JP3664557B2 - Flow-through cell - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a flow-through cell, particularly for immunoassay suitable for quantifying trace amounts of hormones, tumor markers, drugs, enzymes, cytokines, nucleic acids, etc. in an aqueous solution of a sample by fluorescence method or electrochemiluminescence method. It relates to a flow-through cell.
[0002]
[Prior art]
In the field of immunoassay, fluorescent methods, chemiluminescent methods, and electrochemiluminescent methods are used as analytical methods for measuring the presence and concentration of a very small amount (10 ^-14 mol or less) of an analyte to be measured in a sample. ing. The fluorescence method detects light emitted from the sample by photoexcitation, and the electrochemiluminescence method detects light emitted from the sample when a voltage is applied to the sample. In these methods, a luminescent reagent is bound to a substance to be measured such as a hormone by an antigen-antibody reaction, and luminescence derived from the luminescent reagent is quantified. In general, measurement is performed while a sample is passed through a flow-through cell. On the other hand, in the chemiluminescence method, for example, a luminescent reagent is mixed in a test tube, and the light emitted from the test tube in the 4π direction is reflected by the inner surface of the sphere to be collected. Done in
[0003]
For measurement methods that require light irradiation or voltage application to the measurement sample, such as fluorescence and electrochemiluminescence methods, the flow-through method is more advantageous than the batch method in terms of shortening the measurement time, and the device configuration Therefore, there is an advantage that an increase in apparatus cost can be suppressed and maintenance can be easily performed.
[0004]
[Problems to be solved by the invention]
The shape of the flow-through cell that has been conventionally used is a planar type, and the concentration of light emission generated in the flow-through cell is limited to only one surface direction. In particular, in the case of a flow-through cell used for electrochemiluminescence, flat electrodes are often used due to the principle restriction of having an electrode inside the cell and the requirement to keep the potential distribution on the electrode surface uniform. Has been. Conventionally, the photomultiplier tube used for detecting the amount of emitted light is large in size and has a single light receiving surface, which is one of the reasons for adopting the planar cell shape. Therefore, in the immunoassay using the conventional flow-through cell, the luminescence cannot be collected efficiently, and a very small amount of the analyte to be measured cannot be detected with high sensitivity.
[0005]
The present invention has been made in view of such problems of the prior art, and an object thereof is to provide a flow-through cell capable of efficiently collecting a small amount of light emission from a sample and detecting it with high sensitivity. And
[0006]
[Means for Solving the Problems]
In the present invention, the light emission surface of the flow-through cell from which light emission from the sample is emitted has a curved three-dimensional shape, and the wall surface other than the light emission surface is used as a light reflection surface and the light emission generated from the sample is reflected. Shaped so that it can be condensed on the light exit surface. After the light emitted from the sample is reflected by the light-reflecting cell wall, it is efficiently applied to the photodetector placed outside the flow-through cell via the light exit surface. The above-mentioned purpose is achieved by allowing the light to be condensed.
[0007]
That is, the present invention provides a sample inflow portion into which a sample fluid flows, a sample outflow portion, a sample channel connecting the sample inflow portion and the sample outflow portion, and a light exit surface that is a part of the wall surface of the sample channel The light exit surface has a substantially axisymmetric three-dimensional shape, and the wall surface of the sample channel facing the light exit surface has a shape substantially similar to the light exit surface. The wall surface of the sample channel other than the above is a light reflecting surface.
[0008]
The wall surface of the sample channel used as the light reflecting surface can be a wall surface on the side in contact with the sample fluid, or can be an external surface not in contact with the sample fluid. When the outer surface of the sample channel is a light reflecting surface, the cell wall portion needs to be made of a light transmissive material. If the material that makes up the flow-through cell does not deteriorate due to a chemical reaction as in the fluorescence method, a light transmissive material such as an acrylic plate can be used. When the surface in contact with the solution is corroded, a polymer material resistant to corrosion, such as polymethyl methacrylate, can be used as the light transmissive material.
