JP2007040812A - Capillary electrophoretic device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve a problem wherein intensity gets insufficient as excitation light for fluorescence detection, when irradiating a migration path with light from a light emitting diode. <P>SOLUTION: The plurality of light emitting diodes is pulse-driven while shifting timing, the lights therefrom are converged in the one migration path by a lens. Alternatively, the lights from the plurality of light emitting diodes are emitted to the migration path to detect fluorescence emitted from a sample in the vicinity of a sample elution end in the migration path. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  In the present invention, a sample containing biomolecules such as fluorescently labeled DNA is introduced into a thin migration path, electrophoretically separated, and these molecules are detected by irradiating light to excite fluorescence. The present invention relates to an analyzer.

Electrophoresis is one of the basic means for separating biomolecules. In electrophoretic separation of DNA and proteins, plate-like gels have long been used as a separation medium, but in recent years, a separation medium packed in a tubular migration path having an inner diameter of 10 μm to 100 μm has been widely used as a separation medium. It came to be. The method using the former medium is called slab gel electrophoresis (SGE), and the method using the latter medium is called capillary electrophoresis (CE). In CE, in addition to so-called tube capillaries, substrates with thin channels are used as the support for the separation medium. Electrophoresis using the latter support is sometimes referred to as microchip electrophoresis, but is not particularly distinguished in this specification and is included in CE. In electrophoresis, separation can generally be performed at higher speed as a larger electric field strength is used. However, too high electric field strength degrades separation performance due to the generated Joule heat. In CE, the sectional area of a single unit of separation is smaller than SGE. As a result, CE can use higher field strengths than SGE without any reduction in separation. That is, CE can be separated faster than SGE. On the other hand, in CE, since the dimension in the cross-sectional direction of the separation medium (the optical path length when the photometric detection method is used) is smaller than that of SGE, good sensitivity cannot be obtained by absorptiometry. Therefore, in applications that require high detection sensitivity, such as DNA sequencing, detection based on fluorescence measurement, which is more sensitive than absorbance measurement, is used. CE based on fluorescence measurement has become the mainstream technology for DNA analysis because it can achieve both high-speed separation and high-sensitivity detection.
There are various reported examples of CE based on fluorescence detection (for example, Patent Document 1 and Non-Patent Documents 1 and 2). In Patent Document 1, DNA is electrophoretically separated in a tube-shaped capillary, and detection is performed by irradiating a beam output from an argon ion laser to excite fluorescence. In Non-Patent Documents 1 and 2, fluorescence is detected by irradiating light emitted from a light-emitting diode (LED). In Patent Document 2, DNA that is electrophoresed in a capillary is irradiated with a laser beam, and fluorescence emitted from the DNA is transmitted to the end of the capillary using the capillary itself as a waveguide, and the fluorescence emitted from the end of the capillary passes through a liquid tank. Is detected. Hereinafter, a method of detecting the fluorescence emitted from the capillary end through the liquid tank is referred to as end detection. In Patent Document 3, a plurality of LEDs are caused to emit pulses.

JP-A-9-152418

WO 00/04371 JP 2001-93321 A Electrophoresis 2002, vol.23, pp. 2445-2448. Electrophoresis 2004, vol.25, pp. 3796-3804.

  In CEs that require high sensitivity such as DNA sequencing, a laser has generally been used as a fluorescence excitation light source as disclosed in Patent Document 1. However, lasers are more expensive than incoherent light sources such as incandescent lamps, fluorescent lamps, and light emitting diodes (hereinafter referred to as LEDs). Furthermore, in fluorescence detection in DNA sequencing, not only is the sensitivity high, but four types of phosphors having different emission wavelengths must be excited simultaneously. Due to the brightness and ease of handling of phosphors, four types of phosphors with emission wavelengths of 520-700 nm are mainly used. Argon ion lasers with a wavelength of 488 nm or 515 nm are widely used as light sources that can excite them efficiently. Has been used. An argon ion laser is a kind of gas laser, and is particularly expensive and short-lived among lasers. Furthermore, argon ion lasers are not only expensive, but also generate a lot of heat and require large heat dissipation. For these reasons, many conventional DNA sequencers are expensive, require heat exhaust ducts, and require laser replacement as needed.

  On the other hand, LEDs with a wavelength of up to 500 nm are commercially available today. LEDs have a smaller spectrum width and a larger power per unit wavelength than incandescent light sources and fluorescent lamps, and are considered to be suitable for use as fluorescence excitation light sources. LEDs are low in price and have a long life, and generate little heat. Therefore, it is possible to construct a CE system that is inexpensive and easy to handle by using the LED as a fluorescence excitation light source. Here, in Non-Patent Document 1 in which an LED is used as an excitation light source, one LED is used as a light source, and the LED is pulse-driven to eliminate low frequency fluctuations and DC background, but the amount of irradiation light is insufficient. It is assumed that In addition, although it is described that one high power LED is used as a light source, the brightness of an LED having a wavelength of around 500 nm is still about five orders of magnitude smaller than that of a laser, and is in a narrow region such as a capillary lumen. It is assumed that the power when irradiated is not yet sufficient for applications requiring high sensitivity such as DNA sequencing. An object of the present invention is to improve the excitation intensity when a low-intensity light source such as an LED is used as a CE excitation light source, and to improve the sensitivity of fluorescence detection.

