JP2011242263A - Detection method of protein and cartridge for chemical reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a detection method of protein that allows a protein to be detected with high sensitivity and a cartridge for chemical reaction for the detection method.SOLUTION: The detection method of protein for detecting the protein by addressing is provided. The method comprises a step of binding DNA for addressing to the protein to be detected and a step of addressing the protein to be detected using hybridization by binding complementary DNAs with each other. According to the detection method of protein, the addressing of the protein to be detected is performed using the hybridization by binding the complementary DNAs with each other, and therefore, an antigen-antibody reaction is not required to be performed on a chip, the reaction can be performed in a solution which allows an antigen and an antibody preliminary free movement in a three-dimensional manner, and the protein can be detected with high sensitivity.

Description

  The present invention relates to a protein detection method for detecting a protein by addressing and a chemical processing cartridge for performing chemical processing by transferring contents by deformation when an external force is applied.

  A method for detecting an antigen protein by immobilizing an antibody on a substrate and binding a target antigen is known. In this method, a secondary antibody to which a fluorescent molecule is attached is previously bound to a target antigen in a solution, and this solution is dropped onto a substrate on which the primary antibody is immobilized. Since the antigen specifically binds to the primary antibody on the substrate, the presence or absence of the target antigen (protein) can be examined based on the fluorescence emission position by observing the washed substrate with fluorescence.

Japanese Patent Laid-Open No. 2004-184212

  However, in the above method, the reaction between the antigen and the antibody is performed on a substrate on which the primary antibody is immobilized. For this reason, when the reaction field is in the vicinity of the substrate, the probability that the antigen and the antibody are associated with each other is low. Further, the fluidity of the solution is poor on the substrate surface, and the reaction speed and sensitivity are lowered. For this reason, the detection sensitivity is lowered and a long time is required for detection.

  Moreover, although the technique which detects biopolymer with a biochip is known (refer patent document 1), protein cannot be detected with this method.

  An object of the present invention is to provide a protein detection method capable of detecting a protein with high sensitivity, and a chemical reaction cartridge for such a detection method.

  The protein detection method of the present invention includes a step of binding a detection target side DNA for addressing to a detection target protein in the detection method of a protein that detects the presence of a specific protein by addressing, and the detection target protein. A step of binding a fluorescent label; and a step of addressing the protein to be detected using a substrate on which a probe DNA that complementarily binds to the DNA to be detected is fixed.

  According to this protein detection method, since the protein to be detected is addressed using hybridization based on the binding of complementary DNAs, it is not necessary to perform the antigen-antibody reaction on the chip, and the antigen and antibody are preliminarily used. Can be carried out in a solution that can move freely in three dimensions, and proteins can be detected with high sensitivity.

  In the step of binding the detection target DNA to the detection target protein, an antibody that binds between the detection target protein and the detection target DNA may be used.

  In the step of binding a fluorescent label to the protein to be detected, an antibody that binds between the protein to be detected and the fluorescent label may be used.

  The step of binding the detection target DNA to the protein to be detected and the step of binding a fluorescent label to the protein to be detected may be simultaneously performed in a solution.

  In the step of binding the detection target side DNA to the detection target protein and the step of binding the fluorescent label to the detection target protein, an ELISA method, a sandwich ELISA method, or a competitive reaction type ELISA method may be used.

  As a method for fixing the probe DNA on the substrate, a covalent bond method, an electrostatic bond method, a method using an avidin / biotin bond, a method using a gold / thiol bond, a film forming method using photolithography, or an ink jet method may be used. .

  In the step of addressing the protein to be detected, DNA detection by hybridization may be performed simultaneously.

  In the step of addressing the protein to be detected, the protein chip to which the probe DNA is immobilized may be used for addressing.

  The cartridge for chemical reaction of the present invention includes a sample storage portion for storing a sample containing a protein to be detected and a detection target side DNA for addressing in the chemical reaction cartridge for detecting the presence of a specific protein by addressing. Mixing a first solution storage section for storing a first solution, a second storage section for storing a second solution containing a fluorescent label, the sample and the first and second solutions; A binding unit that binds the detection target side DNA and the fluorescent label to a detection target protein; and a substrate on which a probe DNA that binds complementarily to the detection target side DNA is fixed, and addressing the detection target protein And a detection unit that performs the above.

