JP4113735B2 - Protein solid phase chip and solid phase sample preparation device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
This invention relates to a chip contact and solid-phase sample preparation device for protein immobilization, in particular, to an apparatus for detecting a protein of trace amounts contained in the sample.
[0002]
[Prior art]
Various proteins are mixed in clinical specimens and excision samples of diseased sites. As a typical method for detecting a specific protein contained in a trace amount in the sample, there is an enzyme-linked immunosorbent assay (ELISA method).
An immunoconcentration method is known as a simpler and quicker ELISA method. In this method, a specimen is dropped on a porous membrane (membrane) having a solid-phased antibody immobilized on the surface in advance to cause an antigen-antibody reaction on the membrane, and a detection reagent is further dropped to cause a color reaction. Next, the cleaning solution is dropped to clarify the coloration (see, for example, JP-A-1-2233521 and JP-A-2000-329766).
[0003]
[Problems to be solved by the invention]
However, preparing a porous membrane in which antibodies are immobilized in advance is necessary to prepare as many types of porous membranes or solid phase plates as possible when measuring multiple items. There is a problem that decreases.
The present invention has been made in view of such circumstances, and a plurality of types of samples are simultaneously solid-phase formed on a single porous membrane, so that multiple items of protein in the same sample can be quantified, there is provided a protein quantitatively efficient solid phase sample preparation device and chip Contact for protein solid phase can be measured.
[0004]
[Means for Solving the Problems]
This invention comprises a first region having a first through-hole of the multiple, the first template to have a a recess is provided outside the first region, the second through the plurality corresponding to the first through hole comprising a second region having a bore, and a second template for chromatic and ridge-shaped convex portion surrounding the second region, and a porous film, by press-fitting the convex portion into the concave portion, first and second template is fixed, the corresponding first and second through holes become coaxial to each other, the porous membrane is sandwiched in contact with the first region and the second region, each of the first through-hole and the holding porous membrane Thus, a chip for protein solid phase in which a well is formed is provided.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
In the protein solid-phase chip of the present invention, the first and second templates each have a contact region in contact with the porous membrane, and the concave portion and the convex portion are formed so as to surround each contact region. The second through hole is preferably drilled in each contact area. As a result, the porous film is uniformly pressed onto the flat surface, and a good well is formed by the plurality of first or second through holes.
[0006]
The plurality of first and second through holes are preferably oval rather than circular. Thereby, the arrangement density of the first and second through-holes can be made higher than the circular shape.
The first and second through holes are preferably arranged in a matrix.
This facilitates automatic dispensing of the sample into the well formed by the first or second through hole.
It is preferable that the convex portion and the concave portion have a fit fit that can be extracted and disassembled, for example, a fit tolerance of p6 / H7.
Accordingly, the convex portion can be press-fitted into the concave portion so as to be detachable.
[0007]
The present invention also relates to an assembling apparatus for assembling the protein solid phase chip, wherein the first and second templates are stacked so that the concave portion and the convex portion are opposed to each other, and the first member accommodated. And a chip assembling apparatus comprising a pressing member that pressurizes the second template to press-fit the convex portions into the concave portions to fix the templates to each other.
[0008]
Furthermore, the present invention provides a decomposition apparatus for decomposing the protein solid-phase chip, wherein the second template is added in a direction in which the second template is separated from the support member that supports the first template and the supported first template. A chip disassembling apparatus including a pressing member for pressing is provided.
[0009]
The present invention also provides a chip container for horizontally storing the protein solid phase chip loaded with the porous membrane with the first template facing upward, and the first through hole and the porous material from the lower side of the second template. A solid phase sample comprising a membrane and a suction port for sucking air through the second through-hole, and allowing the protein to be solid-phased on the porous membrane by suction from the suction port when the protein-containing liquid is accommodated in the first through-hole A preparation device is provided.
[0010]
Hydrophobic porous membranes used by being sandwiched between the first and second templates are those that can be hydrophobically bonded to proteins, specifically PVDF (polyvinylidene fluoride) hydrophobic membrane, nylon Examples include (charge-treated) membranes, nitrocellulose, and the like.
