the applied voltage while confirming the value of the potential gradient generated in each flow path, “potential gradient display” (202) is input to the display mode input unit 101 on the screen. Then, the graphic screen corresponding to the microchip is selected and displayed, and the input screen shown in FIG. 4 is displayed (203). In order to prompt the input of the voltage value on this input screen, the voltage input units 111 to 114 displayed on the window frame.
A suitable voltage value (electrical characteristic parameter) is input as a numerical value (204). It is checked whether all four voltage values have been input (205), and if not, a warning display prompting input is made (206). When the input of all the voltage values necessary for the calculation is completed, a potential gradient that is another electrical characteristic parameter is calculated using equations (2) to (6) (206). Then, the input voltage value and the calculated potential gradient value are combined on one graphic screen (207) and displayed on the display as voltages 111 to 114, potential gradients 121 to 124, and potential directions 121A to 124A. (208). The analyst can intuitively and easily know whether the input voltage value is appropriate by checking the potential gradient and the potential direction as information other than the voltage value. For example, the potential gradient is directly related to the migration speed, and if the speed is too fast or too slow, problems such as insufficient separation or sample diffusion occur, and sufficient measurement cannot be performed. Therefore, it is determined whether the migration speed, that is, the calculated potential gradient is an appropriate value, and if it is determined that it is not appropriate, the applied voltage value is changed (209). Then, since the potential gradient at each part of the flow path changes, it is determined again whether the potential gradient after the change is appropriate. Thereafter, this operation is repeated (204 to 209) to obtain the optimum four voltage value pairs. . When a set of four voltage values is determined in this way, it is stored in the memory of the computer (21
0) Perform electrophoresis at the determined voltage value. By the way, in the case of using the microchip having the cross-shaped electrophoresis channel as described above, the sample is at the intersection 1.
When reaching 06, the migration direction is switched from the short flow path to the long flow path. That is, in a first analysis, the sample changes from the port 103S to the port 103W in the first state (sample injection), and switches when the sample reaches the intersection 106, and the port 103B to the port 103D.
It is necessary to sequentially execute three states: a second state (electrophoretic separation) toward the direction of (3), followed by a third state (electrophoresis stop) in which the electrophoresis is stopped for detection. Therefore, each voltage from the first state to the third state must be determined by the procedure described above and stored in the memory of the computer. FIGS. 8A, 8B, and 8C are examples in which optimum conditions for voltage values in each of the above-described states were designed using a microchip having electrophoresis channels intersecting in a cross (optimum conditions here). This means the optimum condition for performing analysis in which priority is given to sharpening separation by electrophoresis rather than increasing the signal intensity, and it goes without saying that the optimum condition varies depending on the purpose of the analysis). FIG. 8 (a)
In the sample injection step shown in FIG. 5, a potential gradient from 103S to 103W is applied so that the sample moves from the port 103S, which is the sample introduction channel (shorter channel), toward the port 103W. At that time, a weak potential gradient toward the intersection 106 is created in the separation channel so that the sample does not diffuse into the channel from the ports 103B to 103D, which is the separation channel (longer channel). Stop. Based on the above consideration, the voltage to be applied was determined as shown in FIG. At this time, the amount of the buffer solution flowing into the intersection 106 needs to be equal to the amount of the buffer solution flowing out of the intersection 106. However, if the width of each flow path is the same, the value of the potential gradient is proportional to the migration amount. Since there is a relationship close to proportionality, it can be grasped by checking the potential gradient for sample injection and diffusion prevention. Subsequently, in the electrophoresis / separation step shown in FIG. 8B, the sample existing at the intersection 106 is introduced downstream of the separation channel (between the intersection 106 and the port 103D). Apply a potential gradient. Further, a potential gradient toward the intersection is also applied to the upstream side of the separation channel (between the port 103B and the intersection 106) in order to supply the buffer solution. Furthermore, the sample introduction channel (end 1
From 03S to the intersection 106, the intersection 106 and the end 10
In order to prevent the sample remaining between (3 W) from flowing into the separation channel, a potential gradient is applied to these channels in the direction of separating the sample from the intersection. In this case, since the sample flows in three directions, the amount of sample flowing downstream of the separation channel is reduced and the signal intensity is weakened, but the separation characteristics by electrophoresis can be sharpened. Based on the above consideration, the voltage applied to each port was determined as shown in FIG. Subsequently, when the electrophoresis is stopped as shown in FIG. 8C, when the separation is completed, the movement is stopped by setting all potential gradients to zero, and the separation state is detected by the detector. In this embodiment, the voltage value generated at the intersection 106 is also calculated and displayed at the same time. By displaying the voltage value at the position of the intersection 106, it is easy to determine how much to increase or decrease when inputting the voltage value to be applied to each port by trial and error. FIG. 9 shows another embodiment of the present invention. The potential gradient is displayed on the previous screen, but in this example, the electrophoretic current is displayed. Since the electrophoretic current is also related to the electrophoretic velocity in the same manner as the potential gradient, the same effect can be obtained by displaying this. In this case, the electrophoretic current can be obtained by designating “channel current display” in the mode input unit 124. The electrophoretic currents I1, I2, I3, and I4 flowing through the respective channels can be