WO2000014527A1 - Electrophoresis system - Google Patents

Abstract

An electrophoresis system which uses a plurality of sensors disposed along a migration path in detecting signals that have conventionally been detected by one sensor and which can be formed into a microchip in its entirety by integrating a rectangular capillary tube constituting the migration path with an optical sensor array, thereby enabling a low-cost, fine device to monitor separate conditions concurrently and to analyze an electrophoresis quickly.

Description

 Description i Electrophoresis system Technical field

 The present invention relates to a system for performing an electrophoretic analysis. In particular, the present invention relates to an electrophoresis system in which an electrophoresis unit and a detection mechanism for mechanically reading an electrophoresis result are combined. Background art

 Electrophoretic analysis is a basic analytical operation indispensable for analyzing and separating biological components such as proteins and nucleic acids. Electrophoretic analysis applies the phenomenon that a substance to be analyzed moves in an electric field based on the difference in charge state. By manipulating the charge state under various environmental conditions, it is possible to know not only applications such as detection and quantification of substances, but also physical properties such as molecular weight and isoelectric point. Alternatively, it can be used as a means for collecting a target substance for use as a sample for another analysis operation.

Among electrophoretic analyzes, the most popular new analysis technique is capillaries. A capillary is generally used as a capillary made of silica and having an inner diameter of 10 to 100 10m and a length of 10 to 100 cm. The medium is filled with a gel or various liquids as a medium. Figure 3 shows the principle of capillary electrophoresis and the relationship between the signal detected by one sensor (or detector) and time. In electrophoretic analysis, the effect of convection in the electrophoresis medium due to the heat generated by energization (Joule heat) has a significant effect on the separation results. The heat generated by energization can be a fatal limitation of electrophoretic analysis. For this reason, gel media has been used in conventional electrophoretic analysis to prevent convection. However, by utilizing the special conditions of the inside of the capillaries, the heat can be dissipated effectively. Also, compared to a plane where it is difficult to maintain a uniform temperature, Temperature control can be performed easily with cabillaries. Taking advantage of these advantages, capillary electrophoresis has become widespread as an inexpensive, high-precision analyzer that does not necessarily require a gel medium, has excellent resolving power, and has excellent resolution.

 Especially in the analysis of isoelectric point, electrophoretic electrophoresis can be performed in liquid, so electrophoresis time has been greatly reduced. In conventional gel electrophoresis, it took a long time for the protein to converge to the isoelectric point in the gel medium. In this way, capillary electrophoresis has become very popular, supported by many advantages, but some problems remain. In the known capillary electrophoresis system, the detection of the analyte was performed at one point. For example, in the case of protein detection, it is common that only one optical system for measuring absorption at 280 nm is attached near the end of the migration path. In such a configuration, it goes without saying that the component to be analyzed cannot be detected unless it is migrated to the position where the sensor is disposed. Although the electrophoresis time of capillary electrophoresis is short, there is a case where rapid processing becomes difficult in a system that always requires electrophoresis to a part of the sensor. Alternatively, depending on the charged state of the analyte, there may be cases where the sensor does not swim to a part of the sensor.

 One characteristic of capillary electrophoresis is the presence of electroosmotic flow. Normally, the inner wall of the capillary is negatively charged, attracting the cations in the running buffer. When a voltage is applied to the capillary, the cations move to the cathode. At this time, since the cations move while dragging water ions, the entire solution flows to the cathode. This flow is called an electroosmotic flow, which is an offset flow that does not depend on the sample, and can be controlled to some extent by the pH of the buffer and the coating of the inner wall. Positively charged samples migrate faster with the same beads, and negatively charged samples migrate slower.

Thus, in capillary electrophoresis, positive, negative, and neutral samples can be separated at once by using the offset flow called electroosmotic flow. However However, when the negatively charged sample is I>>, the sample moves to the anode side and does not reach the sensor (Le _B is the speed of the negatively charged substance, Le _e . _F is the speed of the electroosmotic flow) . The only way to solve this problem is to provide an electroosmotic flow that can move even the sample with the highest I to the cathode side, but this is not always possible. Even if this was possible, a positively charged sample would reach the sensor in a short time if fast electroosmotic flow was used, making it impossible to separate two samples with similar mobility. there is a possibility. As one expression of the degree of separation, the relationship shown in Equation 1 is known.

 (Equation 1)

Here, N is the number of theoretical plates, Δ is the difference between the moving speeds of the two samples, and So is the average value of the moving speeds of the two samples. From this, it can be seen that to increase Rs as much as possible, it is necessary to decrease the value. However, increasing the electroosmotic flow increases the value, and as a result, Rs decreases, which is not preferable.

One of the major features of capillary electrophoresis is that isoelectric focusing in a liquid medium can be performed quickly. However, since the protein converged to the isoelectric point cannot be detected by a sensor fixed at one point, an operation for electrophoretically moving the protein to the sensor has conventionally been required. Also, in known capillary electrophoresis, a sample can be supplied only from the end of the migration path, so if a protein that converges to the opposite end in the event of isoelectric focusing becomes an analyte, Requires a long running time. In cases where the isoelectric point cannot be predicted, it is a reasonable approach to start electrophoresis near the center, but supply of the sample to the center is inexpensive due to the characteristics of the capillaries. It is difficult to configure the mechanism.

 In addition, although the use of cavities has facilitated heat dissipation and temperature control, the current size is close to the limit due to restrictions on the production of cavities. Also, in terms of operability, a thinner than the current situation is likely to break, which is not practical. Therefore, it can be seen that the limit has been reached in terms of shortening the electrophoresis time due to miniaturization and high-sensitivity analysis using a smaller amount of sample.

 In addition to capillary electrophoresis, new electrophoretic analysis methods have been proposed. For example, an electrophoresis analysis using a microchannel formed on a flat substrate as an electrophoresis path was attempted. Glass and polymers are often used for microchannels. In this method, since a microchannel is used, a more minute analysis system than a capillary can be constructed by combining semiconductor chips (Science, Vol.261, PP895-897, 1993; Proc. Natl. Acad. Sci. USA, Vol.91, pp.11348-11352, 1994; Anal.Chem., Vol.69, pp.3451-3457, 1997) However, signal detection is still performed at one point, and therefore, capillary electrophoresis. It leaves the same issues as the system. As a very small system, there is known a system in which a detection IC is formed using a semiconductor process and a plastic flow path is formed thereon (International Conference oc Microelectromechanical Systems -MEMS96-, pp.491-496, 1996; International Conference on Solid -State Sensors and Actuators -Transducer97-, pp.503-506, 1997). As a technique for forming a plastic flow channel, application of a new technology such as stereolithography (BME, Vol. 12, No. 4, pp. 66-75, 1998) has also been reported. However, the principle of single point detection at the outlet of the flow channel has been adopted as the conventional detection system.

