JP2017203665A - Photocleavable microcapsules, sensor using the same and a measuring method of substance to be measured using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensor capable of amplifying a signal from the sensor by a molecule recognition element, a photocleavable microcapsule used therefor, and a measuring method of substance to be measured using the same.SOLUTION: The photocleavable microcapsules has a multilayered structure including, from the inner side to the outer side; a skeleton layer 1; a lipid bilayer film 2; an outer shell layer 3; and an antibody modify layer 4 in this order. The skeleton layer 1 is a lamination of polycation and polyanion high molecule electrolyte laminated alternately. The lipid bilayer film 2 consists of 2 molecule film of cationic amphiphile. The outer shell layer 3 is composed of TiO2 functioning as a photocatalyst. The antibody modify layer 4 is a self-assembled monolayer film binned with an antibody 5.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a photocleavable microcapsule, a sensor using the same, and a method for measuring a substance to be measured using the same, and is suitable for highly sensitive measurement of a biomarker.

  High-sensitivity measurement of biomarkers includes chromatography, enzyme-linked immunoassay (ELISA), and electrophoresis, but it requires a lot of time and labor and cost for complicated work. Biomarker visualization devices require rapid and sensitive measurement. Since chemical reaction is a diffusion phenomenon, the smaller the amount of sample, the more advantageous is rapid and high sensitivity. Multiple flow tanks such as dispensing tanks, mixing tanks, reaction tanks, and waste liquid tanks on a small substrate are connected to the flow path. A biosensor that employs a fine flow path system connected by the most suitable.

  A biosensor is composed of a part that recognizes only a target substance (molecular recognition element) and a part that converts recognized information into a signal (signal conversion element), and the characteristics of both elements influence the sensor performance. Usually, the signal conversion element also serves as signal amplification, and there is almost no report about signal amplification on the molecular recognition element side.

On the other hand, as a technique related to the present invention, an electrochemistry in which a ferrocene compound is encapsulated in a microcapsule, the ferrocene compound released by breaking the capsule is used as a mediator, and its oxidation current is measured amperometrically. There is a sensor (Patent Document 2).
Also known is a microcapsule technology in which a surface of a microcapsule composed of a lipid bilayer is coated with a SiO ₂ —TiO ₂ film and photocleavage is caused by utilizing the photocatalytic ability of TiO ₂ (Non-patent Document 1). ).

JP 2009-210417 A JP 2011-145079 A

Tunable UV-Responsive Organic-Inorganic HybridCapsules Kiyofumi Katagiri, Kunihito Kournoto, Shogo IseyaMototsugu Sakai. Atsunori Matsuda, and Frank Caruso

  An object of the present invention is to provide a sensor capable of amplifying a sensor signal on the molecular recognition element side, a photocleavable microcapsule used therefor, and a method for measuring a substance to be measured using the same.

  The photocleavable microcapsule of the present invention is a microcapsule whose surface is modified with a molecular recognition probe capable of specifically binding to a substance to be measured, and the inside of the microcapsule is detected by a light receiving element. A substance for enabling is included, and the shell wall of the microcapsule includes a photocatalyst and a material that is decomposed by light irradiation to the photocatalyst.

  In the photocleavable microcapsule of the present invention, since the molecular recognition probe capable of specifically binding to the substance to be measured is modified on the surface, the molecular recognition probe is not subjected to contact with the liquid to be measured. It can be combined with a measurement substance to form a microcapsule-measurement substance complex. For this reason, when the liquid to be measured containing this complex is brought into contact with a fixed part on which the substance to be measured is immobilized, the microcapsule is fixed to the substance to be measured immobilized on the fixed part via the molecular recognition probe. And a reaction in which microcapsules bind to a substance to be measured existing in a measurement solution via a molecular recognition probe. Furthermore, a substance for enabling detection by a light receiving element is included inside the microcapsule, and the shell wall of the microcapsule includes a photocatalyst and a material that is decomposed by light irradiation to the photocatalyst. Therefore, if light was irradiated to the microcapsule fixed to the fixing part or the microcapsule not fixed to the fixing part but distilled off, the shell wall of the capsule was broken and encapsulated by the action of the photocatalyst. A number of substances are released. And since many of this emitted substance is a substance for enabling the detection by a light receiving element, the signal of a sensor can be amplified according to the quantity of the released substance.

