JP4518015B2 - BF separation device - Google Patents

Description

  The present invention relates to a device for measuring a test substance contained in a specimen.

  In recent years, it has become possible to measure various substances due to advances in analysis, analysis, and inspection techniques. In particular, in the field of clinical testing, the development of measurement principles based on specific reactions such as biochemical reactions, enzyme reactions, or immune reactions has made it possible to measure substances in body fluids that reflect disease states.

  Among them, a field of clinical examination called point of care testing (POCT) has attracted attention. In POCT, simple and quick measurement is the first, and efforts are being made to shorten the time from when a sample is collected until a test result is obtained. Therefore, what is required for POCT is a simple measuring principle, a measuring device that is small, portable, and easy to operate.

  With recent advances in technological development, for example, small measuring devices that can be easily measured, such as blood glucose sensors, have been developed. The ripple effect of POCT enables quick and accurate diagnosis by acquiring quick measurement results, reducing test costs, reducing the burden on the subject due to the small amount of specimens such as blood, and infection. It is possible to reduce the amount of radioactive waste. Currently, the shift to POCT is rapidly occurring in clinical tests, and POCT-compatible measuring instruments are being developed to meet the needs.

  Among them, as a measuring device applied to the POCT measuring device, many use an immunoassay principle. Immunoassays mainly include methods that do not require BF separation, such as turbidimetric methods, specific methods, and latex agglutination methods, and immunocomplexes such as enzyme immunoassay methods, fluorescence measurement methods, and luminescence measurement methods. It is roughly divided into measurement methods that require separation from reactants (BF separation: BF is an abbreviation for Binding / Free). Reagents are placed on a filter paper such as an immunochromatograph from an automated instrument. A supported dry chem device is provided.

  In particular, an immunoassay method that requires BF separation is an important step because the S / N ratio is deteriorated unless a step such as washing is sufficiently performed. Recently, an immunoassay device including a highly systematized BF separation process using magnetic fine particles has been proposed.

  It is a method of performing a binding assay for an analyte of interest in a sample comprising the following steps: (a) preparation of the following composition: (i) the sample (ii) a labeled compound capable of inducing electroluminescence An assay fulfillment material containing bound components, and (iii) a plurality of magnetically sensitive suspended particles capable of specifically binding to the analyte and / or the assay fulfillment material; (b) incubating such composition with the particles Forming a complex containing the labeled compound; (c) introducing the composition into an assay cell; (d) applying a magnetic field to the particles to collect the complex on the electrode surface; A voltage is applied to the electrode during or after the collection to induce luminescence in the labeled compound in the collected complex; and (f) emitted at the electrode surface Measured Minessensu measures the presence of the target analyte in the sample; however, is to place vertically beneath the electrode surface oriented horizontally a plurality of magnets alternating oriented north-south poles.

What is shown here is one that uses electrochemiluminescence, but is one effective BF separation method even for chemiluminescence or enzymatic reaction.
Japanese National Patent Publication No. 6-508406

  However, a measuring instrument that is automated in each measuring process often seen in so-called clinical testing equipment, represented by a BF separation process by fixing magnetic particles using a magnet or the like, has a complicated configuration and is expensive. . Moreover, since all the processes are automated, the equipment becomes large. Recently, there are desktop types, but they still occupy most of the tabletops.

  An object of the present invention is to easily perform BF separation in a device for measuring a test substance contained in a specimen.

A device for measuring a test substance in a specimen having a plurality of chambers and a flow path connecting the chambers installed on a turntable, wherein the device is provided in at least one of the plurality of chambers. The first specific binding substance that specifically binds to the test substance and the test substance are mixed to form a bound complex and an unbound substance, and the bound complex and the unbound substance are separated. There is a BF separation chamber, and the BF separation chamber has a carrier sensitized with either the test substance or a similar substance, or a second specific binding substance that specifically binds to the test substance. After the carrier is allowed to act on the mixed solution of the bound complex or the unbound substance , the bound complex and the unbound substance are separated by separating the carrier by centrifugal force under the rotation condition.