とかける。ここでN_Aはアボガドロ数Cは計測体積における分析対象分子のモル濃度，Vは計測体積，σは分析対象分子の吸収断面積，Iは励起光の強度，hnは励起フォトンのエネルギー， Qは分析対象分子の蛍光量子収率，Fは検出光学系の集光効率collection efficiency，hは検出器の量子効率 Δt₁は検出器の積分時間である。バックグラウンドの電子数Bは背景光による光電子数とダークカウントの和であり，

とかける。ここでC_Bはバックグラウンド相当濃度，Δt₂は検出器出力のサンプリング間隔である。蓄積電荷の転送時間が無視できる場合にはΔt₁＝Δt₂とみなせる。検出器がインターラインまたはフレームトランスファーのＣＣＤであって，蓄積電荷の転送時間≦サンプリング間隔である場合には，Δt₁＝Δt₂＝サンプリング間隔とみなせる。バックグラウンドノイズの二乗平均N₀及びノイズの二乗平均Nはそれぞれ，

で与えられる。ここでNrは検出器のリードアウトノイズである。より一般的には，濃度Cは時刻tと位置r = (x,y,z)の関数，励起光の強度Iと集光効率Fは位置r の関数，結果としてSは時間の関数となり，（数1），（数2）は

となる。

を分子検出効率(molecule detection efficiency, MDE)と呼ぶ。MDEを用いて（数７），（数８）を書き換えると

以下では簡単のため，C,I,Φは位置と時間に依存しないと仮定し，（数1）〜（数4）に基づいて議論する。蛍光検出の感度を高めるということは，一定の濃度Cに対してなるべく高いS/Nを得るということである。明らかにSが大きいほどS/Nもまた大きい。そこで一定のCに対し，（数1）の右辺をいかに大きくするかについて考える。N_A,hは完全な定数であり，Qは分析対象分子によって決まり，分析対象分子によって最適な励起波長がほぼ決まってしまい，同時にσ，nもまた決まってしまう。したがって装置的パラメータでS向上に寄与できるのはV,η，Φ，I,Δt₁となる。 In order to explain the principle of the invention, the basics of fluorescence detection will be described first. The number of photoelectrons S generated in the detector by fluorescence is

Call it. Where N _A is the molar concentration of analyte molecules in the Avogadro number C is measured volume, V is the measurement volume, absorption cross section σ is analyzed molecules, I is the intensity of the excitation light, hn is the energy of the excitation photons, Q is The fluorescence quantum yield of the molecule to be analyzed, F is the collection efficiency of the detection optical system, h is the quantum efficiency of the detector, and Δt ₁ is the integration time of the detector. The number of electrons B in the background is the sum of the number of photoelectrons from the background light and the dark count.

Call it. Here C _B is the background equivalent concentration, Delta] t ₂ is the sampling interval of the detector output. If the accumulated charge transfer time can be ignored, it can be considered that Δt ₁ = Δt ₂ . When the detector is an interline or frame transfer CCD and the transfer time of accumulated charges ≦ sampling interval, it can be considered that Δt ₁ = Δt ₂ = sampling interval. The background noise root mean square N ₀ and noise root mean square N are

Given in. Here, Nr is the readout noise of the detector. More generally, concentration C is a function of time t and position r = (x, y, z), excitation light intensity I and collection efficiency F are functions of position r, and S is consequently a function of time, (Equation 1) and (Equation 2) are

It becomes.

Is called molecule detection efficiency (MDE). Rewriting (Equation 7) and (Equation 8) using MDE

In the following, for the sake of simplicity, it is assumed that C, I, and Φ do not depend on position and time, and discussion is based on (Equation 1) to (Equation 4). Increasing the sensitivity of fluorescence detection means obtaining as high S / N as possible for a certain concentration C. Obviously, the greater the S, the greater the S / N. Therefore, consider how to enlarge the right side of (Equation 1) for a given C. N _A , h is a perfect constant, Q is determined by the molecule to be analyzed, and the optimum excitation wavelength is almost determined by the molecule to be analyzed, and at the same time, σ and n are also determined. Therefore, it is V, η, Φ, I, Δt ₁ that can contribute to the improvement of S in terms of apparatus parameters.