  According to this cartridge for chemical reaction, since the protein to be detected is addressed using hybridization based on the binding of complementary DNAs, it is not necessary to perform the antigen / antibody reaction on the substrate of the detection unit in advance. It can be carried out in a solution of a binding site where antigen and antibody can freely move three-dimensionally, and protein can be detected with high sensitivity.

  In the binding unit, the detection target side DNA may be bound to the detection target protein in a solution.

  In the binding part, the detection target DNA may be bound to the detection target protein using an antigen / antibody reaction.

  The detection unit may simultaneously perform DNA detection by hybridization.

  A protein chip on which probe DNA is fixed may be attached to the detection unit for addressing.

  According to the protein detection method of the present invention, since the protein to be detected is addressed using hybridization based on the binding of complementary DNAs, it is not necessary to cause the antigen-antibody reaction on the chip, and in advance the antigen Can be carried out in a solution in which the antibody can freely move in three dimensions, and the protein can be detected with high sensitivity.

  According to the cartridge for chemical reaction of the present invention, the protein to be detected is addressed using hybridization based on the binding of complementary DNAs, so there is no need to cause the antigen / antibody reaction to be performed on the substrate of the detection unit. In addition, it can be carried out in advance in a solution of a binding site where the antigen and the antibody can freely move three-dimensionally, and the protein can be detected with high sensitivity.

The figure which shows the operation procedure which carries out the fluorescence labeling of the sample. It is a figure explaining the operation procedure of hybridization, (a) is a figure which shows the structure of a protein chip typically, (b) is a figure which shows the mode of hybridization typically. It is a figure which shows the structure of the cartridge for chemical reaction of a present Example, (a) is a top view of a cartridge, (b) is the BB sectional drawing of Fig.3 (a).

  Hereinafter, the protein detection method according to the present invention will be described with reference to FIGS.

  FIG. 1 shows an operation procedure for fluorescently labeling a protein to be detected. As shown in FIG. 1, a sample containing a protein (antigen) 41 to be detected, a secondary antibody 43 fluorescently labeled with a fluorescent molecule 42, and a primary antibody 45 bound to a DNA 44 to be detected are buffered. Mix in 46. Since the protein (antigen) specifically binds to the primary antibody and the secondary antibody, when a plurality of types of specific proteins (antigens) are present in the specimen, each protein (antigen) 41 is It binds to the corresponding primary antibody 45 and secondary antibody 43. For this reason, as shown in FIG. 1, a fluorescent molecule 42 is bound (fluorescently labeled) to the detection target protein (antigen) 41 via the secondary antibody 43, and the detection target side via the primary antibody 45. DNA 44 is bound.

  FIG. 2A is a diagram schematically showing the structure of the protein chip, and FIG. 2B is a diagram schematically showing the state of hybridization.

  As shown in FIG. 2A, on the substrate 51 of the protein chip 5, the site where the probe DNA 52 is fixed is two-dimensionally arranged.

  As shown in FIG. 2 (b), when the mixed solution prepared by the operation procedure of FIG. 1 is dropped onto the substrate 51, the detection target DNA 44 in the mixed solution is a probe having a base sequence complementary to the DNA. Each specifically binds (hybridizes) to DNA 52. Therefore, by selecting the type of probe DNA 52 to be immobilized on the substrate 51 and its fixing position, the detection target side DNA 44 existing in the specimen, in other words, the detection target side DNA 44 is specifically determined according to the position. The type of the bound protein 41 to be detected can be specified (addressing).

  The presence or absence of the hybridization can be confirmed with a fluorescence reader. By reading the position of the site on the protein chip 5 that emits fluorescence, the presence or absence of the protein (antigen) 41 present in the sample and its presence The type can be identified.

  Next, the chemical reaction cartridge according to the present invention will be described with reference to FIG. This cartridge is for performing protein detection according to the protein detection method of the present invention.

  FIG. 3A is a plan view of the cartridge of this embodiment, and FIG. 3B is a cross-sectional view taken along the line BB of FIG.