  With this configuration, BF separation can be easily performed in a device for measuring a test substance contained in a specimen.

  According to the BF separation device of the present invention, the BF separation step, which is an important pretreatment step of the immunoassay method, can be performed only by controlling the rotation, so that a simpler measurement device can be provided. In addition, since the rotation control is utilized, it is possible to carry out other pretreatment steps such as blood cell separation in addition to this, and furthermore, the fluid transfer / stop can be controlled by rotation. The measurement operation can be simplified only by spotting a specimen, and an effective POCT apparatus can be provided.

  As an embodiment of the present invention, first, a mechanism of a BF separation device will be described. A device for measuring a test substance in a specimen, comprising a plurality of chambers and a flow path connecting the chambers installed on a turntable, wherein at least one of the plurality of chambers The first specific binding substance that specifically binds to the test substance and the test substance are mixed to form a bound complex and an unbound substance, and the bound complex and the unbound substance are There is a BF separation chamber for separation, the BF separation chamber comprising a carrier sensitized with a second specific binding substance that binds only to either the bound complex or the unbound substance, the bound complex or unbound A BF separation measurement device capable of separating the bound complex and the unbound material by allowing the carrier to act on the mixed liquid of the binding substance and then separating the carrier by centrifugal force under rotation conditions. The scan is.

  This makes it possible to control rotation without the need for a BF separation step having a special reagent or a complicated mechanism associated therewith, such as the magnetic use of magnetic fine particles and the use of biotin / avidin interaction found in large-scale automated equipment. BF separation can be carried out only with this.

  The most important point of BF separation shown in the device of the present invention is the centrifugal force accompanying rotation and the use of a carrier that can be collected in the outer circumferential direction by the centrifugal force.

  In general, the binding reaction between the test substance and the first specific binding substance is based on an equilibrium reaction, and in the reaction system of the binding reaction, a binding complex of the test substance and the first binding substance, There are three types of substances that are the unbound substance test substance and the first binding substance that remain without being used in the bound complex. In this case, when detecting the amount of binding, a second binding substance containing a substance capable of emitting some detection signal, which can bind to either the first binding substance or the test substance, is used. If the body and the unbound substance are not separated, the S / N ratio becomes worse and accurate measurement cannot be performed.

  Therefore, the device of the present invention uses a carrier having a function of capturing a bound complex or unbound substance, captures either the bound complex or unbound substance on the carrier, and then rotates the device. Then, it is separated by collecting it near the bottom by centrifugal force. After separation, if rotation is stopped and a structure that can be introduced into the flow path coming out from the inner peripheral side of the separation boundary surface by a capillary phenomenon or the like is constructed, it can be rotated further and separated from the separation boundary surface. The liquid on the inner peripheral side can be transferred to another chamber, and BF separation can be completed easily.

  Therefore, the conditions of the carrier of the present invention, such as the weight and material, are selected so that they can settle by centrifugal force.

  Furthermore, if the sedimentation can be performed with a small centrifugal force, the time for the separation process can be shortened and the load on the rotating device or the like can be reduced. In addition, since the carrier needs to have a function of capturing the bound complex or unbound substance, it is necessary to sensitize the substance to be captured and the specific binding substance.

  Considering the above points, for example, latex particles, metal colloids, carriers having chemical bond functional groups, and the like are preferable. In view of the ease of the influence of centrifugal force, the particle size is preferably larger. However, as the size of the carrier increases, the surface area of the carrier in the capture system decreases, so the amount of specific binding substance sensitized to the carrier decreases. Therefore, the optimum conditions for sedimentation and trapping amount due to centrifugal force should be selected with due consideration of the amount of target analyte.

  After the BF separation step in the BF separation chamber of the present invention, as one embodiment, a flow path is provided that can transfer the supernatant after the carrier is separated by centrifugal force to another chamber. Thereby, subsequent detection can be implemented simply.

  That is, another chamber to which the supernatant liquid is transferred contains a substance for detecting the BF-separated substance in the supernatant liquid. Alternatively, as another embodiment, the carrier collected near the bottom surface of the chamber after the carrier is centrifuged may be transferred to another chamber. That is, a substance for detecting the substance separated by BF trapped on the carrier is included.