[0009]
The light reflecting surface can be formed by using a mirror surface treatment such as plating or vapor deposition with a metal material such as Al, Ag, Au or the like, or using the metal itself for the wall surface when deterioration due to a chemical reaction does not occur. When the surface in contact with the sample solution is corroded, it can be formed by plating or vapor-depositing a metal material resistant to corrosion such as Pt.
[0010]
The light emission surface can be a cylindrical surface, and the sample flow channel can be a flow channel for flowing the sample fluid flowing in from the sample inflow portion in two directions around the cylindrical surface. In this case, a small photomultiplier tube or a small photocell such as a photodiode that has been developed and improved in recent years as a photodetector is adopted, and the photodetector is arranged in a space surrounded by a cylindrical surface. Thus, it is possible to efficiently collect and detect light emitted from the sample.
[0011]
In addition, if a conical light reflecting surface having a central axis substantially coincident with the central axis of the cylindrical surface is disposed inside the space surrounded by the cylindrical surface, light emitted from the sample is disposed outside the flow-through cell. It can be efficiently guided to the detector for detection.
The light exit surface may be a dome-shaped indentation surface such as a hemispherical surface or a paraboloid, and in this case as well, it may be inserted near the indentation formed by the dome-shaped light exit surface or partially in the indentation. By arranging the photodetector in this manner, it is possible to efficiently detect light emission from the sample reflected and collected by the light reflecting surface substantially similar to the light emitting surface.
[0012]
In addition, a magnet can be disposed at least outside the wall surface of the sample channel facing the light exit surface. By arranging a magnet outside the flow-through cell, it is possible to employ a method of improving measurement accuracy by performing B / F separation using magnetic beads. The magnet may be an electromagnet or a permanent magnet. Magnetic beads captured on the wall of the flow-through cell by the magnetic force of the magnet, after the analysis is completed, by turning off the current to the electromagnet, or mechanically moving the permanent magnet away from the flow-through cell to weaken the magnetic force, Drain from the flow-through cell.
[0013]
When the flow-through cell is used in fluorescence analysis, a means for irradiating light to the inner wall surface of the flow channel adjacent to the magnet is provided in order to irradiate the luminescent reagent bound to the substance to be measured with excitation light. The light irradiating means guides light from a light source such as a halogen lamp with an optical fiber to the object to be measured through a light transmitting window provided in a part of the light reflecting surface or through a light transmitting light emitting surface. The excitation light is irradiated to the labeled luminescent substance.
[0014]
When the flow-through cell is used in the electrochemiluminescence analysis method, a working electrode and a counter electrode for applying a voltage to the luminescent substance labeled with respect to the measurement object are arranged on the inner wall of the sample flow path. At this time, a thin film of a metal material resistant to electrochemical corrosion such as Pt is plated or deposited on the inner wall of the sample flow path to form a working electrode, and a thin film of a metal material such as Pt is also plated or deposited in the vicinity thereof. Thus, the light reflecting surface and the electrode can be combined with a metal material thin film such as Pt.
[0015]
According to the present invention, it is possible to increase the effective solid angle when light emitted from a luminescent substance labeled with respect to a measurement object enters a photodetector to 2π or more, and weak light emitted from a sample solution. The measurement sensitivity can be improved by collecting and measuring the light efficiently.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 and FIG. 2 are schematic diagrams when a “donut type” flow-through cell, which is an embodiment of a flow-through cell according to the present invention, is applied to a fluorescence analysis method. FIG. 1 is a partially broken perspective view in which one of a pair of magnets arranged outside the cell is removed to make the structure easy to see, FIG. 2A is a plan view, and FIG. 2B is a side view. FIG.
