JP4260620B2 - Flow cell method - Google Patents

Description

  The present invention relates generally to the control of fluid flow across a surface, particularly a sensing surface, within a flow cell of an analytical device. More particularly, the present invention relates to the use of laminar flow cell technology to position fluid flow over a desired surface area within the flow cell.

  Flow cells are now widely used in various analytical systems. Typically, a flow cell has an inlet opening, a flow channel and an outlet opening. The sample fluid to be examined is introduced through the inlet opening, passes through the flow channel, and exits the flow cell through the outlet opening. Within this flow channel, the sample fluid can be analyzed. The flow cell may have more than one inlet opening, and optionally more than one outlet opening, allowing the desired manipulation of the flow pattern within the flow cell.

  In one type of flow cell, the flow channel comprises a sensing surface. This sensing surface is usually a layer of material on which a recognition element for the analyte in the sample is immobilized, which is typically a biochemical affinity partner for this analyte. When an analyte interacts with a recognition element, a physical or chemical change that can be detected by a detector (eg, optical detector, electrochemical detector or calorimetric detector) occurs on the sensing surface. The flow channel may comprise two or more sensing surfaces with different recognition elements.

  The sensing surface within the flow cell can be functionalized or sensitized in situ, ie, within the flow cell. WO 90/05305 discloses a method for functionalizing a sensing surface having functional groups on a surface by passing a reagent solution containing a bifunctional ligand or a multifunctional ligand over the sensing surface. ing. The ligand has the function of immobilizing the ligand on the sensing surface and at least another function exposed on the sensing surface to interact with the analyte.

  WO 99/36766 allows for different ligands to be immobilized on separate sensing areas within a single flow cell channel and allows controlled sample delivery to such sensitizing areas Disclosed are methods and systems that use dynamic addressing techniques. In one embodiment, a so-called Y-cell (which has two inlet ports and one outlet port) is used, where the sample fluid (or in the case of sensitization of the sensing surface, a sensitization). Laminar flow) is provided proximate to the laminar flow of the non-sensitizing fluid (e.g., the reference fluid) so that the fluids have an interface relative to each other on the sensing surface Flowing. By adjusting the relative flow rates of these two fluids, this interface can be positioned laterally so that the sample fluid (or sensitizing fluid) contacts the desired separate areas of the sensing surface. In a modification, a so-called ψ-shaped cell (which has three inlet ports) is used to sandwich the sample fluid (or sensitizing fluid) between two non-sensitizing fluid streams. However, the disadvantage of the method and system described in WO 99/36766 is that selective contact between the desired fluid and different areas of the sensing surface is only possible laterally, i.e. the flow between the flow cell ends. It is possible only in the direction crossing the extension of the road.

  WO 97/01087 discloses a flow cell having an inlet opening and an outlet opening for a sample. A further inlet opening for the reference fluid is provided, and this opening is positioned so that the reference fluid flow is opposite the sample in the flow channel. In this way, the sample fluid can be held away from the blocked volume occupied by the flowing reference fluid without the use of structural partitions in the flow channel. Typically, a detection layer containing a sensitivity recognition element for the analyte extends the entire length of the flow cell channel, and a non-existent region of the flow channel sample can be used to generate a reference signal. However, the flow cell of WO 97/01087, respectively, has a fixed longitudinal extension of the sample area and the non-sample area, the outlet opening being the inlet opening for the sample and the inlet opening for the reference fluid It is necessary to be arranged between the parts.

  It would be desirable to be able to selectively and variably control the extension of fluid flow in the longitudinal or normal direction of the flow cell. In this context, it is also desirable to be able to use a conventional flow cell (eg, the Y-shaped cell or the Ψ-shaped cell described above).

  The present invention fulfills these needs and provides further related advantages.

(Summary of the Invention)
Briefly, the present invention relates to the control of fluid flow on a surface in a flow cell using laminar flow cell technology that includes countercurrent positioning fluid across a variable flow channel region extending from one end of the flow cell to the other. More specifically, the interface between a laminar flow of a desired fluid and a laminar counterflow of another fluid (sometimes also referred to as a “blocking fluid” below) allows the fluid to flow by controlling the laminar flow of each fluid. It can be positioned at a desired distance from the inlet opening.

  Accordingly, a first aspect of the present invention relates to a method of operating an analytical flow cell device, the device comprising an elongated flow cell having a first end and a second end, at least two at the first end. An opening or port and at least one opening or port at the second end. The laminar flow of the first fluid is introduced at the first end of the flow cell, the laminar counter flow of the second fluid is introduced at the second end of the flow cell, and the laminar flow of each fluid is , At the first or second end of the flow cell, drained (regardless of other fluid flow). The position of the interface between the first fluid and the second fluid in the longitudinal direction of the flow cell is adjusted by adjusting the relative flow rates of the two fluids (in other words, the first fluid and the second fluid). By adjusting the ratio of the first fluid flow to the mixed fluid discharge flow).

  The first flow cell end and the second flow cell end are typically at the upstream and downstream ends, respectively, of the flow cell relative to the normal flow direction when sample fluid passes through the flow cell. is there.

  Preferably, the flow cell channel has at least one sensing surface on a wall surface in the flow cell disposed between the first (or upstream) end and the second (or downstream) end. The term “sensing surface” as used herein should be interpreted broadly. The term refers to, for example, sensing (or detecting) an interaction as well as a surface or surface layer that can interact with a fluid in contact with the surface or an analyte present in the fluid itself. Includes surfaces that can be chemically or physically sensitized to allow, as well as surfaces that can be chemically or physically activated to allow subsequent sensitization, for example.