As an analysis method that requires two-dimensional analysis of electrophoresis results, such as DNA base sequence determination, a method of analyzing electrophoresis patterns by scanning cavities is known. However, such a method requires a high-precision optical system for scanning with a minute light beam. In addition, it becomes a complicated system consisting of lenses and prisms, and it is difficult to supply inexpensive devices commercially. More swimming An electrophoresis system that performs detection at a plurality of positions along a moving path is also known (JP-A-10-132783). In the system disclosed in this prior art, a certain range of signals must be integrated in order to achieve a high S / N ratio while performing optical measurement with a short optical path length. Therefore, it is necessary to stop the power supply after the electrophoresis and perform signal measurement and data analysis, which makes it impossible to perform measurement during electrophoresis. In addition, optical components such as lenses are indispensable, and it is difficult to realize an inexpensive system with a complicated structure. Disclosure of the invention

 An object of the present invention is to provide a signal detection principle that enables more rational and rapid separation while taking advantage of the characteristics of the capillary electrophoresis such as rapidity and high accuracy. Specifically, it is an object of the present invention to provide a novel system and analysis method for improving electrophoretic analysis, which had been subjected to various restrictions due to the arrangement of sensors, based on a more rational detection principle. It is.

 A second object of the present invention is to provide an inexpensive electrophoresis system that can be more easily reduced in size than the currently widely used capillary electrophoresis system.

 The present inventors focused on the arrangement of the sensor and the physical shape of the capillary as factors that limit the possibility of electrophoretic analysis, and conducted research on a new configuration. As a result, they have found that a rational detection system can be configured by providing sensors at a plurality of positions corresponding to the migration path, and completed the present invention.

 Furthermore, the present invention provides an electrophoresis system having a very fine structure using current processing technology by using an optical sensor array in which fine sensors are arranged in a matrix in a plane as a plurality of sensors. I found what I could do. That is, the present invention relates to the following electrophoresis system and electrophoresis analysis method.

[1] An electrophoresis system including at least the following components, wherein a signal can be detected by a sensor during electrophoresis. a) an electrophoresis unit for separating an analyte by electrophoresis; andb) a plurality of sensors combined with the electrophoresis unit and arranged on a migration path of the analyte to detect a signal of the substance. ,

 [2] The electrophoresis system according to [1], wherein the sensor is an optical sensor in which a plurality of detection elements are arranged on a plane.

 [3] The electrophoresis system according to [2], wherein the optical sensor in which a plurality of detection elements are arranged on a plane is integrated with the electrophoresis unit.

 [4] The electrophoresis system according to any one of [1] to [3], wherein migration paths in the electrophoresis unit are arranged in parallel.

 [5] The electrophoresis system according to [4], wherein the migration paths arranged in parallel have a single continuous capillary structure.

 (6) The detection element according to (4) or (5), wherein the detection element constituting the sensor detects a signal from one migration path and is not affected by a signal of an adjacent migration path arranged in parallel. Electrophoresis system.

 [7] The electrophoresis system according to [1], wherein an analysis target sample application site is provided at an arbitrary part of the migration path.

 [8] The electrophoresis system according to [7], wherein an analysis target sample application site is provided in an intermediate portion of a migration path of the electrophoresis unit.

 [9] An electrophoretic analysis method including at least the following steps a) to c).

a) Perform electrophoresis of the analyte

b) The signals of the substance are detected at a plurality of positions in parallel with the electrophoretic separation by a plurality of sensors arranged at positions corresponding to a path in which the analyte is migrated by the electrophoretic separation operation.

c) determining the physicochemical properties of the substance based on the signals detected at the plurality of positions and the position information at which the signals are detected.

In the present invention, the electrophoresis system refers to an electrophoresis unit and an electrophoresis path. And a sensor for detecting the signal. The electrophoresis unit according to the present invention includes an electrophoresis path and an energization mechanism necessary for electrophoresis. As these components, those used in known electrophoresis systems can be applied as they are. For example, an electrophoresis unit using a microchannel is an advantageous embodiment of the present invention. The electrophoresis unit has a structure in which fine grooves are arranged on a substrate and the fine grooves are covered as necessary. The liquid flows through the grooves by capillary action. Both ends of the groove are electrodes, and electrophoresis is started by connecting to a power supply. The arrangement of the grooves is arbitrary, but it is advantageous to form the grooves in a rectangular shape, since a long migration path can be formed with a small area. For example, an effective migration length of 6 cm can be achieved by making the migration path with a width of 0.1 strokes into a rectangular shape folded at 0.35 rotation intervals within the range of 5 thighs. Alternatively, a plurality of migration paths can be arranged in parallel. If the components of the electrophoresis unit are made of a material through which the signal of the substance to be analyzed can pass, the state of the electrophoresis can be detected from outside the electrophoresis unit.

The microchannel has a structure in which a groove (flow path) with a cross section of about several hundreds of zm is provided on the substrate surface, and the groove is sealed with a suitable material. Etching is used to form the grooves (channels). Glass was used as the substrate material. Glass has excellent insulation and heat dissipation properties, but it has disadvantages such as the use of glass or silicon, which requires processing at high heat, to seal the grooves (channels). Recently, attempts have been made to mold microchannels based on structure by using materials such as silicone elastomers instead of glass. Materials include polydimethylsiloxane (hereinafter abbreviated as PDMS, Anal. Chem., Vol. 69, ρ 345 3457, 1997), acryl-based resin 11 & 1.6111. 01.69.2626, 1997), polymethyl Methacrylate (hereinafter abbreviated as PMMA, Anal. Chem., Vol. 69, pp. 4783, 1997) or the like is used. First, a mold to be used for the mask is prepared by etching the silicon wafer. A polymer in solution is poured into this to transfer the structure and solidify the polymer. General flow path Each of these can be several tens of meters in depth, 10-100〃ηι in width, and the effective analysis length can be several tens of cm. If PDMS is used as a material, the channel can be easily sealed by natural adsorption to glass or a PDMS substrate. The temperature required for sealing is 105 ° C for bonding with glass and 108 ° C for PDMS, much lower than the bonding temperature required for glass substrates. It has already been confirmed that electrophoretic separation of DNA fragments and the like is possible within the microchannels created in this way. Microchannels using polymers are advantageous in terms of cost because they can be easily mass-produced (Bunseki August 1998, p.608-609). Also, the depth of the microchannel can be made larger than glass that is difficult to mold by etching.