  The BF separation step by rotation control of the present invention is represented by the following two patterns, for example. The two patterns are a pattern in which the supernatant side is transferred to another chamber after the BF separation step and a pattern in which the carrier side is transferred to another chamber after the BF separation step.

  The former case will be described, for example, in the form shown in FIG. FIG. 1 is a top view of an example of a flow path configuration diagram of the measurement device of the present invention.

  That is, a carrier in which a test substance in a specimen, a first specific binding substance that specifically binds to the test substance, and a substance that specifically captures any of a binding complex and an unbound substance are sensitized A mixing chamber 11, a BF separation chamber 12 for BF separation of the mixed solution in the mixing chamber, and a detection chamber 13 for receiving the supernatant after BF separation, 15 is connected.

  Here, it is important that the carrier collected near the bottom surface of the BF separation chamber 12 by the BF separation is set at a position where it does not flow into the detection chamber 13 through the flow path 15. Therefore, it is set on the upper side (inner peripheral side) from the interface between the solid phase region and the liquid phase region, which is the carrier after BF separation.

  The latter case will be described in the form shown in FIG. 2, for example. FIG. 2 shows a view of an example of a flow path configuration diagram of the measuring device of the present invention as viewed from above.

  That is, a carrier in which a test substance in a specimen, a first specific binding substance that specifically binds to the test substance, and a substance that specifically captures any of a binding complex and an unbound substance are sensitized A mixing chamber 21, a BF separation chamber 22 for BF separation of the mixed solution in the mixing chamber, and a waste liquid chamber 23 for receiving the supernatant after BF separation, and further receiving the carrier side There is a detection chamber 24 for each of which is connected by flow paths 25, 26, 27.

  Here, the liquid phase portion in the BF separation chamber 22 generated by the BF separation flows into the waste liquid chamber 23 through the flow path 26, and then only the carrier flows into the detection chamber 24. In this case, the flow paths 26 and 27 for connecting the respective chambers take a form such as an inverted U-shape that exits from the chamber to the inner peripheral side and then folds back to connect to the chamber installed on the outer peripheral side. It is important that the positional relationship between the folded portions 28 and 29 and the upper surface 20 of the BF separation chamber 22 is important. In this case, the folded portions 29 and 28 and the upper surface 20 are preferably arranged in this order from the inner peripheral side.

  Thereby, by applying a gradient from the high state to the low state, the BF separation, the transfer of the supernatant, and the transfer of the carrier can be sequentially performed.

  In the chamber configured in the measuring device of the present invention, an air passage is important and an air hole is formed.

  The flow path of the present invention rises up to a predetermined position on the inner peripheral side, that is, on the side close to the rotation center, from the upper surface (end surface close to the rotation center) of the chamber, and then the outer peripheral side from the position of the outlet of the flow path from the chamber It is formed so as to go down to the position. Here, the downward direction is a direction in which centrifugal acceleration acts on the fluid in the device by the rotation of the device body, and the upward direction is the opposite direction.

  As a specific flow path form, for example, a flow path such as an inverted U-shape can be adopted. In this case, it is important that the inverted U-shaped apex is located on the inner peripheral side of the upper surface of the chamber. By adopting such a configuration, liquid can be held in the chamber during rotation of the device body.

  Furthermore, by stopping the rotation, when the fluid is released from the centrifugal force, the fluid can be transferred to the place of the next process by capillary action. If the liquid is not held in the chamber during the rotation and is continuously transferred, the inverted U-shaped apex is not positioned on the inner peripheral side from the upper surface of the chamber (at the upper surface position of the chamber or the outer peripheral side thereof). To be positioned).

  On the flow path exiting the chamber, a region having a larger thickness than the flow path (hereinafter referred to as a capillary valve) is provided at an appropriate position. 1 and 2 shown above can be implemented by the combination of basic parts shown here.

  Examples of the basic parts of the BF separation measuring device described above will be described in detail with reference to FIGS. 3 and 4. FIG. 3 shows a first flow path part constituting the chamber and flow path, and FIG. 4 shows a second flow path part in which a capillary valve, which is a thick part, is arranged in the flow path.