  In one embodiment of the first mentioned aspect of the invention, the desired laminar flow is introduced through one port at the first end and through the second port at the same end. Drained and blocking fluid is introduced from the second end of the flow cell. This blocking fluid can be exhausted through the second port at the first end, or it can be exhausted through another port at the second end. Depending on the mutual flow rate of the two fluids, the interface between the two fluids (which extends substantially transverse to the longitudinal extension of the flow cell) is relative to the flow cell at the inlet end. / Can be positioned at a different distance from the exit end.

  One application of this embodiment is for selectively processing a desired portion of the sensing surface extending between the flow cell ends. In a common type of analytical flow cell, the sensing surface extends essentially the entire length between the ends of the flow cell so that only a portion of this sensing surface (usually very small) It defines a detection region (ie, a region that is subject to sensing by a detector) that is often centrally located in the flow cell. This selective treatment of a portion of the sensing surface selectively contacts the upstream portion of the sensing surface (ie, the first end of the flow cell referred to above) with a fluid containing the analyte binding ligand. Can be used to immobilize the ligand to the surface. If the sensing surface has a first detection area located within the immobilized surface area and a second detection area further downstream outside the immobilized area, the upstream detection area is sensitive. The detection area downstream can act as a surface and can act as a reference area.

  Selective treatment of the sensing surface within the flow cell can also be used for partial deactivation of the sensing surface. In this case, the sensing surface can include functional groups. This functional group needs to be activated by an activator to react with the analyte specific ligand to form a reactive group that can be immobilized on the sensing surface. After activating the sensing surface, the selective treatment described above can be used to treat the sensing surface region extending from the inlet port to the vicinity of the detection region with a deactivator to deactivate a portion of the sensing surface. . By inactivating the inlet portion of the sensing surface in this manner, the ligand immobilized on the sensing surface does not bind to the sensing surface region in front of the detection region. If the sample subsequently passes through this sensing surface, the inactivation achieved prevents the analyte from being bound to the sensing surface on its way to the detection region. In other words, analyte depletion in the sample fluid passing through the flow cell to the detection region is minimized.

  Another application of the above-mentioned embodiments is the application where the detection area in the flow cell is brought into contact with the fluid whose interaction with the sensing area is to be studied to obtain a fast displacement of the fluid. This is useful, for example, for reaction kinetic studies. In the first state, the interaction between the test fluid and the countercurrent blocking fluid is positioned so as to be proximate to the detection region but not into or through the detection region. In the second state, countercurrent is reduced or stopped and the test fluid is discharged at the second (downstream) end, rather than the first (upstream) end of the flow cell, blocking the test fluid. Displace at high speed with fluid and allow contact with the detection area.

  In another embodiment of the first mentioned aspect of the invention, a laminar flow of fluid is introduced at the first end and discharged through a port at the second end of the flow cell and the counterflow of blocking fluid Is introduced through another port at the second end of the flow cell and drained with the fluid mentioned first. Depending on the respective flow rates of the two fluids, the interface between the two fluids can be adjusted so that the blocking fluid covers the desired area at the second end of the flow cell. In a manner similar to that described above for the first embodiment, the one or more detection regions located on the sensing surface region covered by the blocking fluid are the flow cell followed by the sample fluid between the ends of the flow cell. When used for analysis passing through, it can be prevented from contacting the ligand-containing fluid and thereby forming a reference region.

  Another aspect of the invention relates to a method for analyzing a fluid sample for an analyte. In accordance with the basic concept of the present invention, the method includes partially sensitizing a sensing surface in a flow cell using laminar flow of a sensitizing fluid and laminar counterflow of a blocking fluid, and subsequently a fluid sample. Continuously passing over the sensitized and non-sensitized portions of the sensing surface.

  Yet another aspect of the present invention relates to a method of sensitizing a sensing surface, the method using laminar flow of an inert fluid and laminar counter flow of a blocking fluid, as outlined above, Partially deactivating the activated sensing surface.

  Yet another aspect of the present invention relates to an analysis method. The method includes the step of contacting the sensing area in the flow cell with the test fluid at high speed using laminar flow of the test fluid and laminar counterflow of the second fluid in accordance with the basic concept of the present invention (the term “ “Test fluid” is used herein in a broad sense, and the term includes not only fluid that passes through one or more components (analytes) or fluid that can interact with a sensing surface, but also, for example, surface Also includes fluids that do not interact with the sensing surface (eg, buffers) that can still cause changes (eg, dissociation of bound analyte).

  Another aspect of the invention also relates to a sensitized sensing surface made by a method according to the invention.

  In this specification and the appended claims, the singular forms “a”, “an”, and “the” are meant to include a plurality of references unless indicated otherwise. Also, unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.

  It should also be noted that the terms “comprising”, “including” and “having” may be used interchangeably.

(Detailed description of the invention)
As mentioned above, the present invention generally relates to the control of fluid flow in a flow channel of an analytical flow cell device that typically has at least one sensing surface, which uses laminar flow techniques to This fluid flow is controlled so that can occupy a variable portion of the flow channel length between the flow cell ends. WO 99/36766 referred to above (the entire disclosure of which is incorporated herein by reference) is a controlled fluid flow that uses a hydrodynamic addressing technique to pass a flow cell from one end to the other. However, the present invention relates to controlling the longitudinal extent of fluid flow in a flow cell. If desired, the present invention can be used in the appendix of the method and system disclosed in WO 99/36766.