 A technique for forming a flow path by etching a photosensitive glass in addition to the polymer is also known. Photosensitive glass is silicate glass in which metal ions are added and dissolved together with a sensitizer. Exposure to ultraviolet light causes heat development to generate metal colloids, and crystals grow using the colloids as nuclei. Since these crystals are easily dissolved in an acid, etching can be performed by the acid treatment. A taper can be provided in the cross section of the flow path, or a fine and complicated shape can be accommodated in a plane. Microchannels may be created using this processing technology.

In addition, micro channels can be formed by stereolithography using a photocurable resin. According to the stereolithography technology, the migration path can be formed directly on the sensor. Stereolithography is a technology that assumes a shape in which the target structure is sliced into multiple layers, forms each layer by irradiating light, and sequentially stacks the layers to complete the entire structure. The process of irradiating the light along the sliced shape can be automated by the combination control. Laser lithography involves irradiating a laser beam from above the resin tank and stacking the structure on a vertically movable stage, or irradiating the laser beam from below the resin tank. There is a regulated liquid surface method in which each slice layer is formed and laminated between the quartz glass plate attached to the bottom and the stage. Only However, in these general stereolithography methods, the resin surface is in an unregulated state when forming the inner surface of the channel, which is a hollow part, and a rough, low-transparency surface that is unsuitable for optical measurement is formed. . However, in the regulated liquid level method, in addition to a stage that can be moved in the vertical direction, a regulating plate for controlling the liquid level during the production of the hollow part is used to produce a smooth inner surface of the flow channel with excellent transparency. can do. Stereolithography can freely form shapes that are impossible in principle with conventional molding techniques such as injection molding and etching. Furthermore, according to the stereolithography technology, the distance between the sensor and the migration path can be easily reduced.

 Specifically, for example, when an ultraviolet curable acryl resin is irradiated with an ultraviolet laser to form a migration path, the distance between the sensor and the migration path can be made as close as possible. Shorter distances result in sharper images and higher sensitivity. In the embodiment, the distance between the sensor and the migration path is 0.5 Fiber by the stereolithography technique. Moreover, this is not a limitation in molding technology, but a value set based on the positional relationship with other parts. Therefore, in principle, it is possible to further shorten the distance between the sensor and the migration path.

The depth of the microchannel is an important factor in determining the optical path length. By using a polymer as a material, an optical path length of 50-100 / m can be easily realized. If the optical path length is shorter than this range, sufficient signal intensity cannot be expected especially in the case of absorbance measurement. On the other hand, when the diameter is 100 m or more, the separation ability may be reduced in some cases due to an increase in the depth of the migration path. Therefore, the optical path length of 50-100 / m, especially around 70 μm is a desirable optical path length particularly when the absorbance is used as a signal. In most reports on electrophoresis using microchannels on a flat substrate, detection is performed by fluorescence measurement using an excitation laser and a photomultiplier tube. In studies of electrophoresis using microchannels on known flat substrates, absorption measurements are difficult because the channels are not so deep (thus increasing the detection limit concentration C). On the other hand, in a desirable mode of the present invention, The use of microchannels with a depth of about 70Adm makes it possible to provide electrophoresis systems with practical sensitivity. In a commercially available device, absorption measurement is performed using a cavity with an inner diameter of about 70 mm. The effect of the optical path length on the absorbance measurement is described below in detail.

 First, absorbance A is defined as in Equation 2.

 (Equation 2)

A-sCl— ln ^Iout

I in where i _in , and i. _ut is the amount of light incident on and out of the sample, respectively, £ is the molar extinction coefficient, C is the molar concentration, and 1 is the optical path length. When the absorbance is low, the ratio of the amount of sample absorbed to the amount of incident light can be described by Equation 3.

 (Equation 3)

in out mm (exp (-A)) in 1 "1 A ^ eCl mm in The detection limit is determined by the resolution of the detector. For example, when using a detector with IV of maximum amplitude and lmV of minimum resolution a = £ C1 = 1 (T 3 is the detection limit for (fully feasible). If £ = lxl0 ⁴ / Mcm, 1 detection limit concentration when a two 70〃M is be found from equation 4 it can.

(Equation 4)

Depending on the molar extinction coefficient and the inside diameter of the capillary, this performance is the detection limit of a commercially available device. There is no inferiority to that, and it is a performance that can be applied practically enough. However, since deep etching of glass takes time, the optical path length is generally set to about 10 m. With such a short optical path length, the amount of optical absorption is very small and absorbance measurement is difficult, and sufficient signal intensity cannot be obtained unless fluorescence or luminescence measurement is performed.

 Furthermore, by using a polymer as a material, the surface that blocks the optical path can be made flat. When a polymer is used as a material, for example, the cross section of the migration path is usually rectangular, and the plane that necessarily blocks the optical path is necessarily flat. Therefore, problems such as light scattering caused by circular cavities are reduced, and improvement in detection capability can be expected.

 According to a particularly preferred embodiment of the present invention, as shown in the examples, the microchannel, which is an electrophoresis unit, is placed in close proximity directly above the optical sensor array, thereby collecting light. Optical system is not required. Although commercially available equipment is provided with a detection window (about 0.2 to 0.8 cm), the size of the sensor itself corresponds to this detection window when the sensor is arranged along the flow path. This is about several tens of micrometers, but as can be seen from the above equation, the detection resolution is determined by the optical path length and does not depend on the horizontal spread, so signals are detected while performing electrophoresis in an array configuration. There is no problem in particular. In the present invention, conditions under which the optical system can be eliminated will be described below.