In the case of CE, V = πd ² l / 4 where d is the inner diameter of the capillary and w is the width of the detection region. In high-resolution electrophoresis such as DNA sequencing, it is necessary to satisfy l ≦ 0.1 mm and Δt ₁ ≦ 0.1 s, and d ≦ 0.1 mm, preferably d ≦ 0.05 mm, in order to enable the use of high electric field strength. There is a need. That is, there is a limit to increasing the sensitivity by increasing V and Δt ₁ . In principle, η is 1 or less, and η of an actual semiconductor detector for detecting weak light is approximately in the range of 0.7 to 0.9, and there is no significant difference. The remaining parameters are Φ and I, and increasing these products is the basic guideline for improving the fluorescence detection sensitivity of CE with hardware. To increase Φ, it is important to use a bright lens for detection. In the present invention, a method of increasing the intensity I using a low-luminance light source such as an LED will be described.

で与えられる（光源は空気中にあると仮定した）。n は照射点の屈折率，tは照射光学系の透過率であり，常にt≦1 である。（数9）は照射点における輝度は，いかなる照射系を用いても光源の輝度に照射点の屈折率の二乗を掛けた値を超えられない事実を示し，一般化された輝度不変の法則と呼ばれる。
以下ではＬＥＤはランバート面と仮定する。ランバート面に対しては，光源における単位面積あたりの光パワーE₀は輝度Ｂにより

で与えられる。一方，照射点における強度Iは

で与えられる。Ωは照射点から光源を見込む立体角であり，照射レンズの集光立体角とみなすことができる。片側照射である限りΩ≦2πである。複数方向から照射することによりΩをさらに大きくすることが可能であるが，現実には検出のための立体角を残さなければならないので，困難である。照射系が片側かつ軸対称であるならば，照射点における最大入射角をθとして

となる。 The relationship between the luminance B _{0 at the} light source and the luminance B at the irradiation point is independent of the details of the optical system between the light source and the irradiation point.

(Assuming that the light source is in the air). n is the refractive index of the irradiation point, t is the transmittance of the irradiation optical system, and always t ≦ 1. (Equation 9) shows that the luminance at the irradiation point cannot exceed the value obtained by multiplying the luminance of the light source by the square of the refractive index of the irradiation point, no matter what irradiation system is used. be called.
In the following, it is assumed that the LED is a Lambertian surface. For the Lambertian surface, the light power E ₀ per unit area of the light source depends on the luminance B.

Given in. On the other hand, the intensity I at the irradiation point is

Given in. Ω is the solid angle from which the light source is viewed from the irradiation point, and can be regarded as the condensing solid angle of the irradiation lens. As long as the irradiation is performed on one side, Ω ≦ 2π. It is possible to further increase Ω by irradiating from multiple directions, but in reality it is difficult because a solid angle for detection must be left. If the irradiation system is unilateral and axisymmetric, the maximum incident angle at the irradiation point is θ.

It becomes.

Considering fluorescence detection in a DNA sequencer, n is the refractive index of a single-stranded DNA separation medium, and generally n = 1.4. The transmittance of the irradiation system is ideally 1, but it is necessary to insert a filter for removing wavelength components other than the phosphor excitation wavelength, and in reality, it is t to 0.5. Therefore, assume t = 0.5. Although depending on the detection efficiency Φ, if the excitation intensity I is about 10 ⁶ W / m ² , a preferable sensitivity can be obtained as a DNA sequencer. Since the brightness of an argon ion laser with an output of 1 mW is 4 × 10 ⁹ W / m ² / sr, sufficient intensity can be obtained at θ = 0.5 ° from (Equation 9) to (Equation 12). On the other hand, even the highest brightness LED has a brightness of ˜8 × 10 ⁴ W / m ² / sr. Therefore, even if θ is 90 ° as the upper limit, the obtained strength is 5 × 10 ⁵ W / m ² . In reality, θ = 90 ° is difficult to achieve with a normal irradiation detection system due to the limitation of the brightness of the lens and steric hindrance with the detection lens. Assuming about θ = 40 °, the intensity obtained is ˜10 ⁵ W / m ² , which is about an order of magnitude smaller than when using a laser. The improvement in detection efficiency is several times (at most three times), and it is considered that a one-digit decrease in excitation intensity cannot be covered. Therefore, in a CE system using an LED as a fluorescence excitation light source, it is considered that a configuration that increases the excitation intensity several times is necessary in order to obtain sufficient sensitivity for use as a DNA sequencer. As can be seen from (Equation 11), there are two ways to increase the intensity: increasing the luminance of the light source or increasing the solid angle Ω of the irradiation. Just arranging multiple LEDs increases the total output, but does not change the brightness. Also, increasing the current that drives the LED increases the brightness of the LED. However, continuous driving above the rated value may cause a failure due to heat generation. Even if there is no failure, the wavelength and output become unstable due to temperature rise due to heat generation.

Therefore, in the present invention, the irradiation intensity I is improved by the following two means.
Means 1: A plurality of LEDs are pulsed at a predetermined timing to increase the luminance at the irradiation position.
Means 2: Adopting end detection in the detection system and irradiating with a plurality of LEDs increases the irradiation solid angle Ω.

