Patent Description:
Biochemical tests include the use of biochemical reactions such as an antigen-antibody reaction. These biochemical reactions are performed using an analysis chip or the like. The analysis chip includes a microchannel, inside which an antigen capture membrane as a reaction field of a biochemical reaction is fixed. In advancing the reaction, a liquid sample containing the target antigen is supplied to the microchannel through one opening of the microchannel. A solid phase antibody to capture the target antigen is immobilized on the antigen capture membrane, and thus, filling the microchannel with the liquid sample brings the liquid sample into contact with the antigen capture membrane, allowing the target antigen to bind to the solid phase antibody to be captured. After a sufficient time has elapsed for the reaction, the liquid sample is collected from the microchannel, and then, the fluorescent labeling liquid is supplied to the microchannel. The microchannel is filled with the fluorescent labeling liquid to bring the fluorescent labeling liquid into contact with the antigen capture membrane, causing the target antigen captured by the antigen capture membrane to bind to the fluorescent labeling antibody contained in the fluorescent labeling liquid to be fluorescent-labeled. After a sufficient time for the reaction has elapsed, the fluorescent labeling liquid is collected from the microchannel to finish the reaction. Thereafter, the presence or absence, the amount of binding, or the like, of the target antigen to the solid phase antibody that captures the target antigen is determined by surface plasmon resonance (SPR), surface plasmon-field resonance enhanced fluorescence spectroscopy (SPFS), or the like.

In the biochemical test described above, the liquid sample and the fluorescent labeling liquid are supplied from a pipette tip to the microchannel or collected from the microchannel by dispensation or aspiration by a pump. Moreover, there are cases where the channel interior is washed by a washing liquid supplied to the microchannel before the reaction starts or after the individual liquids are collected. In this case, the washing liquid is also supplied to or collected from the microchannel by dispensation or aspiration by the pump via the pipette tip.

<CIT> discloses that an inlet and an outlet to and from a channel having a reagent arranging area are blocked with a thin plate part, a self-restoring sealing material formed of silicone gel having a self-restoring function, and a planar member higher in elasticity modulus than the silicone gel. To supply a reagent into a microchip, syringe needle-shaped fluid discharging means and fluid collecting means are caused to penetrate the silicone gel, and their tips are caused to enter into the reagent arranging area.

<CIT> discloses an arrangement provided with a chip body having a fluid channel and configured such that an end portion of the fluid channel is opened in a surface of the chip body, and a sheet-like seal member which covers at least the opening in the surface of the chip body for bringing the inside of the fluid channel to a sealed state. The seal member is constituted of laminated sheet members including a first sheet member having a ductility and an elasticity capable of forming a hole therein with use of a nozzle member, and a second sheet member having a lower ductility than the first sheet member. The sheet members adjoining each other are adhered to each other by an adhesive or a tackifier. The second sheet member is disposed on the chip body side than the first sheet member.

<CIT> discloses a miniscule fluid volume dispenser tip including a tube having an elongated constant diameter small bore disposed at the outlet end thereof with a fluid reservoir disposed at the opposite internal end thereof, the outlet end of the dispenser tip being sharply cut away to reduce a surface area to which the dispensed liquid may be attach.

In the biochemical testing apparatus using the above-described analysis chip, liquids needed for reaction or measurement such as a liquid sample, a fluorescent labeling liquid, and a measurement liquid are supplied to the microchannel from the pipette tip in mutually different steps, and collected after a lapse of certain period of time. At the time of liquid delivery, a pipette tip is inserted into a pipette tip insertion portion <NUM> illustrated in <FIG>, and the liquid is dispensed into the pipette tip insertion portion <NUM>. At this time, since there is a resistance due to the microchannel, it would be difficult to allow the liquid to enter the microchannel and difficult to perform appropriate liquid delivery without application of pressure to the pipette tip insertion portion <NUM>. Therefore, in Patent Literature <NUM>, an insertion hole hermetic seal <NUM> is attached to the opening of the pipette tip insertion portion <NUM>, and the pipette tip is inserted into the pipette tip insertion portion <NUM> through the insertion hole hermetic seal <NUM> at the liquid delivery, and the pipette tip insertion portion <NUM> is hermetically sealed by bringing the pipette tip into close contact with the insertion hole hermetic seal <NUM>. Thereby, when liquid is dispensed from the pipette tip, the air in the pipette tip insertion portion <NUM> is compressed to increase the air pressure in the pipette tip insertion portion <NUM>, making it possible to deliver the liquid to the microchannel. Similarly, when liquid is aspirated, the air in the pipette tip insertion portion <NUM> expands to decrease the air pressure in the pipette tip insertion portion <NUM>, so as to allow the liquid to return from the microchannel.