Particularly in a migration path having a structure in which migration paths are arranged in parallel as shown in the examples, high-precision analysis can be performed unless optical signals from adjacent electrophoresis paths are picked up. I can't achieve it. That is, a configuration is required in which one detection element detects a signal from one migration path. It is also necessary to avoid the effect of light passing between the migration paths. In such a case, an optical system for condensing light is usually adopted, but in the present invention, the optical system can be omitted by bringing the detection element and the migration path close to each other. In the present invention, the distance between the detection element and the migration path is usually 1.5 band or less, preferably from 0.1 to 1.0, and more preferably from 0.1 to 0.5. By setting mm, highly accurate analysis results can be expected. By bringing it closer to 0.1 mm or less, a more optically desirable state can be obtained, but insulation breakdown may occur depending on the material constituting the migration path.

 The problems in optical measurement when the migration paths are arranged in parallel are roughly divided into diffraction and refraction. Light passing through the aperture spreads due to the Fresnel diffraction phenomenon. In other words, the light that has passed through the migration path spreads before reaching the detection element. Light passing through a medium having a different refractive index such as liquid / resin / air is refracted at each interface. If the detector surface is not perpendicular to the light, it can lead to a risk of reacting to signals that should not be picked up. Therefore, if the migration path and the detection element are as close as possible and the size of the detection element is reduced, the configuration is such that the signal from the adjacent migration path is not affected and only the signal of one migration path is picked up. can do. That is, it is a desirable embodiment of the present invention that the plurality of sensing elements arranged at a plurality of locations on the migration path make each detection element correspond to a signal from one migration path. It is meaningless to indicate a general value because the refractive index differs depending on the material constituting the migration path.However, for example, a structure in which the detection element is in close contact with the electrophoresis unit is ideal when the optical system is omitted. It can be called a structure. The size of the sensing element is also affected by various conditions, but the closer the sensing element is to the migration path, the more the sensing element can be used, the closer to the width of the migration path. Conversely, the further away from the migration path, the easier it is to pick up light that has been refracted or diffracted from portions other than the migration path, so it is desirable to reduce the size of the sensing element with respect to the width of the migration path. If a charge integration type sensor is used as the detection element, there is no danger that a reduction in area will lead to a decrease in sensitivity.

The spread of light due to diffraction can be predicted by a Fresnel approximation. Refraction can be calculated from the refractive index of the medium through which the light passes and the angle of incidence of the light. From these numerical values, if conditions such as the distance between the migration path and the detection element or the size of the detection element are appropriately adjusted, it is sufficiently practical even without an optical system. It is possible to achieve sensitivity and accuracy at an appropriate level.

 By the way, when a sensor having a large number of sensing elements arranged in a plane, such as an optical sensor array, is used, it is not necessary that all the sensing elements constituting the sensor correspond to the migration path. In such an embodiment, by selectively collecting only the signals of the detection elements corresponding to the migration path, an ideal detection mechanism can be realized as a result.

 In the present invention, it is necessary to arrange a plurality of sensors for detecting a signal derived from the substance to be analyzed along the migration path. Moreover, the sensor constituting the present invention must be capable of detecting a signal during electrophoresis. In the present invention, arranging along an electrophoresis path means that a signal of the substance is transferred at a plurality of positions to a region in the electrophoresis unit where an analyte may move by electrophoresis. This means that the sensors are arranged so that they can be detected. The region where the substance to be analyzed may move is, for example, the entire migration path, but in the present invention, it is not necessary to arrange the sensor so as to cover the entire migration path. At least a plurality of sensors may be arranged at arbitrary positions. However, as will be described later, a configuration in which a sensor element can be arranged corresponding to the entire migration path, such as an optical sensor array composed of densely arranged sensor elements, is advantageous in various points. It is.

Further, in the present invention, a sensor capable of detecting a signal during electrophoresis means a sensor capable of detecting a signal at an arbitrary timing and at an arbitrary place even during electrophoresis. Any kind of signal can be used as long as it serves as an indicator of the substance to be analyzed. Specifically, optical signals are generally used, but in some cases, radioactivity or magnetic activity can be used as an index. Optical signals include such things as absorbance at a particular wavelength, refractive index, fluorescence and luminescence. Sensors for detecting these signals are known. For example, to detect optical signals, photodiodes and phototransistors Available. In order to arrange a plurality of sensors along the migration path, it is preferable to assemble a plurality of sensors arranged in advance in the electrophoresis unit. In order to realize this configuration, it is preferable to use an optical sensor array. An optical sensor array is an electronic component that has a structure in which a sensor element (such as a phototransistor) that can extract changes in optical signals as changes in electrical signals is two-dimensionally arranged. . Since each sensor element detects a signal independently, by comparing the obtained signal with its position information, it is possible to know at which position the signal was detected. In the present invention, the correspondence between the signal intensity during electrophoresis that can be obtained by the present invention and the position information at which the signal intensity is detected is referred to as an image in the present invention. The image can be further superimposed with information on the transition of the signal intensity over time, and displayed on a graph, for example, as shown in FIG.

 If the sensor elements constituting the optical sensor array used in the present invention are made finer and integrated in a small area, the entire optical sensor array can be formed into a microchip.

Such electronic parts are commercially supplied as CCD (Charged Coupled Device), CMOS (Complementary Metal Oxide Semiconductor) image sensors, and the like. CCDs, also called semiconductor image sensors, are the mainstream image sensors used in video cameras. On the other hand, the CMOS image sensor discharges the parasitic capacitance for a certain period of time based on the output signal of the photodiode, and then reads it. It has features such as sensitivity adjustment, low power consumption, and integration not only for one sensor element but also for peripheral circuits, and is attracting attention as a new image sensor. From these devices, select as appropriate, taking into account conditions such as the signal to be detected, sensitivity, degree of integration, or size. In other words, the electrophoresis unit to be combined has a shape that allows the sensor element to be arranged at a position along the path where the analyte is migrated, and has the ability to detect signals originating from the analyte with appropriate sensitivity. It is sufficient to select an optical sensor array. For example, as shown in Fig. 1, When the formed microchannel is used for an electrophoresis unit, it is desirable to arrange one sensor element so that the microchannel can be observed with a certain resolution. At this time, if the sensor elements are more precisely arranged with respect to the microchannel, there is no need to strictly define the positional relationship between the two. This is because as long as the migration path is located in a certain area where one sensor element is arranged, it can be captured by any one of the sensor elements. By using a sensor array in which sensor elements are precisely arranged, a system that does not require strict alignment with the electrophoresis unit can be realized. Such a system is advantageous because the electrophoretic unit alone can be easily replaced.