[0017]
The flow-through cell of this embodiment is connected to a cell body 1 that surrounds a cylindrical space 10 provided in the center to form an annular sample channel, and to the sample channel of the cell body 1 A sample inlet 11 and a sample outlet 12 are provided. In the illustrated example, the cylindrical space 10 in the center of the flow cell is closed at the top, and the photodetector 2 such as a small photomultiplier tube is disposed in the cylindrical space 10.
[0018]
The cell body 1 is made of a light-transmitting polymer material such as acrylic or polymethyl methacrylate, and the outer cylindrical surface 13 except for the cylindrical surface 14 of the cylindrical space 10 in which the photodetector 2 is disposed. The upper surface 15 and the lower surface 16 are plated with a metal such as Al, Ag, Au or the like, so that the surface on the sample flow path side is a light reflecting surface. The inner cylindrical surface 14 remains transparent without being plated. A plurality of light transmissive windows 17 are provided on the upper surface 15 of the cell body 1, and excitation light from a light source 5 such as a halogen lamp guided by the optical fiber 4 flows through the light transmissive window 17. It is introduced into the through-cell body 1 so that the inner surface of the sample channel outer surface 13 can be irradiated.
[0019]
Magnets 3 a and 3 b are arranged along the outer periphery of the outer surface 13 of the flow-through cell body 1. The magnetic force of the magnets 3a and 3b can be controlled by turning on / off the excitation current if it is an electromagnet, and by changing the distance from the cell body 1 if it is a permanent magnet. The magnets 3a and 3b used in this example are permanent magnets. The magnets 3a and 3b are arranged at a distance from the cell body 1 in the figure, but are arranged in close contact with the outer wall of the cell body 1 during analysis.
[0020]
Next, a fluorescence immunoassay method using this flow-through cell will be described. For example, in the case of the sandwich method in which an antibody is recognized by sandwiching two types of antigens, a sample includes (1) a substance that binds an antigen recognition site 1 and a fluorescent reagent, (2) a substance that binds an antigen recognition site 2 and biotin, (3) Three kinds of substances, magnetic beads having a diameter of several micrometers, coated with streptavidin on the surface are added to a sample solution containing an antibody to be measured. Thereafter, by incubating the sample solution at 37 ° C., (1) and (2) sandwich the antibody to be measured from both sides by antigen-antibody reaction, and (2) and (3) bind by avidin-biotin reaction. To do. By these reactions, the fluorescent reagent can be bound to the antibody and bound onto the magnetic beads.
[0021]
The sample solution containing the measurement target prepared in this way flows into the flow-through cell main body 1 from the sample inflow portion 11 and flows separately into the left and right flow paths of the cylindrical space 10. At this time, the partition plates 18a and 18b may be provided so that the sample solution is smoothly divided into the left and right channels. The magnetic beads in the sample solution are captured on the inner wall of the outer surface 13 of the cell body 1 by the magnetic force of the magnets 3 a and 3 b arranged outside the flow-through cell body 1. Components other than the magnetic beads are discharged from the sample outflow portion 12. By separating the fluorescent reagent bound to the antibody and the fluorescent reagent not bound to the antibody (B / F separation), immunoassay can be performed without being affected by the free fluorescent reagent not bound to the antibody. Can do.
[0022]
In the case of the fluorescence method, since the label substance needs to be photoexcited, light from the light source 5 such as a halogen lamp guided by the optical fiber 4 is introduced into the cell body 1 through the light transmissive window 17, and the outer wall surface is introduced. The magnetic beads captured on the inner wall of 13 are irradiated. The fluorescence generated from the fluorescent reagent bound on the magnetic beads is reflected by the side surface 13, the upper surface 15 and the lower surface 16 of the cell main body 1 which is a light reflecting surface, and is condensed on the central portion of the cell main body 1 to be light. The light is emitted from the transparent cylindrical surface 14 and is efficiently detected by the photodetector 2.