  First, means 1 will be described. The ratio of the time during which the LED is lit during the fluorescence measurement to the total measurement time is called the duty ratio. In normal continuous driving, the duty ratio is 1 as a matter of course. In general, when the LED is pulse-driven with a duty ratio of 1/10 or less, the LED operates normally even if it is driven with a current that is about 3.3 times the rated current under continuous drive conditions. In this way, if pulse driving is performed at a low duty ratio, heat is efficiently dissipated during the extinguishing time, so that a rise in temperature is suppressed and fluctuations in output and wavelength do not occur. However, in such a pulse drive, the LED brightness during lighting is improved by about 3 times, but since the duty ratio is low, the brightness of the light source is rather lowered in terms of time average. Therefore, in the present invention, a plurality of LEDs are arranged in the field of view of the first lens, the plurality of LEDs are divided into LED groups that are a plurality of subsets, and pulse driving is performed by shifting the timing for each LED group. Make sure that more than a certain number of LEDs are always lit. Light from a plurality of LEDs emitting light in this way is collected by the first lens and irradiated to the same capillary by the second lens. As a result, the luminance at the irradiation position can be higher than the luminance of the single LED light source during continuous driving, even as a time average value. As a result, the intensity at the irradiation position can be improved.

  In the second means, as a result of using the end detection method for detection, the solid angle at the irradiation point can be used only for irradiation. Therefore, irradiation is performed from multiple directions to increase the irradiation solid angle. This method makes it possible to achieve Ω exceeding the upper limit of 2π for Ω on one side.

  Using an inexpensive LED light source, high-sensitivity fluorescence detection at the same level as laser excitation becomes possible. If applied to an electrophoresis system such as a DNA sequencer, a low-cost and easy-to-use system can be realized.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. Examples 1 to 6 are based on the first means, and Examples 7 to 9 are based on the second means. Needless to say, the first and second means may be used in combination.

  FIG. 1 is a block diagram of the first embodiment. Excitation light emitted from the ten LEDs 4.1 to 4.10 fixed to the support 5 is collected by the first lens 7 and converted into a substantially parallel light beam, and then transmitted through the excitation filter 8. The light is condensed on the migration path 1 by the second lens 9. Both ends of the migration path are immersed in the buffer tank 2.1 and the buffer tank 2.2. The electrode 10.1 and the electrode 10.2 immersed in each buffer tank are connected to the negative electrode and the positive electrode of the power source 3, respectively. A voltage is applied to both ends of the migration path through the buffer, an electric field is generated in the migration path, and the analyte molecules injected into the migration path are migrated and separated at a rate proportional to their mobility. The migration path used in this example is a quartz capillary having an inner diameter of 50 μm, and the injected analysis target molecule is a DNA fragment. Since DNA is a negative ion, it is injected from the negative electrode 2.1 side, migrates toward the 2.2 side, the fragment that reaches the irradiation point 20 is irradiated with excitation light, and the fragment emits fluorescence. The The fluorescence emitted from the DNA fragment is converted into a parallel light flux by the third lens 11, and after passing through the radiation filter 12 to remove the scattered light component of the excitation light, it is provided in the vicinity of a part of the migration path. An image is formed on the detector 14 and detected. The detector 14 used in this embodiment is a photomultiplier tube, but may be a photodiode or an imaging device such as a CCD. If it detects with an imaging device, since the light from a capillary lumen | bore and the scattered light from an outer surface can be distinguished, there exists an advantage that the scattered light which could not be removed completely with the filter 12 can be distinguished.