 For example, when detecting a fluorescence signal, an optical sensor array with a phototransistor is used as one sensor element, and the excitation light is guided from a light source having a specific wavelength. The irradiation of the excitation light may be performed by irradiating the entire optical sensor array at the same time, or a method of scanning with a point-like or linear light source may be employed. Alternatively, if the absorbance at a specific wavelength is used as the signal, the light is also emitted at a specific wavelength, and the absorption is detected by a single sensor element.

 By using an optical sensor array, the relative positional relationship between the individual sensor elements can be kept strictly constant. This is because the sensor element on the optical sensor array is fixed on the substrate. Optical sensors and arrays are electronic components that can be mass-produced, so costs can be kept low. Therefore, it is economical to use many sensors.

In addition to the optical detection method, thermo-optical detection, electrochemical detection, or electrical conductivity detection can also be applied. Thermo-optical detection is applied to the measurement of the phenomenon that when a laser is applied to a sample component, the temperature near the component rises locally and the refractive index changes. The change in the refractive index can be detected by another laser. In electrochemical detection, electrolysis is caused using two electrodes, and the analyte is measured based on the oxidation current or reduction current. In electrical conductivity detection, Two electrodes are placed in the library, and the electrical resistance between the electrodes, that is, the electrical conductivity of the sample, is measured. If the sample component is ionic, the electrical conductivity is proportional to the ion concentration. In a preferred aspect of the present invention, the electrophoresis unit and a plurality of sensors are integrated. The term “integration” in the present invention means to fix the relative positional relationship between the two. In this case, depending on the combination of the signal to be detected and the sensor, it is generally advantageous to bring them as close as possible in terms of noise and sensitivity. In particular, when an optical signal is detected by an optical sensor array, a better S / N ratio can be obtained when the image information is configured based on the signal of the sensor element. The integration may be performed at least when the detection is performed by the sensor. Therefore, for example, it is also possible to adopt a configuration in which both are integrated so that they can be separated, and after electrophoresis, only the electrophoresis unit is replaced and a part of the sensor is repeatedly used.

Various functions required for electrophoretic analysis can be added to the electrophoresis system configured based on the present invention. For example, automation can be achieved by equipping the electrophoresis unit with a mechanism that supplies the sample. In order to perform the work of supplying a sample to a micro electrophoresis path such as a micro-channel, it is necessary to work under a microscope. This operation can be omitted by automation. As a means for supplying a sample to a microscopic migration path, for example, an electrophoretic mass transport technique can be employed. Fig. 9 shows an example of the sample supply means. In other words, an arbitrary part of the microchannel (electrophoresis path for separation) is used as an electrophoresis path to constitute a sample supply electrophoresis path. At the end of this electrophoresis path, an electrode for conducting electricity is connected to the other end while contacting the solution containing the sample. When electricity is applied in this state, the sample solution starts to move electrophoretically and is eventually guided into the microphone channel. When the required amount of sample solution has been transferred into the microchannel, electrophoresis for sample supply should be stopped and electrophoresis for separation on the microchannel side should be started. The sample introduction method based on this principle is Since sample introduction can be performed in an open system with all channels open (that is, no on-off valve is required), higher accuracy can be achieved than with a physical sample supply such as a pump.

 The sample supply means shown here can be provided at any part of the microchannel. In capillary electrophoresis, it was common to supply the sample to the ends of the capillary. However, in the present invention, for example, a sample supply means can be provided in an intermediate portion of the microchannel. Here, the middle part does not necessarily mean the true middle point of the microphone channel. Even if the position deviates from the true midpoint, it should be understood as an intermediate part unless it is at the end. By supplying the sample near the center and detecting the movement of the analyte with multiple sensors, the sensor can always detect whether the sample is positively or negatively charged. In addition, from the viewpoint of increasing the degree of separation, a sample is introduced from the center while the electroosmotic flow is suppressed as much as possible (that is, with the value of the equation (1) reduced), and separation is performed based on the movement of only the charge of the sample. The ideal analysis conditions can be realized.

 In order to actually analyze a substance using the electrophoresis system according to the present invention having the above-described configuration, a substance to be analyzed is introduced into the electrophoresis unit, and the signal is transferred along the migration path while performing electrophoresis. Tracking with sensors placed According to the present invention, it is possible to observe the output of the sensor while performing the electrophoresis, or to perform the sensing after the electrophoresis is completed if it is not necessary. In the present invention, various variations are made possible by detecting the substance to be analyzed at a plurality of positions with respect to the electrophoresis unit. The following is a specific description of an embodiment particularly advantageous for the known electrophoresis system.

One of the electrophoretic analyzes that can be particularly advantageously performed in the present invention is isoelectric focusing. FIG. 2 schematically shows a comparison between the conventional method of detecting a signal with a single sensor and the present invention using a plurality of sensors. Isoelectric point electricity Electrophoresis is an analytical method that utilizes the phenomenon in which protein is converged to a position corresponding to its charge in a pH gradient. Therefore, in principle, it is not possible to know where to detect the signal of the protein before performing electrophoresis. In a preferred embodiment of the present invention, monitoring can be performed at a plurality of positions on the migration path using an optical sensor array, so that a signal can be reliably detected regardless of where the migration is performed. Further, since the state of the electrophoresis can be monitored while performing the electrophoresis, it is an advantage of the present invention that the end of the electrophoresis can be quickly determined. In contrast, in conventional isoelectric focusing, it was necessary to take a sufficient electrophoresis time to ensure convergence to the isoelectric point. An advantage of applying the present invention to isoelectric focusing is that the electrophoresis start position can be freely selected. That is, when the above-described microchannel is employed in the electrophoresis unit, electrophoresis can be started from the center thereof. Since isoelectric focusing is performed on substances whose charge is unknown, it is very reasonable to start electrophoresis from the central point of electrolysis. The introduction method from near the center of the sample enables high resolution electrophoresis without limiting the charge of the component to be analyzed.