  As in WO 99/36766, the configuration and dimensions of the flow cell to be used can vary widely depending on the particular application and / or the particular detection method.

  Exemplary detection methods include, but are not limited to: mass detection methods (eg, piezoelectric methods, optical methods, thermo-optical methods and surface acoustic wave (SAW) methods), and electro-chemical methods (For example, potential difference measurement method, conductivity measurement method, current measurement method and capacitance method). With respect to optical detection methods, typical methods include methods for detecting mass surface concentration (eg, reflective optical methods, including both internal and external reflection methods), resolved angles, wavelengths or phases. (E.g. ellipsometry and transient wave spectroscopy (EWS) (the latter being surface plasmon resonance (SPR) spectroscopy, Brewster angle refractometry, critical refractometry, attenuation total Reflection method (FTR), transient wave ellipsometry, scattered total reflection (STIR), light guide sensor, transient wave-based imaging (eg, imaging at critical angle, resolution at Brewster angle) And, for example, transient fluorescence (TIRF) and phosphorescence based photometry methods, and Wave tube interferometer may also be used.

  SPR spectroscopy can be referred to as an exemplary commercial analytical system to which the present invention can be applied. One type of SPR-based biosensor is sold by Biacore AB (Uppsala, Sweden) under the trade name BIACORE® (hereinafter referred to as “BIACORE instrument”). These biosensors utilize SPR-based mass sensing technology to provide “real time” binding interaction analysis between the surface bound ligand and the analyte of interest. An analytical system comprising a two-dimensional optical detector (eg, SPR detector) based on total internal reflection or total external reflection is disclosed in WO 98/34098, the entire disclosure of which is incorporated herein by reference. Is disclosed.

  However, any instrument or technique that causes the sample to contact the sensing surface in the flow cell under laminar flow can benefit from the present invention.

  With respect to a flow cell suitable for use in the practice of the present invention, such a flow cell can assume many forms, and its design can vary widely depending on the intended application and / or application. Although some exemplary flow cells are disclosed herein for purposes of illustration, any type of flow cell that can contact a liquid sample with a sensing surface under laminar flow conditions may be implemented in the present invention. It should be appreciated that it can be utilized in

  The basic principle of the present invention is schematically illustrated in FIG. The flow cell 1 shown partially in FIG. 1, referred to herein as a “Y-shaped cell”, has two openings 2, 3 at one end of the flow cell (shown here as arms). And one opening 4 at the opposite end. A sensing surface (not shown) is disposed between these two ends. A laminar flow of the desired fluid, indicated by arrow 5, is introduced through one of these two openings (here 2) at the first end and indicated by arrow 6 ( A different) “blocking fluid” laminar counter flow is introduced through the opening 4 at the opposite end of the flow cell. Both fluid laminar flows exit the flow cell through the second opening 3 at the first end. The discharge (typically suction) flow through the outlet opening 3, indicated by the arrow 7, is considerably stronger than the fluid flow 5 through the inlet opening 2. The desired fluid occupies only the first part of the flow cell length (as seen from the first end), as indicated by the hatched region 8 in FIG. The remaining flow cell volume 9, including a small area extending through the outlet opening 3, is occupied by a blocking fluid that prevents further passage of the desired fluid into the flow cell. The spread or extension of the first fluid pulse in the flow cell can be controlled by changing the ratio of the outlet (suction) and inlet flow of the second fluid. Thus, as this ratio increases, more of the flow cell volume is occupied by the second fluid.

  Such variable length extension of the flow pulse can be used for different purposes. In one embodiment, the (eg, bottom) wall of the flow cell supports a material layer that can react with the reagent solution, and only a portion of this material layer is reacted with the reagent using the procedure of the present invention. One application of such a procedure is to provide a sensing region and a reference region arranged sequentially in the normal flow direction within the flow cell, as described below with reference to FIG.

  In FIG. 2, corresponding portions having the same reference numbers as in FIG. 1, the bottom wall of the flow cell supports a substance layer that contains a recognition element (eg, a receptor) for the analyte-specific ligand. The two detection areas 10, 11 are defined along the flow cell by the detection system used (eg optical detection system). Initially, a flow of inert fluid (i.e., fluid that does not react with the material layer) passes through the flow cell, is introduced through opening 4, and is discharged through at least one of openings 2,3. The ligand-containing fluid 5 is then introduced through the opening 2 and discharged through the opening 3 so that the ligand-containing fluid volume 8 extends through the first detection region 10 while the first It does not extend to the second detection region 11, so the interface 12 between the two fluids is located between the detection regions 10 and 11. Thereby, the detection region 10 supports the ligand, but the detection region 11 does not support the ligand. Subsequently, for example when using a flow cell to analyze the sample stream introduced through one (or both) of the openings 2, 3 and discharged through the opening 4 for the analyte, the detection region 10 forms a sensing surface and the detection area 11 forms a reference area.

  Another embodiment that provides a sensing region and a reference region that are sequentially disposed within the flow cell is shown in FIG. The flow cell 20, which can be characterized as a “double Y-shaped cell”, has two inlet arms 21, 22 and two outlet arms 23, 24. In the same manner as in FIG. 2, the bottom wall of the flow cell supports a material layer containing a recognition element for the ligand and the two detection areas are defined by the detection system used. One detection area 25 is located in the center of the flow cell, and the other detection area 26 is located in one of the outlet arms (here 24). A laminar flow of buffer fluid first passes through the flow cell, enters via inlets 21, 22 and outlet 24, and exits via outlet 23. The buffer flow through inlets 21, 22 (not through outlet 24) is then replaced by a laminar flow of ligand-containing fluid. As a result, the ligand-containing fluid occupies the shaded area 27 containing the detection area 25, while the buffer fluid entering via the outlet 24 occupies the “blocked” area 28. Thus, the detection region 25 reacts with and supports the ligand, and conversely, the detection region 26 does not contact the ligand. Subsequently, when using a flow cell for analysis of an analyte-containing sample fluid, the sample fluid is introduced through one (or both) of the inlet arms 21, 22 and through the outlet 24 (and as required). The outlet 23 can also be discharged). The detection area 25 then serves as a sensing area and the detection area 26 serves as a reference area.