[0023]
After one measurement is completed, a cleaning solution for cleaning the inside of the cell is introduced from the sample inflow portion 11 and the magnetic force of the magnets 3a and 3b is weakened (if the magnets 3a and 3b are permanent magnets, the cell By separating from the main body 1), the measured sample solution including the magnetic beads is discharged from the sample outflow portion 12. Thus, since it is a flow-through method, it is possible to continuously measure many sample solutions in a short time.
[0024]
In the above example, the outer surface of the cell body 1 is a light reflecting surface, but the inner surface of the cell body 1 in contact with the sample solution may be made a light reflecting surface by plating or the like. Moreover, the cylindrical space 10 provided in the center part of the cell main body 1 is good also as a hole penetrated up and down as shown in FIG.
[0025]
4 and 5 are schematic views for explaining another embodiment of the flow-through cell according to the present invention. In the previous embodiment, a photodetector such as a small photomultiplier tube is arranged inside a cylindrical space formed in the center of the flow-through cell. On the other hand, what is described here is an example in which a photodiode is used as a photodetector. 4 is a partially broken perspective view corresponding to FIG. 1, and FIG. 5 is a plan view and a side view corresponding to FIG. 4 and 5, the same parts as those in FIGS. 1 and 2 are denoted by the same reference numerals as those in FIGS. 4 and 5, and the detailed description thereof is omitted.
[0026]
In this embodiment, the photodiode 20 is arranged so as to face the columnar space 10 in the center of the cell body 1. A light-reflecting conical surface 25 is added inside the cylindrical space 10. The fluorescence emitted from the light-transmitting cylindrical wall surface 14 is reflected by the conical surface 25, bent in the course, and enters the light receiving surface of the photodiode 20 disposed below the cell body 1. Connected to the photodiode 20 is a signal processing circuit constituted by a low noise IV conversion unit 21, a voltage control unit 22, a BNC connector 23, a high voltage generation unit 24, and the like. The signal from the BNC connector 23 can be processed by a personal computer by A / D conversion.
[0027]
The illustrated magnets 3a and 3b are permanent magnets. The magnets 3a and 3b are arranged at a distance from the cell body 1 in FIG. 5. However, the magnets 3a and 3b are arranged in close contact with the outer wall of the cell body 1 during analysis, and are separated from the cell body 1 during cleaning inside the cell. The
[0028]
FIG. 6 is a schematic view when the flow-through cell of the present invention is applied to an electrochemiluminescence method. Here, an example in which a photodiode is used as the light receiving portion as in FIG. 4 will be described. However, as shown in FIG. 1, light detection such as a small photomultiplier tube arranged in the cylindrical space 10 at the center of the cell body 1 is performed. It is of course possible to use a vessel. In FIG. 6, the same parts as those in FIG. 4 are denoted by the same reference numerals as those in FIG. Further, in FIG. 6, one of the pair of magnets 3 a and 3 b is omitted in the same manner as in FIG.
[0029]
In the case of the electrochemiluminescence method, it is not necessary to carry out photoexcitation unlike the fluorescence method, and therefore it is not necessary to provide the light source 5, the optical fiber 4, and the light transmissive window 17 as shown in FIG. Instead, the working electrode 30 made of Pt or the like is disposed on the inner surface of the outer side surface 13 constituting the sample flow path so as to contact the sample solution, and the counter electrodes 31a to 31d made of Pt or the like are similarly brought into contact with the sample solution. In the vicinity of the working electrode 30.
[0030]
Although the four counter electrodes 31 a to 31 d are provided in the drawing, only the counter electrodes 31 a and 31 b provided on the inner surface of the upper surface 15 or only the counter electrodes 31 c and 31 d provided on the inner surface of the lower surface 16 may be used. Further, the light reflecting surface can be shared by the working electrode 30 and the counter electrodes 31a to 31d provided on the inner surface of the sample flow path.