The pores of the hydrophobic porous membrane are 0.1 to 10 μm, preferably 0.1 to 0.5 μm.
[0011]
When protein is formed in a solid phase on a hydrophobic porous membrane, even if the pores of the hydrophobic porous membrane are larger than the protein, the protein is retained by a hydrophobic bond between the membrane and the protein is hydrophobic. Does not pass through the porous membrane.
In addition, it is preferable to use what performed the initialization process, such as being immersed in methanol, as this hydrophobic porous membrane.
The first and second templates are preferably formed using a chemical resistant resin such as a vinyl chloride resin or a polyethylene resin.
[0012]
The present invention will be described in detail below based on the embodiments shown in the drawings. This does not limit the invention.
Chip for protein solid phase FIG. 1 is a top view of an upper template constituting the chip for protein solid phase of the present invention, FIG. 2 is a cross-sectional view taken along arrow AA of FIG. 1, and FIG. 4 and 4 are cross-sectional views taken along the line BB in FIG.
[0013]
As shown in these figures, the upper template 1 of a rectangular plate (70 mm × 50 mm × thickness 7 mm) has 40 oval through holes 2 arranged in a matrix of 4 rows and 10 columns. Has been. One through hole 2 has a cross-sectional area of about 13 mm ² . A groove (concave portion) 4 having a width of 4 mm and a depth of 4 mm is formed on the lower surface of the upper template 1 so as to go around the circumference of the 40 through holes 2. A rectangular porous membrane contact region 3 is defined inside the groove 4. Further, eight jig through holes 5 are drilled at the bottom of the groove 4.
[0014]
5 is a top view of the lower template constituting the protein solid-phase chip of the present invention, FIG. 6 is a cross-sectional view taken along the line CC in FIG. 5, and FIG. 7 is a cross-sectional view taken along the line DD in FIG.
As shown in these figures, the lower template 11 having a rectangular plate shape (70 mm × 50 mm × thickness 3 mm) is arranged in 4 rows and 10 columns in a matrix at positions corresponding to the through holes 2 of the upper template 1. 40 oval through holes 12 are formed. The through hole 12 has the same shape and cross-sectional area as the through hole 2 (FIG. 1).
[0015]
On the upper surface of the lower template 11, a hook-shaped convex portion 14 having a height of 4 mm and a width of 4 mm that goes around the circumference of the 40 through holes 12 is formed at a position corresponding to the groove 4 (FIG. 1).
A rectangular porous membrane installation region 13 is defined inside the convex portion 14. In addition, six notches 15 are formed on the periphery of the lower template 11. The upper template 1 and the lower template 11 are made of vinyl chloride resin.
[0016]
FIG. 8 is a cross-sectional view showing the protein solid-phase chip 21 of the present invention. The upper template 1 and the lower template 11 are overlapped as shown in FIG. 8, and the convex portion 14 is press-fitted into the groove 4 so as to be detached. Thereby, each through hole 2 and each through hole 12 are coaxial with each other. The inner peripheral surface of the convex portion 14 and the corresponding contact surface of the groove 4 have a fitting tolerance of p6 / H7.
[0017]
In use, a rectangular hydrophobic porous membrane 22 is loaded between the porous membrane placement regions 3 and 13 and is uniformly compressed by press-fitting into the groove 4 of the convex portion 14. As a result, the porous film 22 is partitioned watertightly by the respective through holes 2, and the number of wells (liquid reservoirs) W corresponding to the number of the through holes 2 is formed.
[0018]
Solid phase sample preparation apparatus <br/> Figure 9 is a top view of a solid phase sample preparation device according to the invention, FIG 10 is a E-E in arrow sectional view of Fig.
As shown in these drawings, the apparatus main body 31 is made of an aluminum block and includes a rectangular first recess 32 on the top surface and a rectangular second recess 33 on the bottom of the first recess 32. A rectangular frame-shaped rubber elastic gasket 37 is provided on the bottom surface of the first recess 32 around the periphery of the second recess 33.