  The substance to be encapsulated in the photocleavable microcapsule of the present invention is not particularly limited as long as it can be detected by a light receiving element. Examples of such substances include a catalyst for emitting a luminescent precursor (for example, an enzyme that promotes a reaction that decomposes the luminescent precursor to emit light, such as alkaline phosphatase), and a dye precursor as a dye. Examples include a catalyst for conversion, a light-absorbing substance, and a fluorescent substance.

  In addition, the molecular recognition probe modified on the surface of the photocleavable microcapsule of the present invention is not particularly limited as long as it is a probe that can specifically bind to the substance to be measured. Examples of such a molecular recognition probe include an antibody that specifically binds to an antigen to form a complex, an enzyme that forms a complex with a substrate, a gene encoded by a specific base sequence, and the like.

  The photocleavable microcapsule of the present invention has a structure comprising a skeleton layer, a lipid bilayer membrane that covers the skeleton tank, and an outer shell layer that covers the lipid bilayer membrane and contains a photocatalyst. Can do.

  By using the photocleavable microcapsule of the present invention, a sensor capable of amplifying the sensor signal on the molecular recognition element side can be constructed. That is, the photocleavable microcapsule of the present invention, a fixed part on which a molecular recognition probe capable of specifically binding to a substance to be measured is fixed on the surface, and the fixed part or the liquid to be measured are irradiated with light And a light receiving unit that senses light generated by the action of a substance for enabling detection by the light receiving element.

Using this sensor, the signal of the sensor can be amplified on the molecular recognition element side by the following method. That is,
A first step in which a measurement liquid containing a substance to be measured is brought into contact with a fixing part on which a molecular recognition probe capable of specifically binding to the substance to be measured is fixed on the surface;
A second step of removing the liquid to be measured on the fixed part;
A third step in which the capsule-containing liquid in which the photocleavable microcapsules according to any one of claims 1 to 4 are dispersed is brought into contact with the immobilization part in which the substance to be measured is captured by the molecular recognition probe;
A fourth step of removing the capsule-containing liquid on the fixing part;
A fifth step of irradiating the fixing part or the removed capsule-containing liquid with light to cleave the photocleavable capsule to release a substance enabling detection by a light receiving element;
A sixth step of sensing the intensity of light generated by the action of the substance;
A method for measuring a substance to be measured.

  According to the present invention, for example, in a biosensor, it is possible to amplify the sensor signal on the molecular recognition element side, so that it is possible to perform highly sensitive measurement of a substance to be measured (biomarker such as cancer) it can.

It is a schematic cross section of the photocleavable microcapsule of an embodiment. It is a schematic diagram of alkaline phosphatase encapsulated in the photocleavable microcapsule of the embodiment. It is process drawing which prepares the photocleavable microcapsule of embodiment. 2 is a plan view of a centrifugal sensor 20. FIG. It is process drawing which shows the measurement of the cytokine by the sensor using a photocleavable microcapsule. It is a graph which shows the measurement result by the QCM method of the laminated film used as a model of a skeleton layer. It is the SEM photograph ((A)-(C)) and TEM photograph (D) of the hollow particle which consists of a frame layer. It is the graph which measured the zeta potential for every step at the time of laminating | stacking a cationic polymer electrolyte and an anionic polymer electrolyte. It is a process diagram in the case of forming a TiO ₂ layer on a silicon wafer and further immobilizing antibodies on the surface thereof. On a silicon wafer polyanion / polycation laminated film and TiO ₂ layers is a graph showing the analysis results by EDX of the stacked multilayer structure. It is a photograph which shows the external appearance of a centrifugal immunosensor. It is a model process figure which shows the preparation method of the calibration curve of cortisol. It is a calibration curve when the concentration of cortisol is measured with a centrifugal immunosensor.

  Hereinafter, embodiments embodying the present invention will be described. However, the present invention is not limited to the description of this embodiment and the following examples. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.