  As shown in FIG. 3B, the cartridge of this embodiment includes a substrate 1 in which the protein chip 5 is embedded and an elastic member 2 that is superimposed on the substrate 1.

  On the back surface (lower surface in FIG. 3B) of the elastic member 2, a concave portion having a predetermined shape is formed which is recessed toward the front surface (upper surface in FIG. 3B). This recess creates a space between the cartridge substrate 1 and the elastic member 2, and as shown in FIGS. 3 (a) and 3 (b), a well 21 for receiving a sample and a later-described reagent are accommodated in advance. Wells 22 and 23, a well 24 for mixing the sample and the reagent, a storage unit 25 for storing the protein chip 5, a well 26 for storing the waste liquid, and an injection for injecting the sample into the well 21 A channel 27, a channel 28 connecting the well 21 and the well 24, a channel 29 connecting the well 22 and the well 24, a channel 30 connecting the well 23 and the well 24, the well 24 and the accommodating portion 25. A flow path 31 to be connected and a flow path 32 to connect the accommodating portion 25 and the well 26 are formed.

  The well 22 contains a secondary antibody 43 and a buffer solution labeled with a fluorescent molecule 42, and the well 23 contains a primary antibody 45 and a buffer solution bonded to the detection target side DNA 44.

  Next, a detection method using a cartridge will be described.

  By using an injection needle or the like, a sample containing protein is injected into the well 21 via the injection path 27.

  Next, as shown in FIG. 3B, when the roller 6 is rotated to the right while being pressed against the cartridge, the elastic member 2 is elastically deformed, and the solution in the cartridge is conveyed according to the movement of the roller 6. Therefore, the sample accommodated in the well 21 and the reagent accommodated in the well 22 and the well 23 reach the well 24 via the flow path 28, the flow path 29, and the flow path 30, respectively, and are mixed. . Thereby, the operation procedure shown in FIG. 1 is automatically executed.

  When the roller 6 is further rotated, the mixed solution reaches the accommodating portion 25 through the flow path 31, and hybridization in the protein chip 5 is performed. Excess liquid mixture is contained in the well 26. Alternatively, the washing solution may be stored in a cartridge, and the protein chip 5 after hybridization may be washed using the washing solution. In this case, the cleaning liquid may be reciprocated on the protein chip 5 by reciprocating the roller 6.

  Thereafter, the specimen can be identified as described above by reading the fluorescent position on the protein chip 5 fixed to the cartridge. After removing the protein chip 5 from the cartridge, reading by a fluorescence reader may be performed.

  According to the detection method of the present embodiment, since the antigen-antibody reaction is performed in advance in a solution in which the antigen and the antibody can freely move three-dimensionally, the probability of the association between the antigen and the antibody is high, and highly sensitive detection is possible. It becomes. Further, since the solution can be easily moved, the reaction rate of the antigen / antibody reaction is high, and a trace amount of specimen can be detected in a short time. In general, the larger the value of the product of the solution concentration and the reaction rate, the shorter the time until the antigen / antibody reaction is saturated. In this embodiment, this value can be easily improved.

  A technique for fixing the probe DNA to the substrate has already been established. For this reason, a highly reliable protein chip can be produced at low cost.

  Furthermore, even if the protein (antigen) to be detected changes, only the selection of the antibody and the binding between the antibody and the detection target side DNA 44 need to be considered, and the same probe DNA 52 of the protein chip 5 can be used. Therefore, it is possible to obtain a protein chip at low cost, and it is not necessary to take into consideration a change in hybridization efficiency due to a difference in DNA, and variations between chips are suppressed. In addition, a wide variety of proteins (antigens) can be detected using the same protein chip.

  Various conventional techniques can be applied to the protein detection method of the present invention. For example, as a method for connecting a protein (antigen) serving as a detection target and a fluorescent molecule, an ELISA method, a sandwich ELISA method, a competitive reaction type ELISA method, or the like can be used. The protein chip substrate and the probe DNA can be connected by a covalent bond method, an electrostatic bond method, an avidin / biotin bond method, a gold / thiol bond method, a film formation method using photolithography, or an ink jet method. Etc. can be used.