Furthermore, in order to grasp the positional relationship between the pipette tip and the analysis chip when the pipette tip is inserted into the pipette tip insertion portion <NUM>, there is a need to manage the vertical position of the pipette tip. In particular, the distance between the liquid dispensation port of the pipette tip and a conductor film (gold film) to be a bottom surface of the microchannel greatly influences the residual liquid amount in the channel after the liquid collection, and greatly affects the measurement result. Therefore, in order to control the distance between the liquid dispensation port of the pipette tip and the conductor film with high accuracy, it is preferable to perform end detection of detecting a position of the pipette tip (hereinafter also referred to as an end detection position h0) when the distance between the liquid dispensation port of the pipette tip and the conductor film is minimized, and to perform adjustment of the vertical position of the pipette tip with reference to the detected end detection position h0. In this case, as illustrated in <FIG>, a liquid dispensation port <NUM> of the pipette tip is inserted into the end detection position h0 on the surface of or in the vicinity of the conductor film <NUM>. Thereafter, when the liquid is delivered, a pipette tip <NUM> is moved to a sealed position h2 where the liquid dispensation port <NUM> is separated from the surface of the conductor film <NUM> and the pipette tip <NUM> and the insertion hole hermetic seal <NUM> are in close contact with each other. In a case, however, where a conical end-type pipette tip having an end formed in a conical shape as illustrated is used, an outer diameter D0 of the pipette tip <NUM> at a position of the insertion hole hermetic seal when arranged at the end detection position h0 is larger than an outer diameter D1 at the position of the insertion hole hermetic seal when arranged at the sealed position h2, as illustrated in <FIG>. This end detection causes the diameter of the hole of the insertion hole hermetic seal <NUM> through which the pipette tip <NUM> has penetrated to be increased to the diameter D0. Accordingly, it is difficult, with an outer diameter D1 (smaller than diameter D0) at the position of the insertion hole hermetic seal of the pipette tip <NUM> arranged at the sealed position h2, to close the hole of the insertion hole hermetic seal <NUM> through which the pipette tip <NUM> has penetrated, making it difficult to close the hole of the insertion hole hermetic seal <NUM>. This leads to difficulty in hermetically sealing the pipette tip insertion portion <NUM> and difficulty in sufficiently supplying the liquid to the microchannel <NUM>.

Note that the diameter of the hole of the insertion hole hermetic seal <NUM> does not necessarily stay at the diameter D0 penetrated by the pipette tip at the end detection when the liquid dispensation port <NUM> of the pipette tip is arranged at the sealed position h2 (hereinafter, at the time of liquid delivery after the end detection) depending on the material characteristics of an elastic sheet described below used for the insertion hole hermetic seal <NUM>. For example, with the use of a selected material that shrinks to reduce the diameter of the hole of the insertion hole hermetic seal <NUM> to be smaller than the outer diameter D1 at the position of the insertion hole hermetic seal of the pipette tip <NUM> at the time of liquid delivery after the end detection, it would be possible to solve the problem of insufficient sealability at the time of liquid delivery. In a case, however, where the diameter of the hole of the insertion hole hermetic seal <NUM> is increased to be larger than the outer diameter D1 at the time of liquid delivery after the end detection, the problem of insufficient sealability arises even with the diameter not having been increased to the diameter D0.

In addition, while the liquid is supplied to the microchannel and is collected after a certain period of time, it is difficult to completely collect the liquid from the microchannel. For example, Patent Literature <NUM> discloses a biochemical testing apparatus that inserts, at the time of liquid collection, a nozzle into an insertion hole of a test chip (hereinafter also referred to as an analysis chip) deeper than the depth at the liquid delivery so as to collect the liquid as completely as possible from the microchannel. There is a case, however, a minute amount of liquid remains in the channel even with the use of the biochemical testing apparatus of Patent Literature <NUM>. When the pipette tip <NUM> is inserted into the pipette tip insertion portion <NUM> (refer to <FIG>) for liquid delivery in the next step with the presence of the residual liquid <NUM> of the preceding step in the microchannel <NUM>, the opening of the pipette tip insertion portion <NUM> is hermetically sealed as illustrated in <FIG> after the pipette tip <NUM> is brought into close contact with the insertion hole hermetic seal <NUM>. When the pipette tip <NUM> is further lowered or the liquid is dispensed in this state, pressure would be applied to the inside of the pipette tip insertion portion <NUM> and would cause the residual liquid <NUM> in the previous step to be pushed out to the downstream side of the microchannel <NUM>. When the next step liquid is kept dispensing at this state as illustrated in <FIG>, a bubble <NUM> is sandwiched between the dispensed next step liquid and the residual liquid <NUM> of the preceding step and moves toward the downstream side of the microchannel <NUM>. When the bubble <NUM> stays at a certain position of the antigen capture membrane in the microchannel, steps such as progress of antigen-antibody reaction and detection of surface plasmon excitation fluorescence are hindered, leading to a failure in precise measurement.

In order to achieve sealability at the time of liquid delivery as described thereof, there is provided a test kit as set out in independent claim <NUM>. Moreover, in order to solve the bubble formation problem while achieving the sealability at the time of liquid delivery, there is provided a liquid delivery method as set out in claims <NUM> and <NUM>, as well as a testing apparatus as set out in claim <NUM>. Advantageous developments are defined in the dependent claims.