  FIG. 4 shows a modified design of the embodiment in FIG. Corresponding parts are indicated by the same reference numerals as in FIG. In FIG. 4, the inlet arm is replaced by ports 21, 22 opening in the flow cell 20, and the outlet arm is replaced by ports 23, 24, which are also in the flow cell. By controlling the ratio between the laminar flow of the ligand-containing fluid entering via ports 21 and 22 and the buffer fluid entering via port 24, the blocked area occupied by this buffer fluid is The detection area 26 may be included.

  The procedure of the present invention outlined above can be applied to many other types of flow cells (eg, the so-called “Ψ-shaped cell” having three inlets and one outlet (described in WO 99/36766 referred to above). Is easily understood by those skilled in the art.

  The present invention can be advantageously used with the hydrodynamic addressing techniques disclosed in WO 99/36766 referred to above, as described below.

  5A-5C show a "Y-shaped cell having two inlet ports 31 and 32 (at the end of each inlet arm 31a and 32a) and one outlet port 33 (at the end of outlet arm 33a). ”Type flow cell 30 is illustrated. Three detection regions 34, 45, and 36 are arranged at the center of the flow cell. The entire bottom wall of the flow cell 30 (the entire flow cell surface shown in the figure) supports a material layer containing functional groups that can be activated by an activator. This material layer can be, for example, a carboxymethyl modified dextran gel, where the carboxyl group is activated by, for example, N-hydroxysuccinimide (NHS) and N-ethyl-N-dimethylaminopropylcarbodiimide (EDC). A reactive N-hydroxysuccinimide ester group can be formed.

  Referring to FIG. 5A, in accordance with the hydrodynamic addressing technique described in WO 99/36766 referred to above, a laminar flow of buffer is introduced through the inlet port 31 and a laminar flow of fluid containing an activator is Introduced through the inlet port 32. The layer flow rates of these two fluids are adjusted such that the interface 37 between the two fluids is between the detection regions 35 and 36, and the activator fluid spans the shaded region in FIG. 5A. This means that only the detection region 36 is activated by contact with the activator, and conversely, the detection regions 34 and 35 are not contacted with or activated by the activator.

  The detection region 36 is then reacted with a ligand-containing solution, so that when an analyte-containing sample passes through the flow cell, this detection region can be used as a sensing surface. However, prior to reacting the detection region 36 with the ligand-containing solution, the concept of the present invention is used to inactivate the inlet portion of the flow cell 30 until it is proximate to the detection region 36. In this method, the material on the bottom of the flow cell upstream of the detection region 36 does not contain activation reactive groups and thereby does not bind to the ligand. This in turn means that analyte consumption on the way to the detection region 36 is minimized when the sample flow is subsequently passed through the flow cell from the inlet end to the outlet end.

  To achieve the desired inactivation, now referring to FIG. 5B, a laminar flow of buffer fluid is introduced through the outlet port 33 and exits the flow cell through the inlet port 31. A laminar flow of deactivator is then introduced through port 32 and discharged through inlet port 31 so that a laminar flow of buffer fluid introduced through outlet port 33 is maintained. The ratio of the inlet laminar flow through the inlet port 32 and the outlet laminar flow through the port 31 is such that the interface 37 between the flow of inerting fluid and buffering fluid is positioned proximate to the detection regions 34-36. As a result, the inactivation fluid passes proximal to the detection region but does not spread into the detection region. The flow cell area occupied by the inerting fluid in the flow cell 30 is illustrated by the shaded area 38 in FIG. 5B. The flow cell volume that is blocked by the buffer fluid flow is indicated by reference numeral 39.

  Then, referring to FIGS. 5A and 5B, exactly the same procedure as described above is applied to the detection region 34 to deactivate the flow cell inlet region upstream of the detection region 34, and then a different ligand is applied to the detection region. Can be immobilized. Thereby, the detection areas 34 and 36 form a sensing area, while the intermediate detection area 35 forms a reference area. In the flow cell types of the present invention (eg, Y-shaped cells, Ψ-shaped cells), such formation of many parallel sensing regions by hydrodynamic addressing technology makes effective use of the sensing surface region of the flow cell. It is understood that it is possible.

  In order to analyze a sample solution for a ligand-specific analyte, a laminar fluid flow of sample solution can be introduced through one (or both) of the inlet ports 31, 32 and discharged through the outlet port 33. , All three detection areas 34 to 36 in the flow cell can be directed simultaneously.

  Alternatively, the sample solution can be analyzed using the hydrodynamic addressing technique described in WO 99/36766. Referring to FIG. 5C, the buffer fluid first passes through the flow cell 30. A laminar flow of sample solution is then introduced through the inlet port 32 and a laminar flow of buffer is introduced through the inlet port 31. The laminar velocity of these two fluids is such that the sample flow (shaded area in FIG. 5C) is brought into contact with the detection areas 35 and 36 by placing a fluid interface 37 between the reference area 35 and the sensing area 34. To be adjusted. Thereby, the sample fluid contacts the sensing region 36 and the reference region 35, and an analyte specific for the ligand immobilized on the sensing region 36 binds to the analyte. To address detection regions 34 and 35, the same (or another) sample fluid is introduced via inlet port 31, buffer fluid is introduced via inlet port 32, and interface 37 is detected. Move to a position between regions 35 and 36.