[0019]
The second recess 33 has a cross-shaped groove 34 at the bottom and a suction port 35 at the center of the bottom, and the groove bottom of the groove 34 is inclined so as to become deeper toward the center from the periphery of the second recess. The suction port 35 communicates with a nipple 36 provided for connection to an external suction pump (not shown).
Then, the protein solid phase chip 21 assembled as shown in FIG. 8 is horizontally loaded through the bottom gasket 37 of the recess 32 of the main body 31 of the solid phase sample preparation device. After the protein-containing sample solution is injected or dropped into each well W of the protein solid phase chip 21, a suction pump (not shown) connected to the nipple 36 is activated.
[0020]
As a result, the chip 21 is airtightly adsorbed to the bottom surface of the recess 32 through the gasket 37, and the sample liquid in each well W is sucked through the porous film 22, so that the protein for measurement is applied to the film 22. Solid phase is formed. In this case, a fixing member that presses and fixes the chip 21 against the bottom surface of the recess 32 may be provided in the main body 31.
[0021]
Next, a labeled antibody is injected into each well W, and after binding with a specific protein, a washing process is performed. As a result, an immune complex of the protein to be detected and the labeling substance is prepared on the porous membrane 22. Next, the chip | tip 21 is decomposed | disassembled, the porous membrane 22 is taken out, the sample solid-phased on the porous membrane 22 is optically measured, and the quantity of the target protein is calculated.
[0022]
Chip assembling apparatus FIGS. 11 and 12 are perspective views showing an assembling member 51 and a pressing member 41 constituting the chip assembling apparatus of the present invention. As shown in FIG. 11, the assembly member 51 has a rectangular recess 52 for accommodating and assembling the upper template 1 (FIG. 1) and the lower template (FIG. 5) on the upper surface of an aluminum flat plate. The recess 52 has a flat bottom 54. The assembly member 51 includes two sets of positioning recesses 53a and 53b on the back surface.
[0023]
As shown in FIG. 12, the pressure member 41 includes two pressure clamps 43 disposed symmetrically on the fixed plate 42 and two guide plates 45 disposed in parallel. Positioning pins 45a and 45b project vertically from the two guide plates 45, respectively. In this embodiment, a commercially available pressure clamp 43 (HH450 manufactured by MISUMI Corporation) is used. The pressure clamp 43 includes a toggle mechanism 44. By pressing the handle 46 in the direction of arrow F, the arm 47 rotates in the direction of arrow G, and the rubber head 48 is held at a predetermined position. .
[0024]
A procedure for assembling the chip 21 shown in FIG. 8 in such a configuration will be described.
First, after installing a desired hydrophobic porous membrane 22 (FIG. 8) in the porous membrane placement region 13 of the lower template 11 (FIG. 5), the lower template 11 is recessed 52 of the assembly member 51 as shown in FIG. And horizontally installed on the bottom 54.
[0025]
Next, as shown in FIG. 14, the upper template 1 (FIG. 1) is horizontally inserted into the recess 52 with the groove 4 facing downward, and is superimposed on the lower template 11. Next, as shown in FIG. 15, a rectangular flat plate-like presser plate 54 is inserted into the recess 52 and overlaid on the upper chip 1.
[0026]
Next, the assembly member 51 is fitted on the two guide plates 45 of the pressure member 41 shown in FIG. At this time, two sets of positioning pins 45a and 45b are inserted into the two sets of recesses 53a and 53b on the back surface of the assembly member 51, respectively.
[0027]
Next, when the handles 46 of the two pressure clamps 43 shown in FIG. 12 are pressed in the direction of arrow F, each head 48 rotates in the direction of arrow G, presses the holding plate 54 as shown in FIG. The pressurized state is maintained. Thereby, the convex portion 14 of the lower template 11 is press-fitted into the groove 4 of the upper template 1. Accordingly, the porous film 22 is sandwiched between the upper template 1 and the lower template 11 with a uniform pressure. In this way, the protein solid-phase chip 21 shown in FIG. 8 is completed.
[0028]
Chip disassembly apparatus Figs. 17 and 18 are perspective views showing a chip support member 61 and a pressing plate 62 constituting the chip disassembly apparatus of the present invention.