 The signals detected by the optical sensor array can be processed in any manner depending on the purpose of the analysis. For example, a substance is identified by comparing the mobility of a component to be analyzed in a microchannel with a standard sample, and a quantitative value can be obtained based on the signal intensity. When obtaining a quantitative value, reflect not only the signal intensity but also the area information where the signal was obtained. According to the present invention in which data of a plurality of sensors can be collected even during electrophoresis, it is easy to collect area information.

FIG. 1 shows a preferred embodiment of the present invention. Hereinafter, an embodiment of the present invention will be specifically described based on this diagram. FIG. 1 shows an electrophoresis system according to the present invention in which an optical sensor array (1) and an electrophoresis unit (2) using a microphone channel are combined. Both can fix the mutual positional relationship It can be integrated by means. On the electrophoresis unit (2), a single migration path (3) having a microphone opening channel is provided in a rectangular shape, and constitutes one continuous migration path. The individual optical sensor elements constituting the optical sensor array (1) are fixed on a substrate and output signals corresponding to the optical signals at the positions of the sensor elements independently of each other. In the optical sensor array, the optical sensor elements fixed to the surface are arranged along the turn-back interval of the migration path (3), and are arranged so that the optical signals can be detected at a plurality of sites. . On the other hand, the end of the electrophoresis path (3) is connected to a buffer solution tank (4), and an electrode (6) connected to a power supply (5) is immersed in the buffer solution tank (4). Further, a sample introduction means (7) for introducing a sample to be subjected to electrophoresis is connected to the migration path (3). The sample introduction means (7) itself constitutes an independent electrophoresis path, and the migration path overlaps a part of the migration path of the microphone channel. A sample solution is in contact with the end of the sample introduction means (7), and the sample solution is electrophoretically moved to a portion shared with the microchannel by energization. When the transfer of the required amount of sample is completed, the electrophoresis for supplying the sample is stopped, and the original electrophoretic analysis in the microchannel may be performed again. The movement of a series of sample solutions in the sample supply means using electrophoresis is as shown in FIG. In addition, such a liquid feeding system using electrophoresis can be applied not only to the introduction of the sample but also to the washing of the migration path. In the electrophoresis system according to the present invention shown in FIG. 1, the optical sensor array (1) can be integrated with the electrophoresis unit (2). Also, the separation state of the sample can be optically tracked during electrophoresis. By observing the state of the separation and judging that the electrophoretic separation has been completed, it is possible to use it immediately by stopping it. There is no need to perform long-term electrophoretic separations in order to ensure reliable separation. Or, it may prevent the trouble that some bands move to the end of the electrophoresis path and become inseparable due to excessive electrophoresis time. The electrophoresis system or the electrophoresis analysis method according to the present invention can be applied in a wide range as a general-purpose electrophoresis analysis technique. In particular, it is particularly effective when a large number of samples must be processed quickly and inexpensively. For example, the analysis of glucose in hemoglobin (HbAlc) in blood, which is a diagnostic indicator for diabetes, is expected to require a large number of samples for disease management and screening in an aging society. Analysis item. At present, liquid chromatography and electrophoresis analysis are required to analyze glycated hemoglobin with high accuracy.The electrophoresis system of the present invention, which can perform the analysis at low cost and quickly, is useful. It is. More specifically, a very small amount of blood is collected from a subject using a micro blood collection device and mixed with a hemolysis buffer to prepare a sample for electrophoretic analysis. The trace amount of blood may be collected not only by a special blood sampling device but also by a simple method such as filter paper blood sampling. The obtained hemolyzed sample is introduced into the microchannel of the electrophoresis system according to the present invention, which is filled with an appropriate buffer, by the above-described electrophoretic sample introduction method. This is electrophoresed and analyzed for saccharified mogrobin. The signal that tracks the analyte can be the hemoglobin absorbance. By electrophoresis, normal hemoglobin and glycated hemoglobin are separated, and the ratio between them can be determined. According to the present invention, since the microchannel can be replaced, the running cost can be reduced by replacing only the electrophoresis unit as needed and repeatedly using the sensor array portion. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a perspective view showing an embodiment of the integrated micro electrophoresis system according to the present invention. The electrophoresis unit (2) is integrated with the optical sensor array (1). FIG. 2 is a schematic diagram showing advantages that can be expected by combining a plurality of sensors according to the present invention.

FIG. 3 is a schematic diagram illustrating the principle of capillary electrophoresis. FIG. 4 is a photograph of the integrated microelectrophoresis system according to the present invention.

 FIG. 5 is a photograph of an electrophoresis unit constituting the integrated microelectrophoresis system according to the present invention and a sectional view thereof.

 FIG. 6 is an enlarged photograph of an optical sensor array unit constituting the integrated microelectrophoresis system according to the present invention and a sensor part thereof.

 Figure 7 is an image composed of the output signals of the optical sensor array when the electrophoresis unit was filled with borate buffer.

 FIG. 8 is a graph showing changes in the output signal of the integrated microelectrophoresis system according to the present invention when 0-25 mg / mL blue dextran was introduced. The vertical axis indicates (average output signal of the entire optical sensor array when filled with borate buffer) / (average output signal of the entire optical sensor array when filled with blue dextran). The horizontal axis indicates the concentration of blue dextran (mg / mL).

 FIG. 9 is a schematic diagram showing a procedure for electrophoretically introducing a sample into the integrated microelectrophoresis system according to the present invention.

 FIG. 10 is a diagram showing a temporal change of an image formed from output signals of the integrated micro-mouth electrophoresis system according to the present invention when blue dextran is electrophoresed.

 FIG. 11 is a diagram showing a configuration of an improved electrophoresis chip according to the present invention. It consists of a microchannel made of photosensitive glass and an optical sensor.

 FIG. 12 is a diagram showing a temporal change of an image formed from output signals of the improved micro-mouth electrophoresis system according to the present invention when blue dextran is electrophoresed.

 FIG. 13 is a diagram showing a configuration of an electrophoresis chip using a microchannel manufactured by stereolithography. (A) is a design drawing of the flow channel, and (b) is a cross-sectional view of the electrophoresis chip.

^ o

FIG. 14 is a photograph of an electrophoresis chip produced by stereolithography. (A) is the whole FIG. 2B is an enlarged view of the flow path portion, and FIG. 2C is an enlarged view of the sample injection portion.