According to the present invention, the shape of the pipette tip used in the biochemical testing apparatus is designed in accordance with the dimensions of the analysis chip. With the use of the pipette tip, even when the hole of the insertion hole hermetic seal is increased by end detection of the pipette tip, it is possible to close the hole of the hermetic seal with the outer diameter at the position of the insertion hole hermetic seal of the pipette tip when arranged at the sealed position, enabling achievement of the pressure needed for liquid delivery. This makes it possible to reliably deliver liquids into the microchannel.

The present embodiment relates to a test kit to be used in a biochemical testing apparatus that detects binding of a target antigen by surface plasmon-field resonance enhanced fluorescence spectroscopy (SPFS).

<FIG> is a schematic diagram illustrating a configuration of a biochemical testing apparatus.

As illustrated in <FIG>, a biochemical testing apparatus <NUM> includes an excitation light optical system <NUM>, a measurement unit <NUM>, a photodiode <NUM>, a conveyance mechanism <NUM>, a pump unit <NUM>, an analysis chip <NUM>, a reagent chip <NUM>, and a control arithmetic unit <NUM>.

The excitation light optical system <NUM> includes a laser diode as a light source and emits excitation light <NUM> so as to set an incident angle on a reflection surface <NUM> of the analysis chip <NUM> to an angle θ.

The measurement unit <NUM> includes a photomultiplier tube as a light receiving element and is arranged on an optical path of surface plasmon excitation fluorescence <NUM> and measures light amounts of the surface plasmon excitation fluorescence <NUM> and scattered light <NUM>. The light amount of the surface plasmon excitation fluorescence <NUM> is used to determine the presence or absence of binding of the target antigen or the amount of binding of the target antigen. The light amount of the scattered light <NUM> is used for detecting an incident angle θ of the excitation light. In this case, the angle θ as the incident angle that maximizes the light amount of the scattered light <NUM> is detected as an enhancement angle θr. Alternatively, however, the incident angle θ of the excitation light may be detected using the following photodiode <NUM> as described below. In that case, there is no need to detect the enhancement angle θr using the light amount of the scattered light <NUM>.

The photodiode <NUM> is arranged on the optical path of reflected light <NUM> of the excitation light <NUM> to measure the light amount of the reflected light <NUM>. In this case, the resonance angle that minimizes the light amount of the reflected light <NUM> is detected, so as to be used for specifying the incident angle θ of the excitation light. When the incident angle θ of the excitation light is not to be specified by the light amount of the reflected light <NUM>, the photodiode <NUM> may be omitted and replaced by a light absorber or the like.

The analysis chip <NUM> is attached to the conveyance mechanism <NUM>. The conveyance mechanism <NUM> allows the analysis chip <NUM> to reciprocate between a reaction position B during the progress of reaction and a measurement position A on a measurement optical path. The pump unit <NUM> includes a moving mechanism for moving the pipette tip to a predetermined position, a liquid delivery mechanism for dispensing or aspirating air or a liquid via a pipette tip, and a pressure sensor that detect an air pressure in the pipette tip. The reagent chip <NUM> includes liquid containers that contain individual liquids used in biochemical reactions.

The control arithmetic unit <NUM> includes a control arithmetic block group that controls operation of each of the above-described components.

<FIG> is a cross-sectional view of the analysis chip <NUM>.

As illustrated in <FIG>, the microchannel <NUM> is formed in the analysis chip <NUM>. One end of the microchannel <NUM> is connected to the pipette tip insertion portion <NUM> to which the pipette tip is inserted, while the other end is connected to a stirring tank <NUM> to stir the liquid in reciprocating the liquid in the channel. An insertion hole hermetic seal <NUM> and a stirring tank seal <NUM> are respectively attached to openings of the pipette tip insertion portion <NUM> and the stirring tank <NUM>, not connected to the microchannel <NUM>. The stirring tank seal <NUM> includes a vent <NUM>.

Although not illustrated, the insertion hole hermetic seal <NUM> is formed of a double layer of an elastic sheet and an adhesive sheet, and is formed to allow the elastic sheet to be joined to the periphery of the opening not connected to the microchannel <NUM> of the pipette tip insertion portion <NUM>, via the adhesive sheet. It is preferable that the elastic sheet is a film formed of polyurethane and has a tensile elastic constant of <NUM> GPa to <NUM> GPa, a tensile elongation at break of <NUM>% to <NUM>%, and a tear strength of <NUM> mN to <NUM> mN. The material of the elastic sheet, however, is not particularly limited as long as the pipette tip and the elastic sheet can be brought into close contact with each other. Exemplary materials of elastic sheets other than polyurethane include low density polyethylene (LDPE), linear low density polyethylene (LLDPE), medium density polyethylene (MDPE), nylon, non-stretched polypropylene (CPP), ethylene-vinyl alcohol copolymer (EVOH), silicone, polyvinyl alcohol (PVA) and polyvinyl chloride (PVC). The thickness of the elastic sheet is <NUM>. Note that, the thickness of the elastic sheet is also not particularly limited as long as desired elasticity can be obtained, and may be set appropriately in accordance with the material of the elastic sheet.