<Photocleavable microcapsules>
As shown in FIG. 1, the photocleavable microcapsule of the embodiment has a multilayer structure of a skeleton layer 1, a lipid bilayer membrane 2, an outer shell layer 3, and an antibody modification layer 4 in order from the inside to the outside. . The skeleton layer 1 has a structure in which polycation polymer electrolytes and polyanion polymer electrolytes are alternately laminated, and the lipid bilayer membrane 2 has a hydrophilic group of a cationic type amphiphile facing outward. It consists of a bimolecular film. The outer shell layer 3 is made of TiO _{2 that} functions as a photocatalyst. Furthermore, the antibody modification layer 4 is composed of a self-assembled monolayer (SAM film: Self-Asssembled. Monolayers) composed of carboxydecylphosphonic acid, and an antibody 5 having cortisol as an antigen is present on the carboxy group of carboxydecylphosphonic acid. The phosphonic acid group is fixed by a phosphonic acid ester bond and a hydroxyl group on the surface of TiO ₂ forming the outer shell layer 3.
On the other hand, alkaline phosphatase 6 is dispersed in an aqueous solvent 7 inside the photocleavable microcapsule. As shown in FIG. 2, alkaline phosphatase 6 has an epitope 6a that binds to an antibody.

The photocleavable microcapsule of the embodiment configured as described above can be manufactured as follows according to the process chart shown in FIG.
(1) Formation of skeletal layer 1 First, solid particles 10 serving as nuclei are placed in a beaker, an aqueous solution of cationic polymer electrolyte (or anionic polymer electrolyte) is added and stirred, and then incubated for a certain period of time. At this time, the cationic polymer electrolyte (or anionic polymer electrolyte) is adsorbed on the surface of the solid particles 10. Then, after removing the supernatant by centrifugation, an aqueous solution of anionic polymer electrolyte (or cationic polymer electrolyte) is added and stirred, and then incubated for a certain period of time. Here, the anionic polymer electrolyte (or cationic polymer electrolyte) is adsorbed to the previously adsorbed cationic polymer electrolyte (or anionic polymer electrolyte) by electrostatic attraction. In this way, the cationic polymer electrolyte and the anionic polymer electrolyte are alternately and repeatedly laminated on the solid particles 10 (see FIG. 3B). Finally, the solid particles 10 are dissolved and removed with a solution for dissolving the solid particles 10 to obtain hollow particles 11 composed of a skeleton layer 1 in which a cationic polymer electrolyte and an anionic polymer electrolyte are alternately laminated (FIG. 3 ( c)).

(2) Lamination of lipid bilayer membrane 2 Next, a lipid bilayer is laminated on the hollow particles 11 obtained as described above.
As a material for the lipid bilayer membrane, a cationic amphiphile such as dimethyldioctadecyl ammonium bromide can be used. These cationic amphiphiles are purified and then dissolved in pure water at a temperature higher than the phase transition temperature (for example, 60 ° C.), and MLV (multilayer liposomes, multilamellar vesicles) composed of cationic amphiphiles multilamellar vesicle) Prepare a dispersion. SUV (single-membrane liposome, small unilamellar vesicle) dispersion is obtained by sonicating this MLV dispersion at a temperature above the phase transition temperature. Then, the SUV dispersion is added to the solution containing the hollow particles 11 obtained as described above, and left to stand overnight. Thereafter, the mixture is mixed with a stirrer, and the supernatant is removed to obtain hollow particles 13 on which the lipid bilayer membrane 2 is laminated as shown in FIG.

(3) Encapsulation of alkaline phosphatase 6 A solution in which alkaline phosphatase 6 is dissolved is set to a temperature equal to or higher than the phase transition temperature of the lipid bilayer, and the lipid bilayer 2 prepared as described above is laminated thereon. The hollow particles 13 are immersed. Here, since the lipid bilayer membrane 2 of the hollow particles 13 has a temperature equal to or higher than the phase transition temperature, the permeability of the membrane itself increases, and a solution in which the alkaline phosphatase 6 is dissolved enters the hollow portions of the hollow particles 13. Thereafter, by setting the temperature of the solution to be equal to or lower than the phase transition temperature, the permeability of the lipid bilayer membrane 2 is lowered, and particles 14 containing a liquid in which alkaline phosphatase 6 is dispersed in an aqueous solvent 7 are obtained (FIG. 3 ( e)).