 FIG. 15 is a diagram showing the results of electrophoresis of molecular weight markers.

 FIG. 16 is a diagram showing an electrophoresis pattern reconstructed along the flow channel. The horizontal axis represents the pixel position, and the vertical axis represents the absorbance.

 (Explanation of code)

 (1) Optical sensor array

 (2) Electrophoresis unit

 (3) Electrophoresis path

 (4) Buffer solution tank

 (5) Power supply

 (6) Electrode

 (7) Sample introduction method

 (8) Light source Best mode for carrying out the invention

 Hereinafter, the present invention will be described more specifically based on examples.

1. Creation of an electrophoresis analysis system according to the present invention

 An electrophoresis system according to the present invention having the structure shown in FIG. 1 was created. As an electric swimming unit, a substrate in which micro channels were formed by etching photosensitive glass and a glass seal was applied was created. On the other hand, the optical sensor array was created using CMOS technology.

1-1. Electrophoresis unit

7.342 thigh A 70 流 路 mx 70〃m deep microchannel is provided on a photosensitive glass of xl4.537mm. As shown in Figure 5, we created electrophoresis units arranged in a rectangular shape within the range of 5.57 删 3.23 employment. First, a mask exposing a pattern having a desired shape was applied to the surface of the photosensitive glass, and the surface was irradiated with ultraviolet rays. Next, the photosensitive glass substrate was developed (heat treated) and re-exposed to ultraviolet rays. Areas that have not been exposed to UV light by the heat treatment are not exposed. The re-exposed glass substrate was treated with an acid to dissolve the UV-irradiated portion, and further heated to crystallize. The glass substrate on which the microchannels were formed in the desired pattern was sealed with glass and integrated by heating. At this time, a capillary was attached to the end of the microchannel and the end of the sample supply means as an interface with the outside to form an electrophoresis unit.

1-2. Optical sensor array

 A Ρ + ηρ-type photo-sensitive phototransistor (68〃11 ^ 39 111) is arranged on a silicon wafer in a range of 4.6 删 6.8 thighs at 117 zm intervals both vertically and horizontally and consists of a pixel array of 30 rows x 48 columns. A part of the sensor (Fig. 6, right) was constructed. A part of this sensor was further mounted on a base chip equipped with terminals for power supply and signal output to make an optical sensor array unit (Fig. 6, left). The electrophoresis unit was placed on the obtained optical sensor array unit and fixed with an adhesive to obtain an electrophoresis system according to the present invention (FIG. 4). As shown in Fig. 5, the positional relationship between the two is that the thickness of the glass substrate is 1.0, the gap between the sensor surface and the back of the glass substrate is 0.5 mm, and there is a total of 1.5 intervals. Also, when viewed in plan, the microchannels of the electrophoresis unit are located exactly every third sensor element.

2. Optical recognition of migration path

In the electrophoresis system according to the present invention, it was confirmed how a signal was detected. First, a migration path consisting of microchannels was filled with a borate buffer (transparent) using capillaries. Halogen from above the electrophoresis unit The lamp was used as a light source (8) to illuminate the front surface, and the output signal (mV) of an optical sensor array was recorded. Figure 7 shows an image composed of the output signals of one optical sensor array. The signal in the portion corresponding to the migration path is seen as black. The borate buffer is transparent, but is darkened due to light scattering on the glass material surface as the migration path is formed. Just every third black line appears, clearly showing how it corresponds to the microchannel. If you want to detect an optical signal below the migration path, it is generally not desirable for this part to be dark. However, in the present invention, since the optical sensor and the migration path are close to each other as described above, the image of the migration path can be detected without the aid of an optical component such as a lens.

 The following processing was performed to construct an image from the output signal of the optical sensor array. Each pixel in the optical sensor array is provided with a phototransistor for photoelectric conversion, which outputs a current proportional to the amount of incident light. To sequentially output the current from each pixel, a shift register array was placed in each of the X and Y directions so that only one pixel always provided an output signal. That is, by driving the two shift registers at appropriate timing, the signals of each pixel can be obtained in order. More specifically, the optical sensor array used in this example has 30 rows by 48 pixels. Therefore, each time a signal corresponding to 48 columns is obtained per row, a trigger signal indicating that a transition to the next row should be issued. After converting the obtained current output from the sensor into a voltage, A / D conversion was performed based on the control signal from the sensor, and the data was input to a computer and reconstructed as two-dimensional data.

3. Sample concentration and sensor detection signal

In the electrophoresis system according to the present invention, as a model, instead of borate buffer, 0-25 mg / mL blue dextran (blue, having an absorption maximum of 610 nm) is charged into the electrophoresis path to change the output signal of the optical sensor array. Was observed. Optical density at each concentration The difference between the sensor array output signal (mV) and the output signal when the borate buffer was filled (mV) was averaged, and the relationship between the two was observed. The results are shown in FIG. The output signal of the optical sensor array changed depending on the concentration of blue dextran, confirming that the electrophoresis system according to the present invention enables quantitative analysis.

4. Sample movement detected by an optical sensor array

 It was confirmed that the electrophoresis system according to the present invention can track the movement of the analyte in the migration path in parallel with the electrophoresis. The blue dextran introduced into the migration path filled with borate buffer was migrated, and an image was constructed based on the output signal of the optical sensor array. Blue dextran was electrophoretically introduced into the migration path. Figure 9 shows the principle of sample introduction at this time. That is, a sample introduction path is connected to the sample tank (blue dextran), one electrophoresis path and one anode tank (Fig. 9-1), and electricity is supplied between the sample tank and the anode tank to transfer the blue dextran electrophoretically. Into the electrophoresis path (Fig. 9-2). In this state, the introduced blue dextran starts to move by electrophoresis when electricity is passed through the original electrophoresis path (Figs. 9-13 and 9-14). The change of the output signal at this time was recorded at intervals of 7 seconds.

 FIG. 10 shows an image composed of output signals of an optical sensor array according to the movement of blue dextran. The position of blue dextran became black, and it was confirmed that it moved with the migration time. According to the present invention, the movement of a substance to be analyzed in an electrophoresis path can be simultaneously tracked in parallel.