The LEDs 4.1 to 4.10 are pulse-driven by a power supply 6 connected by flexible electric wires 15.1 to 15.10. Here, each of the plurality of LEDs emits light having substantially the same wavelength. Here, to emit light having substantially the same wavelength means to emit light having different peak wavelengths within a range of deviation of a manufacturing error. Hereinafter, the same conditions are used when a plurality of LEDs are used. In the present specification, the lit LED is represented by a black square, and the unlit LED is represented by a white square. FIG. 2 shows a timing chart of LED driving current pulses. Since each LED is driven at a duty ratio of 1/10, when it is lit, it is driven at 100 mA, which is 3.3 times or more of the rated current value of 30 mA under continuous drive conditions. In addition, by turning on the 10 LEDs at different timings as shown in Fig. 2, one of the LEDs is always lit within the focusing range of the lens 1, so that the capillary is always irradiated with light. Yes. FIG. 3 shows the relationship between the LED drive current and the luminance. The LED used in this example has a recommended operating current of 20 mA and a rated current of 30 mA by the manufacturer, but it can be seen that driving at 100 mA can provide about three times the brightness of the standard operating current drive. Of course, when it is continuously lit at 100 mA, the life is not guaranteed, but it is driven at a duty ratio of 1/10, so a life equivalent to the standard operating current is guaranteed. Of course, there is almost no fluctuation in output power or wavelength due to temperature rise. As a result, in the present embodiment, the excitation light having the same intensity as that obtained when the light from the LED having a luminance of 3 times is continuously lit and irradiated is applied to the irradiation point 20 of the migration path 1. The wavelengths of the LEDs used in this example are substantially 500 nm, and the excitation efficiency for the phosphor is substantially equivalent to that of an Ar ion laser that oscillates at two wavelengths of 488 nm and 514.5 nm. Therefore, it is possible to suitably excite phosphors, ROX, FAM, TAMRA, NED, JOE, TET, VIC, etc. used for DNA modification in DNA sequencing and fragment length analysis. In this embodiment, a high-brightness LED with a luminance of 8 × 10 ⁴ W / m ² / sr is used, and a bright lens with NA 0.9 is used as the irradiation lens, and θ to 40 °, Ω to 1.5 sr. As a result, the intensity at the irradiation point was 3 × 10 ⁵ W / m ² . By adopting an extremely bright camera lens with F number of 0.85 as the detection lens 11, the fluorescence detection sensitivity almost equal to that of the conventional DNA sequencer was achieved. Here, 10 LEDs are arranged, but of course 20 or 30 may be used. If the duty ratio of the LED drive pulse is x, if the number of LEDs is 1 / x or more, at least one LED can always be lit.

  FIG. 4 is an electrophoretogram obtained by injecting, separating and detecting ROX-labeled single-stranded DNA size marker fragment samples using this example. The numerical value on each peak is the fragment length of the DNA fragment belonging to each peak. The concentration of each fragment is 1 nM, which is equivalent to the standard concentration per band of the DNA sequencing sample. Good S / N of 100 or more is obtained for the sample having such a concentration. ROX has almost the same fluorescence characteristics as the dye having the lowest excitation efficiency among the four-color phosphors used for DNA sequencing. That is, this example shows that it has fluorescence detection sensitivity that can also be used as a DNA sequencer. In this example, since a single-color fragment length analysis was performed, only the filter 12 was used as a spectroscopic element. However, a diffraction grating or a prism was inserted between the filter 12 and the lens 13, and an imaging device such as a CCD was used as the detector 14. If used, it can be used as a DNA sequencer capable of multicolor detection.

  FIG. 5 is a block diagram of the second embodiment. Since the configuration of this embodiment is basically the same as that of the first embodiment except for the arrangement of the LEDs, only the vicinity of the LEDs is enlarged and displayed. In this embodiment, 100 LEDs 4.1.1 to 4.10.10 are arranged in a 10 × 10 two-dimensional matrix within the focusing range of the first lens, and 100 LEDs are arranged. Yes. In this embodiment, the LEDs are divided into 10 LED groups composed of 10 LEDs, and the LEDs in each group are turned on simultaneously, and the drive timing is shifted for each group as in the first embodiment. Thus, even when a plurality of LEDs are turned on simultaneously, the same effect as in the first embodiment can be obtained. As an effect unique to the present embodiment, since the LEDs are two-dimensionally arranged, there is an effect that the visual field range required for the first lens 7 can be made smaller than arranging them one-dimensionally. In this embodiment, the two-dimensionally arranged LEDs are turned on simultaneously for each row. However, of course, this is not necessary, and every 10 predetermined arbitrarily distributed LEDs may be turned on simultaneously. Further, although the drive pulses of the respective LEDs are shifted by the pulse width, this is not always necessary. For example, for all 100 LEDs, even if the rising edge of the drive pulse is shifted by 1/10 of the pulse width, 10 LEDs are always lit at the same time. Alternatively, the drive timing may be the same for each two, and the pulse deviation may be 1/5.

  FIG. 6 is a conceptual diagram of the third embodiment. The basic structure of the present invention is similar to that of the first embodiment, but the base (supporting part) 5 on which the LEDs 4-1 to 4-10 are mounted is such that the LED to be lit is always at the center of the field of view of the lens 7. It is scanned by the actuator 21 and reciprocates. In this way, the point where the light on the capillary is irradiated becomes completely the same point, and when used for fluorescence detection in capillary electrophoresis, the width of the detection band can be reduced, resulting in high resolution. Is effective. FIG. 7 is a timing chart of the LED drive current in this embodiment. The period of the reciprocating motion of the substrate 5 is 10T. Each LED emits light when it reaches the center of the field of view of the lens 1. Each LED comes to the center of the field of view of the lens 7 twice during one reciprocation (= 10T), that is, emits light twice, so that the duty ratio is 1/10, so that one light emission time is T / 2. However, the LEDs 4-1 and 4-10 at both ends of the substrate emit light twice, so that light is emitted continuously for a time T once in one round trip. FIG. 8 is a time series of the state of light emission of the LED and the physical movement of the support 5 for fixing the LED in this embodiment. The black-painted LEDs are LEDs that are emitting light, and the white LEDs are LEDs that are unlit. In conjunction with the movement of the substrate 5 by the actuator, the LED that has always come to the center of the visual field of the lens 7 emits light.