  The flow path part 31 shown in FIG. 3 includes a chamber 312 serving as a place for achieving analysis steps such as dilution, mixing, reaction, and detection, and a flow path 311 formed to communicate with the chamber 312 and air. Holes 313 are respectively formed. The chamber 312 has an extended portion 312a that is used when another flow path is connected on the side separated from the portion to which the flow path 311 is connected, and the air holes 313 are arranged so as to be separated from both of them. .

  In this flow path part 31, by setting the apex portion 314 of the inverted U-shaped flow path to the inner peripheral side from the upper surface 315 of the chamber 312, the fluid can be held in the chamber by the centrifugal force generated during the rotation. . In order to keep the fluid flowing continuously, the apex portion 314 of the inverted U-shaped channel may be set on the outer peripheral side from the upper surface 315 of the chamber 312.

  In the case of the present embodiment, since it is necessary to once hold the fluid in the chamber 312, the vertex portion 314 of the inverted U-shaped flow path is set on the inner peripheral side from the upper surface 315 of the chamber 312.

  A flow path part 42 shown in FIG. 4 has a substantially inverted U-shape as a whole, and a flow path 421 whose both ends are inclined toward one side in the short direction of the overall shape, and a flow path 421 disposed on the flow path 421. And a capillary valve 422 forming a space having a larger thickness (cross-sectional area). The capillary valve 422 of the flow path part 42 is a stationary means for stopping the fluid with respect to the fluid transfer utilizing the capillary phenomenon, and by making the thickness (cross-sectional area) of the flow path sufficiently larger than the front and rear flow paths. Then, after the rotation of the device body is stopped, the flow of the fluid flowing through the flow path 421 is stopped by capillary action, and the fluid in a required section can be stopped. In the present embodiment, a capillary valve is provided as appropriate in consideration of the location where the fluid is desired to be stationary, the timing, and the section.

  FIG. 5 and FIG. 6 are diagrams for explaining a manufacturing procedure of a device having the basic flow path parts shown in FIGS. 3 and 4. As shown in these drawings, the inspection device of this example is 3 It has a layer structure. FIG. 5 is a view showing a method for producing the flow path part 31, and FIG. 5B is a sectional view taken along the line II-II in FIG. 5A is a view showing a method for producing the flow path part 12, and FIG. 5B is a sectional view taken along the line III-III in FIG. 1 showing the completed state.

  The reaction of the binding substance of the present invention refers to, for example, a specific binding reaction shown in an antigen-antibody reaction or a specific binding reaction based on a DNA base sequence. Since no signal is output, it is necessary to detect the amount of binding reaction after BF separation in some form.

  For example, as described above, the first binding substance contained in the binding complex is labeled with an enzyme, a chemiluminescent substrate, or the like, and a color development reaction or a luminescence reaction is performed to detect optically. Alternatively, it may be possible to detect by fluorescent measurement by labeling a fluorescent substance. Therefore, in the device of the present invention, it is necessary to provide a detection chamber for carrying out the detection reaction by transferring the substance after the BF separation following the BF separation step in the BF separation chamber. In the above example, for example, a substance that reacts with the enzyme or the chemiluminescent substrate contained in the first binding substance may be held in the detection chamber in a dry state. If the liquid after BF separation is transferred, a color development reaction or a luminescence reaction occurs immediately, and this may be detected optically. In the case of a fluorescent substance, it is only necessary to irradiate excitation light into the transferred detection chamber to emit fluorescence and detect it.

  The measurement method by BF separation of the present invention is more commonly used in immunoassays based on the antigen-antibody reaction principle, and is more general. For example, radioimmunoassay (RIA), enzyme immunoassay (EIA) enzyme immunosolid phase assay (ELISA), chemiluminescence immunoassay (CLIA), chemiluminescence enzyme immunoassay (CLEIA), electrochemiluminescence immunoassay ( ECLIA). There is also a simple immunochromatography method. In these measurement methods, after the separation of BF, the presence or amount of the target test substance can be determined by measuring the amount of binding or the amount of unbound substance by radioactivity detection, color detection, or luminescence detection. Measure.