In addition, while the pipette tip insertion portion <NUM> is hermetically sealed by the insertion hole hermetic seal <NUM>, the term "hermetically sealed" does not mean that the pipette tip insertion portion <NUM> is completely sealed due to the application of the insertion hole hermetic seal <NUM>. For example, the insertion hole hermetic seal <NUM> may be formed by providing a fine through hole to allow penetration of the pipette tip beforehand such that the through hole is closed by insertion of the pipette tip to bring the pipette tip insertion portion <NUM> into a hermetically sealed state. In that case, the through hole includes a first through hole formed in the elastic sheet and a second through hole formed in the adhesive sheet. The size of the first through hole is formed to allow the pipette tip insertion portion <NUM> to be hermetically sealed by insertion of the pipette tip. For example, in the present embodiment, the insertion hole hermetic seal <NUM> has an initial hole having an outer diameter of <NUM>. The second through hole is formed at a position corresponding to the first through hole on the adhesive sheet. At this time, the diameter of the second through hole is preferably longer than the outer diameter D1 at the position of the insertion hole hermetic seal of the pipette tip when arranged at the sealed position h2. This suppresses contact of the pipette tip with the adhesive sheet.

An antigen capture membrane <NUM> to be a reaction field is immobilized inside the microchannel <NUM>. During the progress of the biochemical reaction, liquids such as a liquid sample, a fluorescent labeling liquid or a washing liquid are sequentially supplied to the microchannel <NUM> by the pump unit <NUM> and brought into contact with the antigen capture membrane <NUM>.

A bottom surface of the analysis chip <NUM> includes a conductor film <NUM> and a prism <NUM> provided for generating surface plasmon resonance. The conductor film <NUM> is a thin film formed of gold. Alternatively, the conductor film <NUM> may be formed of a metal such as silver, copper, aluminum, or an alloy containing these metals. The prism <NUM> is a dielectric medium formed of a material transparent to the excitation light <NUM>.

<FIG> is an explanatory diagram for illustrating the reagent chip <NUM>.

As illustrated in <FIG>, the reagent chip <NUM> includes a washing liquid container <NUM>, a specimen container <NUM>, a dilution container <NUM>, a liquid sample container <NUM>, a fluorescent labeling liquid container <NUM>, a measurement liquid container <NUM>, and a waste liquid container <NUM>. The liquid containers <NUM> to <NUM> respectively contain a washing liquid, a specimen, a dilution, a liquid sample, a fluorescent labeling liquid, a measurement liquid, and a waste liquid. In advancing the biochemical reaction, the liquids are supplied from the respective liquid containers <NUM> to <NUM> to the microchannel <NUM> of the analysis chip <NUM> by the pump unit <NUM>. Moreover, the spent liquid collected from the microchannel <NUM> is stored in the waste liquid container <NUM>. On the surface of the reagent chip <NUM> in which the openings of the individual liquid containers <NUM> to <NUM> are formed, an encapsulating seal (not illustrated) is attached so as to cover the openings of the washing liquid container <NUM>, the dilution container <NUM>, the liquid sample container <NUM>, the fluorescent labeling liquid container <NUM>, the measurement liquid container <NUM>, and the waste liquid container <NUM>. Note that it is possible to perform measurement even in a state where the encapsulating seal is not attached.

When a liquid is supplied to the microchannel <NUM>, the pump unit <NUM> moves the pipette tip <NUM> to a predetermined position (any of positions P1 to P6) under the control of the control arithmetic unit <NUM> to be lowered into a predetermined liquid container and aspirates the predetermined liquid. For example, in the case of supplying the liquid sample to the microchannel <NUM>, the pump unit <NUM> moves the pipette tip to a position P4 above the liquid sample container <NUM>, lowers the pipette tip so as to be immersed in the liquid sample to aspirate the liquid sample. Next, after aspirating a predetermined amount of liquid, the pump unit <NUM> raises the pipette tip <NUM>, moves the pipette tip <NUM> to a position P8 above the pipette tip insertion portion <NUM> of the analysis chip <NUM>, and inserts the pipette tip <NUM> into the pipette tip insertion portion <NUM>. The insertion depth of the pipette tip <NUM> into the pipette tip insertion portion <NUM> at the time of liquid delivery is adjusted under the control of the control arithmetic unit <NUM>. The details of the procedure will be described below.

In a case of collecting the liquid from the microchannel <NUM>, the pump unit <NUM> first aspirates the liquid inside the channel. After most of the liquid inside the channel is aspirated, the pump unit <NUM> raises the pipette tip <NUM> to move to a position P7 above the waste liquid container <NUM> of the reagent chip <NUM> under the control of the control arithmetic unit <NUM>. Next, the pump unit <NUM> lowers the pipette tip <NUM> into the waste liquid container <NUM> and dispenses all of the aspirated liquid.

<FIG> is a block diagram of the control arithmetic unit.

As illustrated in <FIG>, the control arithmetic unit <NUM> includes a CPU <NUM>, an excitation light controller <NUM>, a photodiode operation controller <NUM>, a conveyance mechanism controller <NUM>, a pump unit movement controller <NUM>, a liquid supply/collection controller <NUM>, a pipette tip end detector <NUM>, and a measurement control/arithmetic unit <NUM>.