  FIG. 9 is a conceptual diagram of the fourth embodiment. This embodiment is an example using the configurations of the second embodiment and the third embodiment. Out of 1,000 LEDs 4.1.1-4.100.100 arranged in two dimensions, 10 columns × 10 rows = 100 (the array of LEDs orthogonal to the driving direction of the actuator is called a column) The LED is lit at the same time, and the LED mounting area is moved so that the LED in the lighting range is always within the focusing range of lens 1. The rise of the LED drive pulse may be shifted by the pulse width, but in this embodiment, as shown in FIG. 10, the shift of the drive pulse for each LED row is 1 / (pulse width T / 2) Number of columns) = 1/10. As a result, the step of physical movement of the LED mounting base can be set to the width of one LED row, and the 10 rows of LEDs can always be lit in the field of view of the lens 7. (The light emission timing may be the same for every 10 rows, and the drive pulse may be shifted by pulse width T / 2 for every 10 rows. In this case, the actuator step width must be equal to the 10 LED rows, so the load on the actuator In this embodiment, a linear actuator that operates completely smoothly may be used instead of an actuator that performs step driving. Even in this case, the number of LEDs to be lit within the field of view of the lens 7 is 90 to 100, and the average is 95, and the light having a substantially constant intensity within ± 5% from the average is condensed by the lens 7. The capillary is irradiated. In the first embodiment, when the movement of the actuator is smoothed, the number of LEDs emitting light within the field of view of the lens 7 is 1 or 0, and the relative variation is ± 50%, which is a very large variation. As described above, it is effective to irradiate the capillary with light having a substantially constant intensity even in a smooth drive with a small mechanical load on the actuator.

  FIG. 11 shows a fifth embodiment. In this embodiment, the LED fixing support 5 is cylindrical, and this support is rotated around its axis. The driving of each pulse can be the same as in the fourth embodiment, and in this case, the same effect as in the fourth embodiment can be obtained. With this configuration, since the movement of the base is only required in one direction of rotation, the reciprocation as in the fourth embodiment is not required, and the rotation direction of the motor can be made constant. In addition, power is supplied to the LEDs using a power supply means. Specifically, this is performed through a leaf spring 91 fixed to the electric wire 111 connected to the power source 92. When the support 5 rotates and enters the light condensing range of the lens 7, the leaf spring 91 comes into contact with the electrode of the LED and power is supplied to the LED. As an effect of such a configuration, the control of the LED driving pulse becomes unnecessary, and a simple DC power source can be used as the power source 92, and metal fatigue due to the movement of the flexible wire support 5 connecting the LED and the power source. To be dying. In this embodiment, the leaf spring 91 is semi-permanently connected to the power source by soldering, and the leaf spring and the LED electrode are made conductive by detachable mechanical contact. As a semi-permanent connection method, of course, soldering, welding, crimping, joining with silver paste, screwing, connection with a connector, etc. may be used. Conversely, the leaf spring may be semipermanently connected to the LED electrode, and the leaf spring and the power source electrode or the electric wire connected to the power source may be electrically connected by detachable mechanical contact. This method has an advantage that when the plate spring is fatigued by metal, it can be dealt with by replacing the LED mounting substrate 5.

  FIG. 12 shows a sixth embodiment. In this embodiment, a plurality of LEDs are installed on a two-dimensional cylindrical LED fixed support 5 and the support is rotated about its axis. For driving the LED, for example, the configurations of the second and fifth embodiments can be used together. By using multiple LEDs that light up simultaneously, the step width of the rotation of the cylindrical substrate can be reduced, and the motor load can be reduced. In the fifth embodiment, the rotation step is 360 ° / 10 = 36 °, but in this embodiment, 360 ° / 40 = 9 °.