As shown in FIG. 17, the chip support member 61 has a rectangular through window 65 and two sets of positioning recesses 64a and 64b on the back surface. The through window 65 includes six locking claws 63 protruding horizontally from the inner wall. In addition, the holding plate 62 includes eight pushing pins 64 that stand vertically to the surface.
[0029]
A procedure for disassembling the chip in such a configuration will be described. As shown in FIG. 8, the pressing plate 62 is stacked on the chip 21 so that the push pin 64 of the pressing plate 62 is inserted into each of the six jig through holes 5 of the upper template 1 of the chip 21 assembled. The chip 21 is held by the holding plate 62.
[0030]
Then, the pressing plate 62 is horizontally inserted into the through window 65 of the chip support member 61 with the chip 21 facing downward as shown in FIG. At this time, the six locking claws 63 pass through the notch 15 of the lower template 11 and are locked to the peripheral edge of the lower surface of the upper template 1.
[0031]
Next, the chip support member 61 is fitted on the two guide plates 45 of the pressure member 41 shown in FIG. At this time, two sets of positioning pins 45a and 45b are inserted into the two sets of recesses 64a and 64b on the back surface of the chip support member 61, respectively.
[0032]
Next, when the handles of the two pressure clamps 43 shown in FIG. 12 are pressed in the direction of arrow F, each head 48 rotates in the direction of arrow G, presses the pressing plate 62, and the pressure state is maintained. . At the same time, the convex portion 14 of the lower template 11 is pushed out of the groove 4 of the upper template 1 by the eight pushing pins 64. As a result, the lower template 11 is separated from the upper template 1 and falls downward. Then, the porous film 22 is taken out from the dropped lower template 11.
[0033]
Protein measurement Examples of protein measurement using the present invention are described in detail below.
In this measurement example, the amount of expression of CDK1 (Cyclin Dependent Protein Kinase 1) contained in each of 10 samples is quantified using one protein solid phase chip 21 (FIG. 8).
CDK1 is an enzyme protein that controls the progression of the cell cycle.
[0034]
(A) Preparation of reagent First, the following reagent and specimen are prepared.
(1) Loading buffer
TBS (: Tris Buffer Saline)
(Composition: 25 mM Tris (pH 7.4) and 150 mM sodium chloride aqueous solution)
(2) Dilute standard reagent recombinant CDK1 antigen with TBS solution containing 0.005% NP-40 (surfactant) and 2.5μg / 50μL BSA, CDK1 antigen concentration 50ng / mL, 125ng / mL, 250ng / mL, 390ng Prepare 5 concentrations of / mL and 500ng / mL.
(3) Sample (10 samples)
A product obtained by homogenizing specimens collected from cells and tissues, and with a TBS solution containing 0.005% or less Nonidet P-40 (surfactant) so that the total protein amount is 2.5 μg / 50 μL Already prepared.
(4) Control samples (2 samples)
Using leukemia-derived cultured cells HL60 and Jurkat, a TBS solution containing 0.005% or less of NP-40 (surfactant) was prepared so that the total protein amount was 2.5 μg / 50 μL.
(5) Background solution
CDK1 with a concentration of 0.0μg / 50μL (diluted with TBS solution containing 0.005% or less of NP-40 (surfactant) and 2.5μg / 50μL of BSA (bovine serum albumin))
(6) Cleaning solution
TBS (: Tris Buffer Saline)
(Composition: 25 mM Tris (pH 7.4) and 150 mM sodium chloride aqueous solution)
(7) TBS solution of blocking reagent 4% BSA (bovine serum albumin)
(8) Primary antibody reagent 4μg / mL rabbit antibody CDK1 antibody (diluted with 80% blocking agent (Block Ace Dainippon Pharmaceutical))
(9) Secondary antibody reagent 4μg / mL biotinylated anti-rabbit antibody (prepared with 1% BSA and TBS solution)
(10) FITC labeling reagent
10μg / mL FITC (Fluoresceinisothiocyanate) labeled streptavidin diluted with 1% BSA and TBS solution)
(11) Rinse reagent
20% methanol [0035]
(B) Measurement procedure The measurement procedure is as follows.