The CPU controls the entire biochemical test. The excitation light controller <NUM> controls emission of excitation light. The photodiode operation controller <NUM> controls operation of the photodiode <NUM>. The conveyance mechanism controller <NUM> controls the conveyance mechanism <NUM> so as to convey the analysis chip <NUM> between a reaction position B and a measurement position A described below.

The pump unit movement controller <NUM> determines the position and the height of the pipette tip and controls the moving mechanism of the pump unit <NUM> so as to move the pipette tip to the predetermined position and height. The liquid supply/collection controller <NUM> determines the operation of dispensation or aspiration of a liquid, and controls the liquid delivery mechanism of the pump unit <NUM> so as to dispense or aspirate a predetermined amount of liquid. The pipette tip end detector <NUM> detects the end of the pipette tip as described below. The measurement control/arithmetic unit <NUM> performs control related to measurement, such as measurement of the light amount of surface plasmon excitation fluorescence.

The biochemical test is a test method of capturing a target antigen as a detection target by a biochemical reaction, attaching a fluorescent label to the captured target antigen, and determining the presence or absence of the detection target, a captured amount, or the like, on the basis of the light amount of fluorescence from the attached fluorescent label. <FIG> is a flowchart illustrating a procedure of a biochemical test. Hereinafter, a procedure of a biochemical test will be described with reference to <FIG>.

At a start of the test, detection of the end of the pipette tip is performed (step S101). In the detection of the end of the pipette tip, the pipette tip is attached to the pump unit <NUM> under the control of the control arithmetic unit <NUM>. After the pipette tip is attached to the pump unit <NUM>, the pipette tip is moved to the position P8 above the pipette tip insertion portion <NUM> of the analysis chip <NUM>, lowered, and penetrates the insertion hole hermetic seal <NUM> and is inserted into the pipette tip insertion portion <NUM>. Next, the pump unit <NUM> lowers the pipette tip while dispensing the air, and brings the pipette tip close to the surface of the conductor film (gold film) <NUM> being the bottom surface of the pipette tip insertion portion <NUM>. At the same time, the pressure sensor of the pump unit <NUM> detects the air pressure in the pipette tip. The air pressure in the pipette tip increases as the liquid dispensation port of the pipette tip approaches the conductor film <NUM>. The pipette tip end detector <NUM> of the control arithmetic unit <NUM> compares a registered value of the air pressure in the pipette tip in the vicinity of the conductor film surface registered beforehand with the measured value of an air pressure within the pipette tip detected by the pressure sensor of the pump unit <NUM>. When the measured value detected by the pressure sensor starts to become equal to or higher than the registered value registered beforehand, the pipette tip end detector <NUM> stores a vertical position of the liquid dispensation port of the pipette tip at that time as the end detection position h0, and then stops the descent of the pipette tip. At that time, the liquid dispensation port of the pipette tip is positioned on the surface or in the vicinity of the conductor film <NUM>. In addition, the outer diameter of the pipette tip at that time at the position of the insertion hole hermetic seal is defined as a diameter D0 (refer to <FIG>).

Next, the measurement liquid is supplied to the microchannel <NUM> (step S102). At this time, the analysis chip <NUM> is arranged at the reaction position B. The measurement liquid also serves as a washing liquid. As described above, the antigen capture membrane <NUM> to be a reaction field to which the solid phase antibody is immobilized is fixed in the microchannel <NUM>. The antigen capture membrane <NUM> has coating of a protective layer provided to maintain a solid phase antibody capture capability for a long period. Therefore, after the measurement liquid is supplied to the microchannel <NUM>, the measurement liquid is caused to reciprocate within the channel to remove the protective layer. The washing described above may be performed by using a dedicated washing liquid instead of the measurement liquid. In a case where the microchannel <NUM> and the antigen capture membrane <NUM> are clean and the preservation protective layer or the like is not coated on the antigen capture membrane <NUM>, there is no need to perform washing.

After the removal the protective layer, succeeding enhancement measurement is performed while the microchannel <NUM> is filled with the measurement liquid without the measurement liquid being collected (step S103). At this time, the analysis chip <NUM> is arranged at the measurement position A on the measurement optical path, the enhancement angle θr that maximizes the light amount of the scattered light <NUM> or the angle of the resonance angle that minimizes the light amount of the reflected light <NUM> is detected as the incident angle θ of the excitation light. After the enhancement measurement is finished, the measurement liquid is collected from the microchannel <NUM> by aspiration by the pump unit <NUM> so as to be stored in the waste liquid container <NUM> of the reagent chip <NUM>.

Next step to perform is a primary reaction to bind the target antigen with the solid phase antibody (step S104). At this time, the analysis chip <NUM> is arranged at the reaction position B, and the liquid sample is supplied to the microchannel <NUM>. The liquid sample is obtained by diluting the specimen collected directly from an examinee with dilution. When the microchannel <NUM> is filled with the liquid sample, the liquid sample comes in contact with the antigen capture membrane <NUM>, and the target antigen contained in the liquid sample binds to the solid phase antibody immobilized on the antigen capture membrane <NUM> and is then captured. After the lapse of a sufficient time for the reaction, the liquid sample is collected from the microchannel <NUM> to be stored in the waste liquid container <NUM> of the reagent chip <NUM>.