FIG. 13 is a conceptual diagram of the seventh embodiment. A voltage is applied between the buffer tank 2.1 and the buffer tank 2.2 by the power source 3, and the molecule to be analyzed is electrophoresed in the migration path (capillary in this embodiment). Around the irradiation point 20, 100 LEDs 4.1.1 to 4.10.10 are arranged on a cylindrical surface (in the figure, the cylinder is shown as 131 in the cross-sectional view). That is, the cylinder is installed so as to surround the capillary, and the LED is installed on the inner wall surface facing the capillary of the cylinder. Multiple LEDs are lit at the same time. FIG. 14 is an enlarged view of the vicinity of the irradiation point in the present embodiment. Before and after the irradiation position, light shielding portions 30a and 30b are provided around the capillary. As a result, the irradiation width is not widened and the electrophoretic resolution is not lowered. FIG. 15 is an enlarged view of a cross section around the irradiation point by the cross section A. FIG. A cylindrical filter 8 is inserted between the LED and the capillary. This filter 8 removes a component whose wavelength matches the emission wavelength of the fluorescent light from the emitted light of the LED. Analyte molecules in the migration path are excited by the emitted light from these LEDs and emit fluorescence. The fluorescence emitted at the irradiation point propagates in the migration path 1 by total internal reflection and is transmitted to the end face. The fluorescence emitted from the end face is converted into a parallel light beam by the condenser lens 11 through the buffer tank 2.1, and after the light other than the fluorescence is blocked by the filter 12, it is formed on the photoelectric surface of the CCD camera 14 by the imaging lens 13. Imaged. The detector 14 is installed in the vicinity of the sample elution end of the capillary and at a position facing the sample elution end. As in this embodiment, as a result of detecting the fluorescence after transmitting it to the end face, it is possible to use all the solid angles at the irradiation point for irradiation. In this embodiment, 100 LED chips each having a size of 0.2 mm × 0.2 mm are arranged on a cylindrical surface having a diameter of 0.73 mm and a length of 2.27 mm with a clearance of 0.03 mm centered on the irradiation point. Therefore, the solid angle Ω = 2.1π where the light source is expected at the irradiation point, and a solid angle exceeding the limit of conventional one-side irradiation is realized. In the present embodiment, a high-brightness LED with a brightness of 8 × 10 ⁴ W / m ² / sr under standard operating conditions is used in continuous operation under standard conditions, and a filter with a transmittance of about 0.5 is used. The strength of 5.4 × 10 ⁵ W / m ² can be obtained. As a result, it is possible to obtain sufficient fluorescence detection sensitivity by using a camera lens of F number 1.2 as the detection lens.
In the present embodiment, the LEDs are arranged on the cylindrical surface, but of course, they may be arranged on a spherical surface including the irradiation point of the capillary. A parallel plane between two capillaries may be used. In either case, it is possible to realize an irradiation solid angle close to 4π, which is the absolute limit of the parallel plane solid angle.

FIG. 16 is a schematic diagram of the eighth embodiment. The basic configuration of this embodiment is the same as that of the seventh embodiment, and FIG. 16 is a cross-sectional view taken along a plane orthogonal to the capillary at the irradiation point. In this example, the light from the LEDs 4.1 to 4.4 is collimated by the lenses 7.1 to 7.4, and the spectral purity is increased by the filters 8.1 to 8.4. Squeeze with 9.4 and irradiate capillary 1 from 4 directions. Looking at one direction, in this embodiment, one light is emitted from each direction. Of course, a plurality of LEDs may be arranged in the field of view of the lens 7.1-7.4 in each direction as in the first embodiment. good. Since irradiation was performed from four directions, the total irradiation solid angle could be 4 times Ω = 6 sr. As a result, the LED was continuously driven under standard operating conditions to achieve an irradiation spot intensity of 4 × 10 ⁵ W / m ² . As an effect of the present embodiment, there is a merit that the number of LEDs used can be reduced. In this embodiment, irradiation is performed from four directions, but in general, irradiation may be performed from the n direction with n being an arbitrary integer. In this embodiment, the optical axis of the irradiation light is arranged in a plane orthogonal to the capillary, but it is of course possible to irradiate this plane from an oblique direction.

  FIG. 17 is a schematic diagram of the ninth embodiment. In this embodiment, the light from the LEDs 4.1 to 4.8 is introduced into the optical fibers (light transmission means) 14.1 to 14.8 and then irradiated to the capillary 1. One end of the optical fiber is installed in the vicinity of the LED, and the other end is installed in the vicinity of the capillary 1. As a result, in this embodiment, there is an effect that the LEDs can be arranged flexibly. In addition, in this embodiment, light from one LED is introduced from each optical fiber. However, a plurality of LEDs are arranged in the field of view of the lens 7.1-7.4 in each direction to emit light from the plurality of LEDs. Of course, it may be introduced into each optical fiber.

  FIG. 18 is a conceptual diagram of the tenth embodiment. In this embodiment, the electrophoresis system and the detection system are the same as those in the seventh embodiment. The difference is that a plurality of LEDs 4.1-4.10 are pulse-driven at different timings as in the first embodiment, condensed by the lens 9, and irradiated onto the capillary 1. Even if the light is condensed by the lens 9, light from different light emitting points is condensed at a slightly shifted position. In other words, the emitting position changes as the emitting LED changes over time. In the first embodiment, since the direct irradiation point is observed, a photomultiplier tube having a detection surface larger than the movement of the irradiation position is used as the detector 14. In this embodiment, the cross section of the capillary with a fixed light emitting point can be regarded as a single point, so that the detection surface is small as in the avalanche photodiode, but the quantum efficiency is higher than that of the photomultiplier tube, and the dark current is high. It is possible to use a highly sensitive detector with a small.