[1] Prepare one PVDF membrane as the hydrophobic porous membrane 22 and immerse it in methanol.
[2] Immerse the membrane of [1] in the loading buffer of (1).
[3] Set the membrane of [1] on the lower template 111 and place the upper template 1 on it.
[4] Assemble the chip 21 with the membrane sandwiched by using the chip assembly device.
[5] Set the chip 21 to the solid phase sample preparation device 31 (FIG. 9). Here, as shown in FIG. 20, the 40 wells W of the chip 21 are grouped in advance into the standard series group Gs, the background group Gb, the control sample groups Gj and Gh, and the sample groups G1 to G10. To do.
[6] Dispense each of the standard reagents of (2) having 5 levels of concentration into 2 wells W of group Gs, 50 μL per well.
[7] Dispense 50 μL per well of each of the 10 types of specimens of (3) into 2 wells W of groups G1 to G10.
[8] Dispense 50 μL of each of the two types of control specimens in (4) into two wells W in groups Gj and Gh.
[9] Dispense 50 μL of the background solution of (5) into each well W of group Gb.
[10] After dispensing, suction from the suction port 35 at a negative pressure of 250 mmHg for about 30 seconds, thereby immobilizing the protein on the membrane.
[11] Dispense 50 μL of the washing solution (6) into all wells W of the chip 21 and suck and wash at a negative pressure of 500 mmHg for about 15 seconds.
[12] Dispense 50 μL of blocking solution (7) into all wells W of chip 21 and leave it for about 15 minutes.
[13] Suction for about 15 seconds at a negative pressure of 500 mmHg.
[14] Dispense 50 μL of the primary antibody reagent (8) into all wells W of the chip 21 and leave it for about 30 minutes.
[15] Suction for about 15 seconds at a negative pressure of 500 mmHg.
[16] Dispense 50 μL of the washing solution (6) into all wells W of the chip 21 and suck and wash at a negative pressure of 500 mmHg for about 15 seconds. Repeat this 5 times.
[17] Dispense 50 μL of the secondary antibody reagent (9) into all wells W of the chip 21 and leave it for about 30 minutes.
[18] Suction for about 15 seconds at a negative pressure of 500 mmHg.
[19] Dispense 50 μL of the washing solution (6) into all wells W of the chip 21 and suck and wash for about 15 seconds under a negative pressure of 500 mmHg. Repeat this 3 times.
[20] Dispense 50 μL of FITC labeling reagent (10) into all wells W of chip 21 and leave it for about 30 minutes.
[21] Suction for about 15 seconds at a negative pressure of 500 mmHg.
[22] Dispense 50 μL of the cleaning solution (6) into all wells W of the chip 21 and suck and wash for about 15 seconds at a negative pressure of 500 mmHg. Repeat this 5 times.
[23] The chip 21 is removed from the device 31.
[24] Disassemble the chip 21 with the chip disassembly device and take out the membrane.
[25] Lightly rinse the removed membrane with the rinse reagent (11).
[26] Dry in the greenhouse for 20 minutes,
[27] Measure fluorescence intensity with a fluorescence reader.
[0036]
(C) Results Assuming that the average fluorescence intensity of the background solution solid-phased in the group Gb in FIG. 20 is the background fluorescence intensity Ib for correction, and the value four times the standard deviation is the detection lower limit Imin, From measured values
Ib = 176.3 (count / mm ² )
Imin = 35.4 (count / mm ² )
It becomes.
Therefore, Ib is subtracted from the fluorescence intensity Is of the standard reagent solid-phased in the standard series group Gs to calculate the net fluorescence intensity Iso, and a calibration curve shown in FIG. 22 is created. Here, the amount that 1 ng / 50 μL of the standard reagent (2) can recognize CDK1 contained in the specimen is 1 U / 50 μL.
For 10 samples and 2 control samples, calculate the average fluorescence intensity for each 2 wells, and subtract Ib to calculate the net fluorescence intensity. Based on the calculation result, the concentration curve of FIG. 22 is used to calculate the CDK1-containing concentration of each sample. The calculation results are shown in FIG.