Next, the inside of the flow channel is washed (step S105). In order to remove nonspecifically adsorbed target antigens, a washing liquid is supplied to the microchannel <NUM> and is reciprocated to wash the microchannel <NUM>. After completion of washing, the washing liquid is collected from the microchannel <NUM> to be stored in the waste liquid container <NUM> of the reagent chip <NUM>.

Next, a secondary reaction for fluorescent labeling is performed (step S106). In this case, the fluorescent labeling liquid is supplied to the microchannel <NUM>. When the microchannel <NUM> is filled with the fluorescent labeling liquid, the fluorescent labeling liquid and the antigen capture membrane <NUM> come into contact with each other, and the fluorescent labeling antibody contained in the fluorescent labeling liquid binds to the captured target antigen, so as to attach a fluorescent label to the captured target antigen. After the lapse of a sufficient time for the reaction, the fluorescent labeling liquid is collected from the microchannel <NUM> to be stored in the waste liquid container <NUM> of the reagent chip <NUM>. Thereafter, the washing liquid is supplied into the microchannel <NUM>, and the inside of the channel is washed (step S107) similarly to step S104.

Finally, in order to determine the presence or absence of binding or the amount of binding of the target antigen to the solid phase antibody, the intensity of the fluorescent label attached to the target antigen, that is, the light amount of fluorescence is measured (step S108). At this time, with the analysis chip <NUM> being arranged at the measurement position A, the excitation light <NUM> from the excitation light optical system <NUM> is emitted to the reflection surface <NUM> of the analysis chip <NUM>. The excitation light <NUM> is reflected by the reflection surface <NUM>, and at the time of reflection, an evanescent wave leaks from the reflection surface <NUM> to the conductor film <NUM> side. The electric field of the leaking evanescent wave resonates with the surface plasmon of the conductor film <NUM> and is enhanced. The enhanced electric field excites the fluorescent label attached to the target antigen captured by the antigen capture membrane <NUM>. From the excited fluorescent label, surface plasmon excitation fluorescence <NUM> is emitted. The measurement unit <NUM> measures the light amount of the surface plasmon excitation fluorescence <NUM> and determines the presence or absence or the amount of binding of the target antigen.

<FIG> are explanatory diagrams for illustrating operation of the cylindrical end-type pipette tip <NUM> (hereinafter also simply referred to as a pipette tip). Hereinafter, the operation of the pipette tip <NUM> will be described with reference to <FIG>.

While the pipette tip <NUM> is formed of polypropylene (PP), the material is not particularly limited. Note that it is preferable that the pipette tip <NUM> is formed of a material having water repellency, chemical resistance, or inhibiting effect on protein adsorption. Exemplary materials of pipette tips other than polypropylene include polystyrene (PS), polyethylene (PE), low density polyethylene (LDPE), and fluororesin (PFA).

The pipette tip <NUM> includes, at an end portion to be inserted into the pipette tip insertion portion <NUM> at liquid delivery, a cylindrical intermediate portion 4003a having an equal outer diameter and a conical dispensation portion 4003b positioned closer to the liquid dispensation port side than the intermediate portion 4003a. The outer diameter of the cylindrical intermediate portion 4003a having an equal outer diameter is <NUM>. From the viewpoint of ensuring the rigidity of the pipette tip, it is preferable that the pipette tip <NUM> is formed to have the outer diameter of the liquid dispensation port <NUM> of <NUM> or more.

During the pipette tip end detection performed in the above-described step S101, the pipette tip is inserted up to the end detection position h0 as illustrated in <FIG>. With this operation, the diameter of the hole of the insertion hole hermetic seal <NUM> penetrating through the pipette tip is increased to the outer diameter D0 of the pipette tip at the position of the insertion hole hermetic seal at that time. While the liquid is delivered using a multi-stage liquid delivery method to be described below in individual succeeding steps, the liquid dispensation port of the pipette tip <NUM> is arranged at the sealed position h2 when a liquid is supplied to the microchannel, as illustrated in <FIG>. With a configuration in which the cylindrical end-type pipette tip as illustrated has the cylindrical intermediate portion 4003a having an equal outer diameter, both the outer diameter D0 of the pipette tip at the position of the insertion hole hermetic seal when the pipette tip is arranged at the end detection position and the outer diameter D1 of the pipette tip at the insertion hole hermetic seal when the pipette tip is arranged at the sealed position are equal to the diameter D of the cylinder. With this configuration, even when the hole of the insertion hole hermetic seal is increased to the diameter D0 by end detection of the pipette tip, it is possible to close the hole of the insertion hole hermetic seal with the outer diameter D1 at the position of the insertion hole hermetic seal of the pipette tip when arranged at the sealed position, enabling dispensation of the liquids with ensured sealability.