  The hydrodynamic addressing technique described in WO 99/36766 is also used with the present invention to provide two or three parallel sensing regions (eg, as in FIGS. 5A-5C) and one or more located downstream. It will be readily appreciated that a sensing surface having a plurality of reference regions (eg, as in FIGS. 2-4) may be provided.

  In certain types of flow cells, compressing a plate or chip having one or more sensing surfaces (hereinafter referred to as a sensor unit) in contact with an element or block having one or more open channels therein Thus, one or more flow cells are formed. Such flow cell devices are described, for example, in WO 90/05245 (the disclosure of which is hereby incorporated by reference) and are also used in the above-mentioned commercial BIACORE equipment. By using a detachable sensor unit similar to this, sensitization according to the invention (including optical activation and deactivation) after removal of the sensor unit, for example in one or more flow cells, Analysis using a sensor unit in another analysis device (which can of course also be another flow cell device) is possible.

  The present invention can also be used to cause a rapid change or shift of fluid in contact with one or more sensing areas in a flow cell. WO 99/36766 discloses a rapid shift of the fluid in contact by lateral movement of the interface between two different fluids flowing through the flow cell, but the present invention describes the flow between fluids in the longitudinal direction of the flow cell. This shift is possible due to the movement of the interface. This can be explained by referring to FIG. 5B. Assume that sensing regions 34 and 36 support a ligand that can specifically react with an analyte in the sample. Sample laminar flow is introduced through port 32 and buffer laminar flow is introduced through port 33. Both sample flow and buffer flow are drained through port 31, and the ratio between sample flow and buffer flow is adjusted to the position of interface 37 near detection area 34-36 but not internal. As a result, the sample stream 38 (shaded area in FIG. 5B) does not contact this area. The sample and buffer flow rates are then adjusted to move the interface 37 to another (upstream) side position (not shown in FIG. 5B) of the detection region 34-36, thereby allowing the sample flow to be reduced. Touch the detection area. Alternatively, rather than moving interface 37 as described above, the interface closes port 31 to fill the entire flow cell with sample, stop buffer flow, and drain sample flow through port 33. Can be removed.

  It is understood that the rise and fall times are limited only by the movement of the interface from a first position that is not in contact with the detection region to a second position where the sample flow is in contact with the detection region. The sample volume required to move the interface from the first position to the second position is part of the capacity of the flow cell itself. Thus, instead of shifting from buffer flow to sample flow using a valve at some distance from the sensing area, the interface can be moved only in a portion of the volume of the flow cell. Since the rise time is proportional to the replaced capacity, 10 times the decrease in capacity will reduce the rise time by about 10 times. Similar benefits are achieved with shorter fall times.

  Such rapid rise and fall times are necessary when measuring fast reaction kinetics (eg, when studying association and dissociation). In one embodiment, the analyte may pass over the sensitized sensing area. The sample stream can then be moved out of contact with the sensitized sensing area and the dissociation rate can be detected. Alternatively, the sample stream can be rapidly moved over the sensitized sensing area, thereby allowing for the detection and analysis of association kinetics.

  A modified embodiment of the present invention to achieve rapid fluid shift is described below with reference to FIGS. 6A and 6B. This embodiment uses a flow cell 40 similar to the flow cell in FIGS. 3 and 4, which has two openings 41, 42 at one end and two openings 43, 44 at the other end. . Each opening 41-44 is associated with a respective valve (not shown) that opens and closes these openings. A plurality of (here, three) detection regions 45 a to 45 c are located in the center of the flow cell 40.

  In FIG. 6A, for example, a laminar flow of sample fluid (indicated by arrow 46) is introduced through opening 41 and, for example, a laminar flow of buffer (indicated by arrow 47) is introduced through opening 43. Is done. Both fluid streams are exhausted through opening 42 as indicated by arrow 50. Sample stream 48 (shaded area in FIG. 5A) and buffer stream 49 together form a combined stream with interface 51 therebetween. In this state of the flow cell 40, the detection regions 45a-c are in contact with the buffer flow only.

  The state of the flow cell 40 is then changed to that shown in FIG. 6B by closing the valve associated with the opening 42 and opening the valve associated with the opening 44. This causes the sample flow and buffer flow to exit the flow cell through the opening 44 as a combined flow, as indicated by arrow 52, and the interface 51 moves toward the opposite side of the flow cell 40, resulting in Only the sample flow (shaded area in FIG. 6B) contacts the detection areas 45a-c. Thus, by opening the valve associated with the opening 44 and closing the valve associated with the opening 42, or vice versa, a rapid shift of fluid in contact with the detection regions 45a-c can be effected. Depending on the detailed design of the flow cell 40, it is also usually necessary to adjust the individual flow rates of the buffer flow and the sample flow, respectively.

  In order to achieve the desired fluid shift as fast as possible, the interface 51 between the two fluid laminar flows is preferably where subsequent rapid contact with the sample is desired (eg in the sample For studying the association of the analyte to the surface-bound ligand), it is located near the row of detection regions 45a-c in FIG. 6A, but without contact and subsequent rapid contact with the buffer is desired. (E.g., to study the dissociation of the analyte from the surface bound ligand), it is close to the row of detection regions 45a-c but on the opposite side.