obtained from the above-described expression (2) and the following relational expression. I1 = (HV1-V0) / R1 (A) (7) I2 = (HV2-V0) / R2 (A) (8) I3 = (HV3-V0) / R3 (A) (9) I4 = (HV4-V0) / R4 (A) (10) In the above embodiments, voltage values are inputted, and thereby the state of migration such as potential gradient and migration current is grasped. In contrast, other parameters are calculated, but the potential gradient value of each channel is directly input, and the applied voltage value to each port that satisfies the conditions is calculated and displayed. May be. In this case, 121 to 124 are displayed in a window frame and become a potential gradient input unit, and 111 to 114 are display units for the calculated results. In this case, it is necessary to determine the voltage value at any one place, but if the lowest voltage value of the four ports is 0 V, the other three voltages are also uniquely determined. be able to. That is, the voltage values HV1 to HV4 of each port are obtained from the following equations using the intersection voltage V0, the potential gradients G1 to G4, and the flow path lengths L1 to L4. HV1 = V0 + G1 (11) HV2 = V0 + G2 (12) HV3 = V0 + G3 (13) HV4 = V0 + G4 (14) Here, the lowest voltage among HV1 to HV4. Let Vx be Vx with Vx = 0. V obtained
By substituting 0 into the remaining three equations, four voltages V1 to V4 (any one of which becomes zero potential) can be determined. Further, as shown in FIG. 10, all of the voltages 111 to 114, potential gradients 121B to 124B, and potential directions 121C to 124C of the respective ports are formed into a window frame shape and can be solved by the above formula. By inputting an arbitrary set of voltage / potential gradients, the voltage value and potential gradient value of the remaining places where no input is made may be calculated and displayed. Embodiments of the present invention are listed below. (1) The microchip electrophoresis apparatus according to (1), comprising at least two or more electrophoresis channels intersecting each other. (2) The microchip electrophoresis apparatus according to claim 1 or 2, wherein the voltage is applied to the electrophoresis channel from at least four or more locations. (3) The input electrical characteristic parameter is a voltage applied to the migration flow path, and the calculated electrical characteristic parameter is a potential gradient or a migration current generated in the migration flow path. Item 4. The microchip electrophoresis apparatus according to Item 3. (4) The input electrical characteristic parameter is a potential gradient or electrophoretic current generated in the migration channel, and the calculated electrical characteristic parameter is a voltage applied to the migration channel. Item 4. The microchip electrophoresis apparatus according to Item 3. (5) The microtic electrophoresis apparatus according to claim 2, wherein the voltage value at the position of the intersection of the electrophoresis channel is also displayed. (6) The microtic electrophoresis apparatus according to any one of claims 1 to 3, wherein the screen synthesized by the screen synthesis means is a graphic screen. As described above, in the present invention, the potential gradient and the electrophoretic current, which are information necessary for grasping the electrophoretic state, are simultaneously displayed on the screen. However, the optimum voltage value can be set easily.
In particular, in electrophoresis using a microchip having a flow path that crosses a cross, parameter settings in a plurality of steps must be optimized. However, if a simulation according to the present invention is performed, an optimum design can be easily performed.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a configuration of a microchip substrate used in a microchip electrophoresis apparatus. FIG. 2 is a diagram showing a hardware configuration of a microchip electrophoresis apparatus used in the present invention. FIG. 3 is a diagram showing another hardware configuration of the microchip electrophoresis apparatus used in the present invention. FIG. 4 is a diagram showing a screen for displaying a migration state of a microchip electrophoresis apparatus that is an embodiment of the present invention. FIG. 5 is a configuration block diagram for displaying the screen. FIG. 6 is a graph showing the relationship of electrical characteristics on a microchip calculated by the present invention. FIG. 7 is a flowchart for setting parameters on a screen displayed according to the present invention. FIG. 8 is a diagram showing an example in which optimum condition design is performed. FIG. 9 is a view showing a screen for displaying a migration state of a microchip electrophoresis apparatus according to another embodiment of the present invention. FIG. 10 is a diagram showing a screen for displaying a migration state of a microchip electrophoresis apparatus that is another embodiment of the present invention. [Explanation of Symbols] 10: Microchip 3S, 3W, 3B, 3D: Port 4: Electrophoresis channel (sample introduction side channel) 5: Electrophoresis channel (sample separation side channel) 6: Intersection 32: CPU 34- 37: High voltage power supply 38: Computer 39: Keyboard 40: Display 103S, 103W, 103B, 103D: Port 104: Migration channel (sample introduction side channel) 105: Migration channel (sample separation side channel) 106: Intersection 111 to 114: voltage input units 121 to 124: potential gradient display units 121A to 124A: potential direction display units 121B to 124B: potential gradient input units 121C to 124C: potential direction input unit 126: intersection voltage display unit 126B: intersection voltage input Part

Claims (1)

(57) [Claims] [Claim 1] An electrophoresis channel is formed on a microchip, a sample is introduced from an opening provided on the electrophoresis channel, and a voltage is applied to the electrophoresis channel to electrically In the microchip electrophoresis apparatus for performing electrophoresis, the microchip electrophoresis apparatus has a control unit having a screen for inputting an applied voltage value or / and a parameter value for grasping a migration state, and an applied voltage from the screen, When the necessary electrical characteristic parameter value of the parameters for grasping the electrophoresis state is input, the applied voltage and the parameter for grasping the electrophoresis state are not yet determined based on the input electrical characteristic parameter value. The electric characteristic calculation means for calculating a certain other electric characteristic parameter value, and the calculated electric characteristic parameter value and the inputted electric characteristic parameter value are simultaneously displayed. Microchip electrophoresis apparatus characterized by comprising a screen combining means for displaying on.