Note that the outer diameter D0 of the pipette tip at the position of the insertion hole hermetic seal when arranged at the end detection position need not be exactly the same as the outer diameter D1 of the pipette tip at the position of the insertion hole hermetic seal when arranged at the sealed position. Even when the diameter D0 and the diameter D1 are slightly different, the sealability at the time of liquid delivery can be achieved as long as the hole of the insertion hole hermetic seal that has been increased to the diameter D0 can be closed with the outer diameter D1.

In addition, the intermediate portion 4003a of the cylindrical end-type pipette tip need not be cylindrical. This is because the hole of the insertion hole hermetic seal that has been increased to the diameter D0 can be closed by the outer diameter D1 as long as the outer diameter D1 at the insertion hole hermetic seal position when arranged at the sealed position is formed to be equal to or larger than the outer diameter D0 at the insertion hole hermetic seal position when arranged at the end detection position and the outer diameter of any portion between the outer diameter D1 and the outer diameter D0.

In addition, in a case where a through hole through which the pipette tip penetrates is provided in the insertion hole hermetic seal <NUM> beforehand, the through hole is formed to be smaller than the outer diameter D1 of the pipette tip at the insertion hole hermetic seal when arranged at the sealed position in order to ensure the sealability at the time of liquid delivery as described above.

When an encapsulating seal is attached to the surface of the reagent chip <NUM>, the pipette tip <NUM> penetrates through the encapsulating seal to be inserted into the washing liquid container <NUM>, the dilution container <NUM>, the liquid sample container <NUM>, the fluorescent labeling liquid container <NUM>, the measurement liquid container <NUM>, or the waste liquid container <NUM>. At that time, a hole through which the pipette tip <NUM> has penetrated is opened in the encapsulating seal. When the hole through which the pipette tip <NUM> has penetrated is widely opened in the encapsulating seal, the liquid contained in each of the liquid containers <NUM>, and <NUM> to <NUM> might spill. In order to prevent the liquid from spilling from each of the liquid containers <NUM>, and <NUM> to <NUM>, the pipette tip <NUM> preferably has an outer diameter of <NUM> or less from any of portions coming in contact with the encapsulating seal when the liquid dispensation port <NUM> is inserted up to the bottom surface of each of the liquid containers <NUM> and <NUM> to <NUM> to the liquid dispensation port <NUM>.

In the above-described biochemical test procedure, particularly in the primary reaction in step S104, the secondary reaction in step S106, and the light amount measurement in step S108, bubbles attached to the antigen capture membrane <NUM> might hinder the progress of individual steps, specifically, by suppressing the reaction or by scattering of the excitation fluorescence, leading to a failure in performing precise measurement. <FIG> are explanatory diagrams for illustrating a multi-stage liquid delivery method. As will be described below, it is possible to suppress the occurrence of bubbles as described above by this multi-stage liquid delivery method, enabling precise and stable measurement without inhibiting reaction or the like by bubbles.

As illustrated in <FIG>, at the time of liquid delivery, the pipette tip <NUM> is first inserted into a comparatively shallow ventilation position h1 so as not to seal the pipette tip insertion portion <NUM>. This configuration ensures an air escape between the pipette tip <NUM> and the insertion hole hermetic seal <NUM>. Next, a first amount of liquid is dispensed at this ventilation position h1. In this state, since the air escape is ensured in this state, dispensation of the liquid would apply no pressure to the inside of the pipette tip insertion portion <NUM>, and thus, the residual liquid <NUM> in the preceding step is not pushed out toward the downstream side of the microchannel <NUM>. Furthermore, as illustrated in <FIG>, the dispensed first amount of liquid and the residual liquid <NUM> of the preceding step are integrated, and bubbles are not sandwiched between the dispensed first amount of liquid and the residual liquid <NUM> of the preceding step, and thus, the generation of bubbles is eliminated.

Next, as illustrated in <FIG>, the pipette tip <NUM> is inserted into a sealed position h2 deeper than the ventilation position h1 so as to be in close contact with the insertion hole hermetic seal <NUM>, and a second amount of liquid is dispensed. In this state, since the pipette tip <NUM> is brought into close contact with the insertion hole hermetic seal <NUM>, the pipette tip insertion portion <NUM> is hermetically sealed and the air escape path is eliminated. Therefore, when a liquid is dispensed, pressure is applied to the inside of the flow path, enabling the liquid to be supplied to the microchannel <NUM>.

In the present embodiment, as described below, by further performing the operation according to the above-described multi-stage liquid delivery method, it is possible to obtain a further effect of suppressing generation of bubbles and ensure reliable liquid delivery into the microchannel. This makes it possible to achieve accurate and stable measurements without inhibiting the progress of biochemical reactions or generation of surface plasmon excitation fluorescence by bubbles.

Before pipette tip insertion portion <NUM> is hermetically sealed to deliver the liquid in the multi-stage liquid delivery method described above, a portion of the liquid is dispensed at the ventilation position h1 where an air escape path is ensured. In this case, by further providing the conical dispensation portion 4003b on the side closer to the liquid dispensation port side than the cylindrical intermediate portion 4003a having an equal outer diameter, the outer diameter D2 of the cylindrical end-type pipette tip at the insertion hole hermetic seal position when arranged at the ventilation position h1 is smaller than the diameter D of the cylinder as illustrated in <FIG>. Accordingly, it is also possible to ensure the air permeability of the pipette tip insertion portion <NUM>. This ensures an air escape path to enable the liquid to be dispensed without pushing out the residual liquid <NUM> of the preceding step, leading to achievement of liquid delivery without generation of bubbles.