5. Improvement of electrophoresis chip using microchannel made of photosensitive glass

5-1. Improvement of photosensitive glass

The channel shape is the same as that described in the first embodiment. However, the thickness of the bottom glass was 0.4 strokes. Due to the bottom glass, the distance from the channel to the sensor was 830 m (0.83 thighs). Due to the production process, the bottom glass has There is cloudiness in the minute, but this time, the thickness of this part has been reduced from 0.93 thighs to 0.33 ridges, so that a clearer image can be obtained.

5-2. Improvement of optical sensor array

 The optical sensor array described in Example 1 was modified as follows.

Number of pixels: 1 4 7 x 1 4 7

 The size of one pixel: 39 / mx 39yam

Photoelectric conversion method: Charge integration method using photodiode

Absorbance detection limit: 0.0012

 One pixel is smaller than the phototransistor sensor described in the first embodiment. In addition, the reduction in noise has reduced the absorbance detection limit. This is due to the use of a kind of integral type photoelectric conversion method in which the photodiode generates a current generated by light irradiation in a parasitic capacitance.

5-3. Electrophoresis experiment

 A microchannel was fabricated using photosensitive glass, and then integrated onto the optical sensor to produce an integrated electrophoresis chip (Fig. 11). In this improved electrophoresis system, it was confirmed that the movement of a single substance by electrophoresis could be detected in parallel with the electrophoresis. As a sample, 4.9 nl of pull-dextran (25 mg / ml) was filled in the migration path, and the change in the output signal of the optical sensor array was observed (FIG. 12). The buffer used was a 50 mM phosphate buffer (pH 7). The detection wavelength was 620 nm and the applied voltage was 50 V / cm.

 As a result, it was possible to clearly detect the movement of the sample as a spot. The detection was performed using the absorption method. The moving speed was about 0.1 mm / sec.

6. Electrophoresis system using micro-channel fabricated by stereolithography 6-1. Electrophoresis chip

 Fig. 13 shows the design drawing and Fig. 14 shows a photograph of the fabricated chip. Using a method called stereolithography (a method of curing a resin one layer at a time by laser irradiation and scanning to create a three-dimensional shape, electric processing technology 21 [68], p8-12, 1997) A channel made of acrylic resin was directly produced. The channel has a cross section of 100〃mx 100〃m and an effective length of 4.5cm. Since it was fabricated directly on the sensor, the distance between the channel and the sensor surface could be reduced to 0.5 mm. In addition, the transparency of acrylic resin is very high, and a good image is expected. Note that a sensor of 147 × 147 pixels was used as the optical sensor.

6-2. Electrophoresis experiment

 As a sample, two kinds of blue molecular weights were used. The buffer used was a mixture of ethylene glycol and Tris buffer. The detection wavelength was 620 dishes and the applied voltage was 18 V / cm.

 As a result of the electrophoresis experiment, it was confirmed that the two samples migrated on the two-dimensional image and separated over time (Fig. 15). Fig. 16 shows a one-dimensional reconstruction of the migration pattern along the flow channel. Ogura Kakura for industrial use

According to the present invention, the progress of electrophoretic analysis can be tracked quickly and in parallel. For example, when the present invention is applied to microchannel electrophoresis, the signal of the analyte can be traced even immediately after the start of electrophoresis. If the separation can be confirmed within a short swimming time, the electrophoresis can be completed at that point.c In a known system that only performs detection at one point, the target substance is detected as a signal. Unless electrophores migrate to the location where the mechanism is located, No information is available. In particular, when applied to isoelectric focusing, a rational analysis system that can start electrophoresis from the center of the pH gradient and constantly monitor the subsequent movement of the analyte can be obtained. In the isoelectric focusing electrophoresis analysis according to the present invention, rapid analysis is possible because the convergence state of the protein can be continuously monitored. Furthermore, the combination with the means for introducing a sample from the central part of the migration path enables detection independent of the charged state of the component to be analyzed.

 In the embodiment of the present invention in which the plurality of sensors are arranged in an optical sensor array, signals can be detected two-dimensionally, and the plurality of sensors can be integrated with the electrophoresis unit at low cost and easily. Can be Furthermore, the electrophoresis unit integrated with the optical sensor array has a single capillary structure arranged in a rectangular shape on a plane, so that the whole system can be integrated on a small chip. This chip functions as a rapid electrophoresis system for trace samples. In addition, in the present invention, a very simple optical system in which a sensor array is disposed immediately below an electrophoresis unit can be provided. In this case, an expensive optical system such as a lens is unnecessary. Also, precise processing is not required. Therefore, it leads to cost reduction of the electrophoresis system. In particular, in a mode in which a microchannel is used for an electrophoresis unit, since an optical system that does not cause light scattering that affects the absorbance measurement is used, an optical system excellent in reliability is realized.

Claims

The scope of the claims

1. An electrophoresis system comprising at least the following components, wherein a signal can be detected by a sensor during electrophoresis.

a) an electrophoresis unit for separating an analyte by electrophoresis; andb) a plurality of sensors combined with the electrophoresis unit and arranged on a migration path of the analyte to detect a signal of the substance. ,

 2. The electrophoresis system according to claim 1, wherein the sensor is an optical sensor in which a plurality of sensing elements are arranged on a plane.

 3. The electrophoresis system according to claim 2, wherein an optical sensor having a plurality of detection elements arranged on a plane is integrated with the electrophoresis unit.

 4. The electrophoresis system according to claim 13, wherein migration paths in the electrophoresis unit are arranged in parallel.

 5. The electrophoresis system according to claim 4, wherein the electrophoresis paths arranged in parallel have a single continuous capillary structure.

 6. The electric device according to claim 4, wherein the detection element constituting the sensor detects a signal from one migration path, and is not affected by a signal of an adjacent migration path arranged in parallel. Electrophoresis system.

 7. The electrophoresis system according to claim 1, wherein an analysis target sample application site is provided at an arbitrary part of the migration path.

 8. The electrophoresis system according to claim 7, wherein an analysis target sample application site is provided at an intermediate portion of a migration path of the electrophoresis unit.

 9. An electrophoretic analysis method including at least the following steps a) to c).

a) Perform electrophoresis of the analyte

b) In parallel with the electrophoretic separation, a plurality of sensors are arranged at positions corresponding to the paths in which the analytes migrate through the electrophoretic separation operation. Detect signal at multiple locations

 C) determining the physicochemical properties of the substance based on the signals detected at the plurality of positions and the position information at which the signals are detected.