  The protein detection method of the present invention is not limited to the above embodiment, and various modifications are possible. For example, instead of preliminarily binding a secondary antibody to a protein (antigen), after addressing by hybridization on the substrate, the secondary antigen is bound to the protein (antigen) on the substrate, thereby fluorescently labeling it. May be. Moreover, you may add a fluorescent marker to the antibody corresponded to the primary antibody of the said embodiment. Furthermore, the hybridized DNA may be fluorescently labeled by applying an intercalator method.

  Alternatively, FRET may be applied to add different fluorescent markers to the primary antibody and the secondary antibody, and site detection using fluorescence energy transfer may be performed.

  Furthermore, in the said embodiment, although the protein chip which performs the detection in a plane was shown, you may use the protein chip which performs the detection in three dimensions. For example, a sample (mixed solution described above) configured to be capable of penetrating the chip during hybridization can be used.

  A site for detecting DNA may also be provided on the protein chip. In this case, for example, it is possible to simultaneously detect DNA of a pathogenic bacterium for a specific infectious disease contained in a body fluid and an antibody made against the pathogenic bacterium with a single chip.

  As described above, according to the protein detection method of the present invention, the protein to be detected is addressed using hybridization by binding of complementary DNAs, so that the antigen-antibody reaction is performed on the chip. This is not necessary, and can be carried out in advance in a solution in which the antigen and the antibody can freely move three-dimensionally, and the protein can be detected with high sensitivity.

  The scope of application of the present invention is not limited to the above embodiment. The present invention can be widely applied when detecting proteins by addressing.

5 Protein chips 21 wells (sample container)
24 wells (joining part)
25 Storage part (detection part)
41 Protein (antigen)
42 fluorescent molecule 43 secondary antibody 44 DNA to be detected
45 Primary antibody 46 Buffer solution 52 Probe DNA

Claims (11)

In a protein detection method for detecting the presence of a specific protein by addressing,
Binding a target DNA for addressing to a target protein;
Binding a fluorescent label to the protein to be detected;
Addressing the protein to be detected using a substrate on which probe DNA that binds complementarily to the DNA to be detected is fixed;
A protein detection method comprising: The method for detecting a protein according to claim 1, wherein in the step of binding the detection target DNA to the detection target protein, an antibody that binds between the detection target protein and the detection target DNA is used. . The method for detecting a protein according to claim 1 or 2, wherein in the step of binding a fluorescent label to the protein to be detected, an antibody that binds between the protein to be detected and the fluorescent label is used. The step of binding the detection target DNA to the detection target protein and the step of binding a fluorescent label to the detection target protein are simultaneously performed in a solution. The method for detecting a protein according to 1. An ELISA method, a sandwich ELISA method, or a competitive reaction type ELISA method is used in the step of binding a detection target DNA to the detection target protein and the step of binding a fluorescent label to the detection target protein. Item 5. A method for detecting a protein according to any one of Items 1 to 4. As a method for immobilizing probe DNA on the substrate, a covalent bond method, an electrostatic bond method, a method using an avidin / biotin bond, a method using a gold / thiol bond, a film forming method using photolithography, or an ink jet method is used. The method for detecting a protein according to any one of claims 1 to 5. In a chemical reaction cartridge that detects the presence of a specific protein by addressing,
A sample container for containing a sample containing the protein to be detected;
A first solution storage section for storing a first solution containing the detection target DNA for addressing;
A second container for containing a second solution containing a fluorescent label;
A binding unit that mixes the sample and the first and second solutions, and binds the detection target side DNA and the fluorescent label to the detection target protein;
A detection unit that has a substrate on which probe DNA that binds complementarily to the detection target side DNA is fixed, and that addresses the detection target protein;
A cartridge for chemical reaction, comprising: 8. The chemical reaction cartridge according to claim 7, wherein the binding unit binds the detection target side DNA to the detection target protein in a solution. 9. The chemical reaction cartridge according to claim 8, wherein the binding unit binds the detection target side DNA to the detection target protein using an antigen / antibody reaction. The cartridge for chemical reaction according to any one of claims 7 to 9, wherein the detection unit simultaneously performs DNA detection by hybridization. The chemical reaction cartridge according to any one of claims 7 to 10, wherein a protein chip on which probe DNA is fixed is attached to the detection unit.

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