  As described above, the detection substance or detection reaction of the present invention relates to an enzyme reaction, a thing related to chemiluminescence, or a thing related to electrochemiluminescence, and can be carried out in a detection chamber transferred after BF separation. .

  The second specific binding substance sensitized to the carrier of the present invention is a substance that specifically binds to the test substance. The measurement flow in this case is shown in FIG. First, the test substance 51 and the first specific binding substance 52 in the specimen are mixed and then allowed to act on the second specific binding substance sensitized to the carrier (FIG. 5a).

  As a result, a sandwich-type binding complex can be formed on the carrier via the test substance (FIG. 5b).

  Then, by rotating, the carrier is collected near the bottom surface in the BF separation chamber by centrifugal force, and the sandwich-type binding complex and other substances can be separated (FIG. 5c).

  After the separation, the carrier side shown above is transferred to another detection chamber and transferred to the detection chamber using the BF separation measuring device of FIG. 2, and the amount of the sandwich-type binding complex is first specific in the detection chamber. Measured by 55 of bound material.

  The second specific binding substance sensitized to the carrier of the present invention is a test substance that binds to the first binding substance or a substance similar thereto. The measurement flow in this case is shown in FIG. First, after the test substance 61 and the first specific binding substance 62 in the specimen are mixed, they are allowed to act on the second specific binding substance 63 sensitized to the carrier (FIG. 6a).

  Thereby, a competitive reaction system is constructed in the mixed system, and the amount of the first binding substance on the carrier is reduced according to the amount of the test substance. Alternatively, the amount of the first binding substance in the liquid phase is increased (FIG. 6b).

  Then, by rotating, the carrier is collected near the bottom surface in the BF separation chamber by centrifugal force and separated into a solid phase and a liquid phase (FIG. 6c).

  After the separation, the carrier side is transferred to the detection chamber using the BF separation measuring device having a pattern for transferring the carrier side to another detection chamber, and 65 of the first binding substance on the carrier is measured in the detection chamber. Alternatively, the liquid phase side is transferred to the detection chamber using a BF separation measuring device having a pattern for transferring the liquid phase side to the detection chamber, and 65 of the first binding substance in the liquid phase is measured in the detection chamber.

  The carrier of the present invention may further contain a substance that can be aggregated. Thereby, the carrier becomes larger due to aggregation, and the BF separation step can be completed in a short time with a small centrifugal force. The substance to be aggregated is, for example, a first specific substance sensitized by another particle or the like.

  Examples of the sample to be measured by the BF separation measurement device of the present invention include a blood sample and a urine sample. In the case of a blood sample, red blood cells are included, and the red blood cells may adversely affect the measurement. In general, in the case of a blood sample, red blood cells are removed by centrifugation or the like prior to analysis / measurement.

  Since the measurement device of the present invention is based on rotation control based on rotation conditions, it can be centrifuged prior to BF separation. As a result, a simple measuring device that can be implemented in a one-step operation can be produced.

  In the BF separation measurement device of the present invention, the test substance to be measured includes plasma components and serum components in blood, or proteins and hormones that are red blood cell components.

  Furthermore, not only in blood, but also proteins, hormones, etc. in urine, tears, sweat, and saliva are targeted. That is, all the objects currently measured in the clinical chemistry measurement field and measured by the binding reaction principle such as immune reaction can be the objects to be measured by the measuring device of the present invention.

  As a form of the manufacturing process of this invention, it shows in FIG. 7, for example. 7A is a view showing a method of producing the flow path part FIG. 3, and FIG. 7B is a sectional view taken along the line II-II in FIG. 3 showing the completed state.

  As a representative example, a method for producing the flow path part of FIG. 3 will be described in detail with reference to FIG.

  When forming the chamber 312 and the flow path 314, the portion 79 corresponding to the chamber 312 and the flow path 311 was cut using the double-sided adhesive sheet 71, as shown in the cutting step C in FIG. This cutting was performed by cutting a layer other than the release paper 74 of the adhesive layer on the lower surface so as to peel off the chamber and the flow path.