In a case where a through hole through which the pipette tip penetrates is provided in the insertion hole hermetic seal <NUM> beforehand, it is preferable that the through hole is formed to be larger than the outer diameter D2 of the pipette tip at the insertion hole hermetic seal position when arranged at the ventilation position in order to ensure the air permeability at the time of liquid delivery as described above. In a case, however, where the diameter of the through hole is increased to be larger than the outer diameter D2 by end detection of the pipette tip described above, the through hole may be formed to have a diameter smaller than the outer diameter D2.

In addition, due to the insertion of the pipette tip up to the end detection position h0 or the sealed position h2, the insertion hole hermetic seal <NUM> extends to the inside of the pipette tip insertion portion <NUM> to form an extending portion. In this case, it is possible to prevent interference of the extending portion with liquid dispensation from the liquid dispensation port <NUM> by arranging the liquid dispensation port <NUM> of the pipette tip <NUM> to be positioned below the extending portion.

Note that it is allowable to form the cylindrical end-type-end pipette tip to include the outer diameter D2 at the position of the insertion hole hermetic seal when arranged at the ventilation position, the size being equal to or smaller than the outer diameter D1 at the insertion hole hermetic seal position when arranged at the sealed position, in at least one position in the intermediate portion 4003a, and there is no need to provide the conical dispensation portion 4003b on the side closer to the liquid dispensation port than the intermediate portion 4003a. For example, it is allowable to provide a recess having the outer diameter D2 in the cylindrical intermediate portion 4003a without providing the conical dispensation portion 4003b.

Moreover, with the dispensation portion 4003b having a simple conical shape, a travel distance of the pipette tip from the ventilation position h1 to the sealed position h2 might be prolonged. To cope with this, the dispensation portion 4003b may be shaped in a substantially funnel shape including a first truncated cone having the outer diameter decreasing at a constant first decrease rate toward the liquid dispensation port direction and a second truncated cone having the outer diameter changing at a second decrease rate smaller than the first decrease rate or not changing (that is, when the second decrease rate is zero), the second truncated cone being formed continuously from the first truncated cone (the shape is cylindrical when the second decrease rate is zero). With this configuration, the travel distance of the pipette tip from the ventilation position h1 to the sealed position h2 can be reduced.

In addition, in a case where the formation of bubbles does not occur or the influence of the bubbles can be ignored, there is no need to deliver liquid using the multi-stage liquid delivery method. In this case, there is no need to provide the conical dispensation portion 4003b having the outer diameter D2 smaller than the cylindrical intermediate portion 4003a.

As described above, the cylindrical end-type pipette tip provided in the present test kit has the cylindrical intermediate portion 4003a having an equal outer diameter at the end portion thereof, in which the outer diameter at the insertion hole hermetic seal position when arranged at the end detection position matches the outer diameter at the insertion hole hermetic seal position when arranged at the sealed position. Accordingly, even when the hole of the insertion hole hermetic seal is increased by end detection of the pipette tip, there is no loss of sealability at the time of liquid delivery. In addition, by further providing the conical dispensation portion 4003b having an outer diameter smaller than the outer diameter at the insertion hole hermetic seal position when arranged at the sealed position on the side closer to the liquid dispensation port side than the intermediate portion 4003a, it is possible to ensure air permeability in execution of liquid delivery using the multi-stage liquid delivery method, leading to achievement of liquid delivery without bubble generation.

Claim 1:
A test kit comprising an analysis chip (<NUM>) and a pipette tip (<NUM>),
wherein the analysis chip (<NUM>) includes a pipette tip insertion portion (<NUM>) hermetically sealable by a seal (<NUM>) and includes a microchannel (<NUM>) connected at a bottom surface of the pipette tip insertion portion (<NUM>),
an end portion of the pipette tip (<NUM>) to be inserted into the pipette tip insertion portion (<NUM>) includes: a first portion configured to be in contact with the seal (<NUM>),
characterized in that the end portion of the pipette tip (<NUM>) further includes: a second portion positioned closer to the liquid dispensation port (<NUM>) side than the first portion, and
an outer diameter of the second portion is formed to be larger than an outer diameter of the first portion and any portion between the first portion and the second portion,
wherein the end portion of the pipette tip (<NUM>) to be inserted into the pipette tip insertion portion (<NUM>) further includes a third portion having an outer diameter smaller than the outer diameter of the first portion, the third portion being positioned closer to the liquid dispensation port (<NUM>) side than the second portion, and the third portion having a funnel shape, and
wherein the seal (<NUM>) comprises a through hole, the through hole having a diameter smaller than an outer diameter of a portion of the second portion of the end portion of the pipette tip (<NUM>) to be inserted into the pipette tip insertion portion (<NUM>), and the through hole having a diameter larger than the third portion of the pipette tip (<NUM>).