[0031]
Next, an electrochemiluminescence immunoassay method using this flow-through cell will be described. For example, in the sandwich method in which an antibody is recognized by sandwiching two kinds of antigens, the sample is processed in the same manner as in the fluorescence immunoassay described above, and a luminescent reagent is bound to the antibody and bound to magnetic beads. The The sample solution containing the measurement target prepared in this way flows into the flow-through cell main body 1 from the sample inflow portion 11 and flows separately into the left and right flow paths of the cylindrical space 10. The magnetic beads in the sample solution are captured on the inner wall of the outer surface 13 of the cell body 1 by the magnetic force of the magnets 3 a and 3 b arranged outside the flow-through cell body 1, and components other than the magnetic beads are extracted from the sample outflow portion 12. Discharged.
[0032]
After capturing the magnetic beads in the cell body 1, if a voltage of about 2.0 to 2.4 V is applied between the working electrode 30 and the counter electrodes 31a to 31d, the luminescent reagent emits light by this voltage application. This light emission from the reagent is reflected by the side surface 13, the upper surface 15, and the lower surface 16 of the cell body 1 that is a light reflecting surface, and is collected at the center of the cell body 1 and emitted from the light-transmitting cylindrical surface 14. However, it is detected efficiently by the photodetector 20. After one measurement is completed, a cleaning solution for cleaning the inside of the cell is introduced from the sample inlet 11 and the magnetic force of the magnets 3a and 3b is weakened, so that the measured sample solution including the magnetic beads can be obtained. The sample is discharged from the sample outlet 12.
[0033]
FIG. 7 and FIG. 8 are schematic views when a “recessed” flow-through cell, which is another embodiment of the flow-through cell according to the present invention, is applied to the fluorescence method. FIG. 7 is a perspective view of an excitation light irradiation optical system, a photodiode as a photodetector, and a magnet to be mounted, FIG. 8A is a plan view of the cell body, and FIG. 8B is a view of the cell body. It is a side view.
[0034]
The flow-through cell of this embodiment has a dome-shaped depression 40 whose main body is made of a light-transmitting polymer material such as acrylic or polymethyl methacrylate and has a hemispherical shape, a paraboloid shape, or the like at the center. Have A sample channel 45 having a substantially uniform thickness is formed around the recess 40. The wall surface of the flow path forming the depression 40, that is, the depression surface 44 is light-transmitting, and the convex curved surface 43 facing the light-transmitting depression surface 44 is plated with a metal such as Al, Ag, or Au on the inner surface or the outer surface. The light reflectivity is given by doing.
[0035]
A magnet 47 is arranged outside the convex curved surface 43 so as to fit into the convex curved surface 43 (in FIG. 7, the magnet 47 is illustrated away from the convex curved surface 43). The photodiode 20 is disposed on the side of the recess 40, and excitation light from the light source 5 such as a halogen lamp guided by the optical fiber 4 is passed through the light-transmitting recess surface 44 into the sample channel 45. It can be introduced.
[0036]
Next, a fluorescence immunoassay method using this flow-through cell will be described. For example, in the sandwich method in which an antibody is recognized by sandwiching two types of antigens, the sample is processed in the same manner as in the fluorescence immunoassay described above, and a fluorescent reagent is bound to the antibody and bound to the magnetic beads. The The sample solution thus prepared flows into the flow-through cell body from the sample inflow portion 41 and flows through the sample flow path 45 along the dome-shaped depression. The magnetic beads in the sample solution are captured on the inner wall of the convex curved surface 43 that forms the sample channel 45 by the magnetic force of the magnet 47 disposed outside the flow-through cell main body 1. Components other than the magnetic beads are discharged from the sample outflow portion 42.