  On the other hand, an air hole 313 is opened in the top cover 76 in advance, and a recess 312C corresponding to the chamber 312 is formed.

  The three layers thus adjusted were bonded through a double-sided adhesive sheet 75 to produce a basic flow path part. Although not described here, the BF separation measuring device of the present invention can be obtained by inserting necessary reagents in a dry state before bonding the three-layer members.

  An apparatus for controlling the rotation of the measuring device of the present invention is shown in FIG. 8 includes a clamper 811 that fixes the device 80 (showing a schematic side shape of the device body), a turntable 813 that supports the device 80, a spindle motor 814 that rotates the turntable 813, And a control device 815 for controlling the rotation of the spindle motor 814. Although not shown here, if an optical system is also configured, it can be used as a detector, and a simple measurement system can be provided as a so-called device reading device.

  The BF separation measuring device according to the present invention can easily carry out the BF separation step in the process of the rotation operation, and is useful for the testing device in the POCT field mainly used in the clinical testing field.

The figure which looked at an example of the flow-path block diagram of the measuring device of this invention from the top The figure which looked at an example of the flow-path block diagram of the measuring device of this invention from the top The figure which shows the relationship between the chamber and flow path which are basic flow path parts of this invention The figure which shows the relationship between the capillary valve which is the basic flow path parts of this invention, and a flow path The figure which showed an example of the BF separation process method of this invention The figure which showed an example of the BF separation process method of this invention Diagram showing fabrication process The figure which shows the device for controlling the rotation of the device

Explanation of symbols

11, 21 Mixing chamber 12, 22 BF separation chamber 13, 24 Detection chamber 14, 15, 25, 26, 27 Channel 23 Waste liquid chamber 28, 29 Folded part 31, 42 Channel parts 311, 421 Channel 312 Chamber 312 a Extension Portion 313 Air hole 314 Vertex portion 315 Upper surface 422 Capillary valve 51, 61 Test substance 52, 62 First specific binding substance 63 Second specific binding substance 71 Double-sided adhesive sheet 74 Release paper 76 Top cover 79 Portion to flow path 311

Claims (8)

A device for measuring a test substance in a specimen having a plurality of chambers and a flow path connecting the chambers installed on a turntable, wherein the device is provided in at least one of the plurality of chambers. The first specific binding substance that specifically binds to the test substance and the test substance are mixed to form a bound complex and an unbound substance, and the bound complex and the unbound substance are separated. There is a BF separation chamber, and the BF separation chamber has a carrier sensitized with either the test substance or a similar substance, or a second specific binding substance that specifically binds to the test substance. hints, after reacted with the carrier in a mixture of binding complexes or unbound material, the rotating conditioning, child separating the unbound material and the binding complex by separating by centrifugal force carriers BF separation measuring device according to claim. The BF separation chamber according to claim 1 is provided with a flow path capable of transferring the supernatant liquid after separation of the carrier by centrifugal force to another chamber under rotation conditions. The BF separation measuring device according to claim 1. 3. The BF separation measuring device according to claim 2, wherein the another chamber into which the supernatant liquid according to claim 2 is transferred contains a substance for detecting a substance separated by BF in the supernatant liquid. . 2. The BF separation chamber according to claim 1, wherein the carrier collected near the bottom surface of the chamber after being centrifuged is transferred to another chamber under rotation conditions. BF separation measurement device. 5. The BF separation measuring device according to claim 3, wherein the other chamber to which the carrier according to claim 4 is transferred contains a substance for detecting the substance separated by BF trapped on the carrier. 6. The second specific binding substance sensitized to the carrier according to claim 1 can form a sandwich-type binding complex with the first specific binding substance via the test substance. The BF separation measuring device described. BF separation measuring device 6 described range claim 1, characterized in that it comprises further a substance capable of aggregating the carriers of claims 1 to 6, wherein. First binding substance of claims 1 to 7 described, BF separation measuring device of claims 1 7, wherein the includes a substance that emits a detectable signal.

Images