  It can be used for an electrophoresis apparatus that is inexpensive and easy to maintain.

1 is a configuration diagram of Embodiment 1. FIG. 2 is a timing chart of LED drive current pulses in the first embodiment. Relationship between LED drive current and brightness. FIG. 5 is an electrophoretogram obtained using Example 1. FIG. The conceptual diagram of Example 3. FIG. 10 is a timing chart of LED drive current pulses in Example 3. The time series of the movement of the LED support body in Example 3. FIG. The conceptual diagram of Example 4. FIG. 10 is a timing chart of LED drive current pulses in Example 4. The conceptual diagram of Example 5. FIG. The conceptual diagram of Example 6. FIG. The conceptual diagram of Example 7. FIG. FIG. 10 is an enlarged view of the vicinity of an irradiation point in Example 7. Sectional drawing by the surface orthogonal to the capillary in Example 7. FIG. The conceptual diagram of Example 8. FIG. The conceptual diagram of Example 9. FIG. The conceptual diagram of Example 10. FIG.

Explanation of symbols

1: Electrophoretic path, 2.1-2.2: Buffer tank, 3: Power supply, 4.1-4.10, 4.1.1-4.10.10: Light emitting diode, 5: Support, 6: Power: 7: Lens, 8, 8, 8.1-8.2: Filter, 9: Lens, 10.1-10.2: Electrode, 11: Lens, 12: Filter, 13: Lens, 14: Detector , 20: detection point, 21: actuator, 30a to 30b: light shielding part, 91.1 to 91.4: leaf spring, 92: power supply, 111: electric wire, 131: cylindrical surface.

Claims (17)

A migration path for running the sample;
A voltage application unit for applying a voltage to the migration path;
A plurality of light emitting diodes;
A power supply unit comprising a pulse generation unit for applying a pulse current to the plurality of light emitting diodes;
A light detection unit that detects light emitted from the sample, and the pulse generation unit controls application of the pulse current so that a certain number of the light emitting diodes among the plurality of light emitting diodes emit light. Electrophoresis device. A first lens disposed between the migration path and the light emitting diode;
A second lens for condensing the light that has passed through the first lens on the detection site on the migration path;
The electrophoresis apparatus according to claim 1, wherein the predetermined number of light emitting diodes are located in a light condensing range of the first lens.   The electrophoretic device according to claim 1, wherein each of the plurality of light emitting diodes emits light having substantially the same wavelength.   A support unit for installing the plurality of light emitting diodes; and an actuator for moving the support unit, wherein the actuator is configured to dispose the support unit so that the fixed number of light emitting diodes are located in the light collection range. The electrophoresis apparatus according to claim 2, wherein the electrophoresis apparatus is moved.   The electrophoresis apparatus according to claim 4, wherein the plurality of light emitting diodes are two-dimensionally arranged on the support portion.   The electrophoresis apparatus according to claim 4, wherein the support portion is a cylinder, and the plurality of light emitting diodes are installed on an outer surface of the support portion.   The electrophoresis apparatus according to claim 4, wherein the cylinder rotates about its axis.   2. The electrophoresis apparatus according to claim 1, wherein the migration path is a capillary, and the light detection unit detects the light in the vicinity of a part of the capillary.   2. The electrophoresis apparatus according to claim 1, wherein the migration path is a capillary, and the light detection unit detects the light in a region near the sample elution end of the capillary.   The electrophoretic device according to claim 4, further comprising power supply means in contact with the support portion, wherein the light emitting diode is supplied with power through the power supply means.   The electrophoresis device according to claim 1, wherein the photodetector is any one of a photomultiplier tube, a photodiode, or a CCD. A migration path for running the sample;
A voltage application unit for applying a voltage to the migration path;
A plurality of light emitting diodes for irradiating the migration path with light;
An electrophoretic apparatus comprising: a detector installed facing the sample elution end of the electrophoretic path.   The electrophoretic device according to claim 12, wherein the plurality of light emitting diodes emit light having substantially the same wavelength.   The said migration path has a 1st light-shielded site | part and a 2nd light-shielded site | part in each of the irradiated site | part irradiated with the said light, and the vicinity of the both ends of the said irradiated site | part. The electrophoresis apparatus according to 1.   13. The apparatus according to claim 12, further comprising a support part installed around the migration path, wherein the plurality of light emitting diodes are installed on an inner wall surface of the support part facing the migration path. Electrophoresis device.   13. The electrophoretic device according to claim 12, further comprising light transmission means having one end portion disposed in the vicinity of the light emitting diode and the other end portion disposed in the vicinity of the electrophoresis path. A pulse generator for applying a pulse current to the plurality of light emitting diodes;
The electrophoretic device, wherein the pulse generator controls the pulse current so that a certain number of light emitting diodes among the plurality of light emitting diodes emit light.

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