Note that those with a net fluorescence intensity equal to or lower than Imin are indicated as N · D (not measurable) in FIG. In the control sample, HL60 is known to be 150 to 250 counts / mm ² and Jurkat is known to be 380 to 550 counts / mm ² , and HL60 and Jurkat in FIG. Is judged to have been done correctly.
[0037]
【The invention's effect】
According to this invention, the hydrophobic porous membrane can be releasably sandwiched between the first and second templates by simply press-fitting the convex portion into the concave portion, thereby forming a plurality of wells. It is possible to provide a protein solid-phase chip that is easy and that can simultaneously solidify a wide variety of samples.
[Brief description of the drawings]
FIG. 1 is a top view of an upper template of a protein solid-phase chip of the present invention.
FIG. 2 is a cross-sectional view taken along the line AA in FIG.
FIG. 3 is a bottom view of the upper template shown in FIG. 1;
4 is a cross-sectional view taken along the line B-B in FIG. 1;
FIG. 5 is a top view of the lower template of the protein solid-phase chip of the present invention.
6 is a cross-sectional view taken along the line CC in FIG.
7 is a cross-sectional view taken along the line DD in FIG.
FIG. 8 is an assembled cross-sectional view of the protein solid-phase chip of the present invention.
FIG. 9 is a top view of the sample preparation device of the present invention.
10 is a cross-sectional view taken along the line E-E in FIG. 9;
FIG. 11 is a perspective view of essential parts of the chip assembling apparatus of the present invention.
FIG. 12 is a perspective view of essential parts of the chip assembling / disassembling apparatus according to the present invention.
FIG. 13 is an explanatory diagram showing a procedure for assembling a chip according to the present invention.
FIG. 14 is an explanatory diagram showing a procedure for assembling a chip according to the present invention.
FIG. 15 is an explanatory diagram showing a procedure for assembling the chip according to the present invention.
FIG. 16 is an explanatory view showing a procedure for assembling a chip according to the present invention.
FIG. 17 is a perspective view of a main part of the chip disassembling apparatus according to the present invention.
FIG. 18 is a perspective view of a main part of the chip disassembling apparatus according to the present invention.
FIG. 19 is an operation explanatory view of the chip disassembling apparatus of the present invention.
FIG. 20 is an explanatory diagram for explaining grouping of wells of a chip in a protein measurement example using the present invention.
FIG. 21 is an explanatory diagram showing the results of protein measurement using the present invention.
FIG. 22 is an explanatory diagram showing a calibration curve in protein measurement using the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Upper template 2 Through-hole 3 Porous membrane contact area 4 Groove 5 Through-hole 11 Lower template 12 Through-hole 13 Porous membrane contact area 14 Convex part 22 Porous film W Well

Claims (4)

A first template for chromatic a first region having a first through-hole of the multiple, and a recess is provided outside the first region,
A second template for chromatic and a second region having a plurality of second through-hole corresponding to the first through hole, and a ridge-shaped convex portion surrounding the second region,
A porous membrane,
By press-fitting the convex portion into the concave portion, first and second templates are fixed, the corresponding first and second through holes become coaxial to each other, the porous membrane is in contact with the first region and the second region A protein solid-phase chip that is sandwiched and has wells formed by the respective first through holes and the sandwiched porous membrane . 2. The protein solid phase chip according to claim 1 , wherein the porous membrane is a hydrophobic porous membrane .   The protein solid-phase chip according to claim 1, wherein the convex portion and the concave portion have a fit tolerance of an interference fit that can be extracted and disassembled. A chip accommodation portion for horizontally accommodating the protein immobilization chip according to any one of 請 Motomeko 1-3 in the above first template, the first through-hole and a porous from the lower side of the second template A solid phase sample comprising a membrane and a suction port for sucking air through the second through-hole, and allowing the protein to be solid-phased on the porous membrane by suction from the suction port when the protein-containing liquid is accommodated in the first through-hole Preparation equipment.

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