[0037]
Excitation light guided by the optical fiber 4 is introduced into the cell body through the light-transmitting recessed surface 44 and is irradiated onto the magnetic beads captured on the inner wall of the convex curved surface 43. Fluorescence generated from the fluorescent reagent bound on the magnetic beads is reflected by the convex curved surface 43 serving as a light reflecting surface, condensed on the side of the dent 40, and emitted from the light transmissive dent surface 44. It is detected efficiently by the diode 20. After one measurement, the cleaning solution for cleaning the inside of the cell is introduced from the sample inflow portion 41 and the magnetic force of the magnet 47 is weakened so that the measured sample solution including the magnetic beads flows out of the sample. It is discharged from the part 42.
[0038]
Here, the case where the method is applied to the fluorescence method has been described. However, the working electrode and the counter electrode are arranged on the inner surface of the sample channel 45 instead of the excitation light irradiation optical system, whereby the flow-through cell shown in FIGS. Can also be applied to the electrochemiluminescence method.
[0039]
【The invention's effect】
According to the present invention, since the measurement cell structure has a shape with excellent light collecting properties, the measurement sensitivity of the fluorescence method and the electrochemiluminescence method can be improved at a low cost.
[Brief description of the drawings]
FIG. 1 is a partially broken perspective view showing an example of a flow-through cell according to the present invention.
2A is a plan view and FIG. 2B is a side view of the flow-through cell shown in FIG.
FIG. 3 is a partially broken perspective view showing another example of the flow-through cell according to the present invention.
FIG. 4 is a partially broken perspective view showing another example of the flow-through cell according to the present invention.
5A is a plan view and FIG. 5B is a side view of the flow-through cell shown in FIG.
FIG. 6 is a partially broken perspective view showing another example of the flow-through cell according to the present invention.
FIG. 7 is a perspective view showing another example of the flow-through cell according to the present invention.
8A is a plan view and FIG. 8B is a side view of the cell main body of the flow-through cell shown in FIG. 7;
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Cell main body, 2 ... Photodetector, 3a, 3b ... Magnet, 4 ... Optical fiber, 5 ... Light source, 10 ... Cylindrical space, 11 ... Sample inflow part, 12 ... Sample outflow part, 13 ... Outer surface, 14 ... Cylindrical surface, 15 ... upper surface, 16 ... lower surface, 17 ... light transmissive window, 18a, 18b ... partition plate, 20 ... photodiode, 21 ... low noise IV conversion unit, 22 ... voltage control unit, 23 ... BNC Connector, 24 ... High voltage generating part, 25 ... Light reflecting conical surface, 30 ... Working electrode, 31a-31d ... Counter electrode, 41 ... Sample inflow part, 42 ... Sample outflow part, 43 ... Convex curved surface, 44 ... Indentation Surface, 45 ... sample flow path, 47 ... magnet

Claims (6)

A flow comprising a sample inflow part into which a sample fluid flows, a sample outflow part, a sample channel connecting the sample inflow part and the sample outflow part, and a light exit surface that is a part of the wall surface of the sample channel In the through cell, the light exit surface has a substantially axisymmetric three-dimensional shape, and the wall surface of the sample channel facing the light exit surface has a surface shape substantially similar to the light exit surface, The wall surface of the sample channel other than the exit surface is a light reflecting surface ,
The flow-through cell, wherein the light exit surface is a cylindrical surface, and the sample flow path branches the sample fluid flowing in from the sample inflow portion in two directions around the cylindrical surface. .   2. The flow-through cell according to claim 1, wherein a conical light reflecting surface having a central axis substantially coincident with the central axis of the cylindrical surface is disposed inside the space surrounded by the cylindrical surface.   2. The flow-through cell according to claim 1, wherein the light exit surface is a dome-shaped indentation surface. The flow cell according to any one of claims 1 to 3 , wherein a magnet is disposed outside the wall surface of the sample channel facing at least the light emitting surface. 5. The flow cell according to claim 4, further comprising means for irradiating light to the inner wall surface of the flow path adjacent to the magnet. The flow-through cell according to claim 4, wherein a working electrode and a counter electrode are disposed on an inner wall of the sample channel.

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