  It is understood that using the above procedure, the “dead capacity” of the flow cell 40 is very low and decreases to a fraction of the flow cell capacity.

In further non-limiting examples that follow to further illustrate the present invention, BIACORE equipment is used. As mentioned above, the BIACORE instrument is based on surface plasmon resonance (SPR). The analytical data is provided in the form of a sensorgram that plots the signal in resonance units (RU) as a function of time. The 1,000 RU signal corresponds to about 1 ng of analyte binding per mm ² . A detailed discussion of the technical aspects of the BIACORE instrument and the SPR phenomenon can be found in US Pat. No. 5,313,264. More detailed information regarding matrix coatings for biosensor sensing surfaces is given, for example, in US Pat. Nos. 5,242,828 and 5,436,161. In addition, a detailed discussion of the technical aspects of the biosensor chip used in combination with the BIACORE instrument can be found in US Pat. No. 5,492,840. The entire disclosure of the above U.S. patents is incorporated herein by reference.

(Example 1)
(Deactivation of the activated inlet area of the flow cell to increase the mass transport of the sensing area)
A BIACORE S51® instrument (Biacore AB, Uppsala, Sweden) was used. This instrument comprises a Y-shaped channel flow cell of the type shown in FIGS. Sensor Chip CM5 (Biacore AB) is used as a sensor chip, which supports a gold surface to which a carboxymethyl modified dextran polymer hydrogel is covalently bound. The optics measures three spots centered on the sensing surface that forms one channel wall of the flow cell.

(material)
Ligand: Biotin-Jeffamine conjugate, Mw 374.5 (in-house), 1.75 mg in 2 mM, 2386 μl of 10 mM borate (pH 8.5)
Analyte: Biotin-antibody (from Biotin Kit, Biacore AB)
Coupling agents: Amine coupling kit (Biacore AB), EDC / NHS (N-ethyl-N-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide)
Driving buffer: PBS pH 7.2
Deactivator: ethanolamine.

(A. Sensitization of sensor chip CM5-Method 1 (prior art))
The sensor chip was first activated by injection of EDC / NHS at 30 μl / min for 420 seconds. The ligand diluted 1: 2 in borate buffer was then injected for 140 seconds at a flow rate of 10 μl / min. After ligand fixation, ethanolamine was injected for 7 minutes at 30 μl / min to inactivate all activation sites that did not bind to the ligand. Analyte diluted 1:10 in PBS was then injected at 20 μl / min for 120 seconds and the analyte uptake at the detection spot was measured.

(B. Sensitization of sensor chip CM5-Method 2 (method of the present invention))
The sensor chip was first activated by injection of EDC / NHS at 30 μl / min for 420 seconds. The flow cell region preceding the detection spot is then followed according to the procedure of the invention as described above with respect to FIG. 5B by injecting a countercurrent of 21 μl / min ethanolamine and 40 μl / min buffer for 60 seconds. Selectively inactivated. The ligand diluted 1: 2 in borate buffer was then infused for 140 seconds at a flow rate of 10 μl / min for 140 seconds and ethanolamine was injected for 7 minutes at 30 μl / min for binding to the ligand. All missing activation sites were inactivated. Analyte diluted 1:10 in PBS was then injected at 20 μl / min for 120 seconds and the analyte uptake at the detection spot was measured.

  (result)

上記の結果から、フローセルにおける活性化された入口領域（検出スポットまで）の不活性化（これは、リガンドの固定を防止する）は、本実施例において、質量の移送を約７０％増加させることがわかる。従って、検出スポット（検出領域／検出容量）の前の全ての活性領域／容量の不活性化は、分析物の移動を最小にすると結論付けられ得る。この不活性化技術の使用は、検出スポットまたは検出領域または検出容量からの任意の距離における「活性表面」を有する入口チャネルの位置付けを可能にすることが、さらに明らかである。

From the above results, inactivation of the activated inlet region (up to the detection spot) in the flow cell (which prevents ligand immobilization) increases mass transfer by about 70% in this example. I understand. Therefore, it can be concluded that inactivation of all active regions / volumes prior to the detection spot (detection region / detection volume) minimizes analyte migration. It is further apparent that the use of this inactivation technique allows the positioning of an inlet channel with an “active surface” at any distance from the detection spot or detection region or detection volume.

FIG. 1 schematically shows an embodiment of the method according to the invention, wherein a flow cell having two openings at one end and one opening at the opposite end is used. FIG. 2 schematically illustrates the method embodiment of FIG. 1 as applied to a flow cell having two detection areas located at different distances from the inlet end. FIG. 3 schematically illustrates another embodiment of the present invention using a flow cell having two openings at each end and having two detection areas located at different distances from the ends. Show. FIG. 4 schematically illustrates a modification of the embodiment of FIG. 5A-5C schematically illustrate an embodiment of the present invention, in which a flow cell with three parallel detection areas is used. 6A and 6B schematically illustrate yet another embodiment of the present invention, in which a flow cell having two openings at each end is used.

Claims (40)

  A method of operating an analytical flow cell device, the flow cell comprising an elongated flow cell having a first end and a second end, at least one port at the first end, and the second end. At least one port in the section, the method introducing a laminar flow of a first fluid at the first end of the flow cell, a laminar counterflow of a second fluid in the flow cell Introducing at a second end; discharging a laminar flow of each fluid at the first end or the second end of the flow cell; and the first fluid and the second fluid. Adjusting the position of the interface between the first fluid and the second fluid by controlling the relative flow rates of the first fluid and the second fluid.   The method of claim 1, wherein the first fluid is introduced through a first port at the first end of the flow cell and drained through a second port at the first end.   The method of claim 2, wherein the first fluid flow and the second fluid countercurrent are both exhausted through a port at the first end.   The method according to claim 1, wherein the flow cell is a Y-shaped flow cell or a Ψ-shaped flow cell.   The flow cell has at least two ports at the second end, and the first fluid flow and the second fluid flow pass through one of the ports at the second end. The method of claim 1, wherein the method is discharged.   6. The flow cell of any one of claims 1-5, wherein the flow cell has at least one sensing surface at a wall surface located between the first end and the second end of the flow cell. the method of.   The method of claim 6, wherein the first fluid can react with the sensing surface and the second fluid does not react with the sensing surface.   The method of claim 6, wherein the second flow can react with the sensing surface and the first fluid does not react with the sensing surface.   The at least one sensing surface has a first distance from the first end of the flow cell at a first distance and a first distance greater than the first distance from the first end of the flow cell. At least one sensing region at two distances, wherein an interface between the first fluid flow and the second fluid flow is the first end from the first end of the flow cell. So that the first fluid is in contact with the first distance sensing area from the first end of the flow cell. And the second fluid is in contact with the detection region at the second distance from the first end of the flow cell.   The method of claim 9, wherein the at least one detection region at the first distance is an analyte binding sensing region and the at least one detection region at the second distance is a reference region.   The at least one sensing surface comprises at least one detection region between the first end and the second end of the flow cell, and the first fluid flow and the second end The interface between the fluid flow is adjusted to be a position between the first end and the at least one detection region so that the first fluid is in contact with the first end. The method of claim 7, wherein the method is in contact with a sensing surface area extending from a portion to substantially the at least one detection area.   The at least one sensing surface is chemically activated by an activator and the first fluid includes an inactivator, and as a result is contacted by the first fluid. The method of claim 11, wherein the surface region is inactivated.   The flow cell comprises at least one detection region between the first end and the second end of the flow cell, and in a first state, the interface is the first end. And in the second state, an interface between the first fluid and the second fluid is located between the at least one detection region and the at least one detection region. 9. A device according to any one of the preceding claims, wherein the first fluid is moved to a position between the second end of the flow cell so that the first fluid is in contact with the at least one detection region. the method of.   The interface is adjusted such that, in the first state, the first fluid contacts a sensing surface region extending from the first end to substantially the at least one detection region. 14. The method according to 13.   The flow cell has at least two openings at the second end and at least one detection region between the first end and the second end; The first fluid is introduced through a first opening at the first end, the second fluid is introduced through a first opening at the second end, and each fluid flow Is discharged at a second opening at the first end of the flow cell so that the interface between the two fluid flows is between the first end and the at least one detection region. And in a second state, the first fluid is introduced through the first opening at the first end of the flow cell, and the second fluid is Through the first opening at the end of the Flow is exhausted through a second opening at the second end so that the interface between the two fluids is between the at least one detection region and the second end of the flow cell. The method according to claim 1, wherein the method is in between.   A change from the first state to the second state stops the fluid flow through the second opening at the first end, and the second at the second end. 16. The method of claim 15, comprising draining the two fluid streams through the openings of the two.   A change from the second state to the first state stops the fluid flow through the second opening at the second end, and the second at the first end. 16. The method of claim 15, comprising draining the two fluid streams through the openings of the two. A method of sensitizing a sensing surface arranged to be passed by a fluid flow in a flow cell comprising the following steps:
Passing a laminar flow of activation fluid through the flow cell to chemically activate the sensing surface;
Passing laminar flow of deactivation fluid and laminar counterflow of blocking fluid over the sensing surface to have an interface to each other and adjusting the flow rate of the two fluids, Selectively contacting and deactivating a predetermined region of the activated sensing surface extending from one end of the flow cell, and by passing a laminar flow of sensitizing fluid over the sensing surface Selectively sensitizing the activated portion of the sensing surface;
Including the method.   The flow cell has a first end and a second end, and the interface between the passivation fluid and the blocking fluid is between the two ends of the flow cell. 19. The method of claim 18, wherein the method extends substantially transverse to the flow cell extension. The sensitizing step is as follows:
Providing a laminar flow of a first sensitizing fluid and a laminar flow of a second fluid adjacent to the sensitizing fluid flow so that the two fluids have an interface with each other Flowing together over the sensing surface, wherein at least the sensitizing fluid can sensitize the sensing surface, and adjusting the relative flow rates of the sensitizing fluid and the second fluid, Locating an interface, so that the sensitizing fluid contacts a discontinuous sensing area of the sensing surface to selectively sensitize the sensitizing area;
20. A method according to claim 18 or 19, comprising:   The flow cell has a first end and a second end, and an interface between the sensitizing fluid and the second fluid is between the first end of the flow cell and the second end. 21. The method of claim 20, wherein the method extends substantially parallel to an extension of the flow cell between the ends of the flow cell.   22. A sensitized sensing surface made according to any one of claims 18-21. A method for analyzing a fluid sample for an analyte comprising:
Sensitizing a detection area on a sensing surface by the method of any one of claims 18-21;
Contacting the sensitized region with the fluid sample, and detecting an interaction between the analyte and the detection region;
Including the method. A method for analyzing a fluid sample for an analyte comprising the following steps:
Providing a flow cell, the flow cell having a first end and a second end, and a sensing surface on a wall surface in the flow cell;
A laminar flow of sensitizing fluid is introduced at the first end of the flow cell, a laminar counter flow of blocking fluid is introduced at the second end of the flow cell, and the laminar flow of each fluid is allowed to flow through the flow cell. Draining at the first end or the second end and adjusting the position of the interface between the sensitizing fluid and the blocking fluid so that the sensitizing fluid is Contacting a first portion of a sensing surface and the blocking fluid contacts a second portion of the sensing surface to selectively sensitize the first portion of the sensing surface;
Introducing a laminar flow of the fluid sample at the first end of the flow cell and discharging a flow of the fluid sample at the second end of the flow cell, so that the sample flow Continuously passing through the sensitized portion of the sensing surface and the non-sensitized portion of the sensing surface, and the sensitized and non-sensitized portions of the sensing surface Detecting the interaction of the analyte with
Including the method. A method of analysis comprising the following steps:
Providing a flow cell, the flow cell having a first end and a second end, and at least one sensing region, the sensing region being on a wall surface in the flow cell; and A step spaced from the first end of the flow cell;
A laminar flow of test fluid is introduced at the first end of the flow cell, a laminar counter flow of a second fluid is introduced at the second end of the flow cell, and the first of the flow cell Or at the second end, discharging each fluid so that an interface is formed between the two fluids, the interface between the ends of the flow cell, Extending substantially transverse to the extension of the flow cell,
In a first state, setting a relative flow rate between the test fluid and the second fluid to locate the interface, so that the test fluid is in contact with the first end and the A step in a position between at least one sensing region;
Changing the relative flow velocity of the laminar flow in a second state, so that the interface is at a position between the at least one sensing region and the second end of the flow cell; Determining the effect of the test fluid on the at least one sensing area;
Including the method.   The interface is positioned such that in the first state, the test fluid extends from the first end to substantially the at least one sensing region but does not contact the sensing region. 26. The method according to 25.   The interface is positioned such that, in the second state, the second fluid extends from the second end to substantially the at least one sensing region but does not contact the region. The method according to 25 or 26. A method of analysis comprising the following steps:
Providing a flow cell, the flow cell having a first end and a second end, and at least one sensing region, each of the first end and the second end being Having two openings, and the sensing region is present on a wall surface in the flow cell spaced from the end of the flow cell;
In a first state, a laminar flow of test fluid is introduced through the first opening at the first end of the flow cell, and through the first opening at the second end of the flow cell, Introducing a laminar countercurrent of two fluids and discharging each fluid stream through a second opening at the first end, so that the laminar flow between the two fluids Is formed at a location between the first end of the flow cell and the at least one sensing region,
In a second state, a laminar flow of the test fluid is introduced through the first opening at the first end of the flow cell and the first opening at the second end of the flow cell. Through which the laminar counterflow of the second fluid is introduced and discharged through the second opening at the second end, so that the two fluid layers An interface between the flow is at a position between the second end of the flow cell and the at least one sensing region;
(I) from the first state said second state Contact and / or from (ii) said second state step of changing to said first state, as well as the at least said change to one of the sensing area Determining the impact,
Including the method.   29. In the first condition, the interface is positioned such that the test fluid extends from the first end to substantially the at least one sensing region but does not contact the region. The method described.   The interface is positioned such that, in the second state, the second fluid extends from the second end to substantially the at least one sensing region but does not contact the region. Item 30. The method according to Item 28 or 29.   The change from the first state to the second state includes closing the second opening at the first end, and opening the second opening at the second end. 31. The method of any one of claims 28-30, comprising:   The change from the second state to the first state includes closing the second opening at the second end, and opening the second opening at the first end. 32. The method of any one of claims 28-31, comprising:   33. A method according to any one of claims 25 to 32, wherein the test fluid contains an analyte and the association of the analyte to a sensing region is determined.   33. The method of any one of claims 25-32, wherein the test fluid is free of analyte and the dissociation of the analyte from the sensing region is determined.   The flow cell device comprises a fluid channel element and a plate member, the fluid channel element having at least one channel in a flat plane, and the plate member having at least one sensing surface on the surface. 35. The plate member is adapted to be removably pressed against the fluid channel element and forms with each channel a flow cell comprising a sensing surface. The method described in 1. 36. The method of claim 35, comprising the following steps:
A method of treating at least one sensing surface by the method of any one of claims 7-21, and moving the plate member from the flow cell device to an analysis device and the treated sensing surface. A process for subjecting to an analysis procedure,
Including the method.   37. A method according to any one of claims 23 to 36, comprising the step of detecting an interaction event in one or more detection areas by means of an optical sensor.   38. The method according to claim 37, wherein the optical sensor is based on transient sensing, in particular surface plasmon resonance. A method of chemically treating a surface region in a flow cell comprising the following steps:
Providing a flow cell having a first end and a second end;
Introducing a laminar flow of processing fluid at the first end of the flow cell;
Introducing a laminar counterflow of blocking fluid at the second end of the flow cell;
Discharging a laminar flow of each fluid at the first end or the second end of the flow cell, so that the two fluids have an interface between the fluids Passing through the flow cell, and adjusting the relative flow rates of the two fluids, so that the interface is located at a predetermined distance from the first end of the flow cell, and Selectively contacting the processing fluid with a wall surface region in the flow cell extending at a predetermined distance from the first end of the flow cell;
Including the method.   40. The method of claim 39, wherein the surface area of the wall is part of a chemically active surface and the processing fluid can inactivate the active surface.

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