(4) Formation of outer shell layer 3 For forming the outer shell layer 3 made of TiO ₂ on the particles 14, a well-known sol-gel method for forming a titanium oxide film can be used. That is, after a titanium alcoholate such as titanium tetraisopropoxide (Ti [OCH (CH ₃ ) ₂ ] ₄ ) is hydrolyzed, a titanium oxide sol solution is obtained, and hollow particles 14 are added to this solution to add TiO ₂ to the hollow particles 14. Then, after removing the supernatant and drying, particles 15 having the outer shell layer 3 made of TiO2 particles formed on the surface are obtained.

(5) Surface modification with molecular recognition probe Finally, the particle 15 is immersed in an aqueous solution of carboxydecylphosphonic acid, and the hydroxyl group on the surface of TiO ₂ constituting the outer shell layer 3 is bonded to the phosphonate ester to form a self-assembled monomolecular film. (SAM membrane: Self-Asssembled. Monolayers), then the antibody modification layer 4 is formed by linking the carboxylic acid group present in the self-assembled monolayer with the antibody 5 having a cytokine as an antigen. A cleavable microcapsule 16 is obtained.

<Measurement of cytokines>
Next, cytokine measurement using the photocleavable microcapsule will be described. FIG. 4 is a plan view of the centrifugal sensor 20 that serves as a reaction field in sensing. The centrifugal sensor 20 is provided with a round hole 21a at the center of a plastic disc 21. The round sensor 21 can be rotated at an arbitrary number of rotations by inserting the round hole 21a into a rotation shaft of a rotation driving device (not shown). it can. The disc 21 is provided with dumbbell-shaped sensor recesses 22 at four symmetrical positions orthogonal to each other. The measurement unit recess 22 is connected to the mixing unit 22a and the measurement unit 22b by a thin groove 22c. On the surface of the mixing portion 22a, an anti-cytokine antibody (however, an antibody having an epitope different from that of the antibody 5 immobilized on the surface of the photocleavable microcapsule) is immobilized. On the other hand, an anti-alkaline phosphatase antibody is immobilized on the surface of the measurement unit 22b. The antibody density required for immobilization is determined by the concentration of the substance to be measured. Cytokines for use as cancer markers are designed to capture photocleavable microcapsules at a concentration of 1-100 pg / mL. Immobilization efficiency can be adjusted by the alkyl chain length of the molecules constituting SAM.

The centrifugal sensor 20 configured as described above is set on the rotary drive device so as to be horizontal. The details of the centrifugal sensor are described in the following document.
1) Tomoki Shimakura and Masaki Yamaguchi: Application of hydrophobic micropatterns to centrifugal fluid valve in flow channel, Journal of Adhesion Science and Technology, 29 (23) (2015) 2565-2575
2) Masaki Yamaguchi, Hiroki Katagata, Yuki Tezuka, Daisuke Niwa, Vivek Shetty: Automated-immunosensor with centrifugal fluid valves for salivary cortisol measurement, Sensing and BioSensing Research, 1 (2014) 15-20
Then, a predetermined amount of a measurement solution containing cytokine is added to the mixing unit 22a (upper part of FIG. 5A). Thereby, the cytokine in the measurement solution binds to the anti-cytokine antibody immobilized on the mixing portion 22a at a ratio corresponding to the concentration (bottom of FIG. 5 (a)). And after driving a rotation drive device and removing a measurement liquid, the dispersion liquid of a photocleavable microcapsule is added. As a result, the anti-cytokine antibody modified on the surface of the photocleavable microcapsule recognizes and further binds to another epitope in the cytokine bound to the anti-cytokine antibody immobilized on the mixing part 22a (FIG. 5 (b) bottom). Then, after driving the rotation driving device to remove the non-fixed photocleavable microcapsules, water is added and light is irradiated with a high-pressure mercury lamp (not shown). As a result, the capsule is broken by the action of the titanium oxide catalyst of the photocleavable microcapsule bonded to the cytokine, and the encapsulated alkaline phosphatase is released (bottom of FIG. 5 (c)). Then, the rotational drive device is driven to move the alkaline phosphatase together with water to the measurement unit 22b (upper part of FIG. 5 (d)). Thus, the alkaline phosphatase that has moved to the measurement unit 22b binds to the anti-alkaline phosphatase antibody immobilized on the measurement unit 22b (bottom of FIG. 5 (d)). Finally, a luminescent substrate that emits light by alkaline phosphatase (ALP) is added to the measurement unit 22b, chemiluminescent light generated by the catalytic action of alkaline phosphatase is received by the photomultiplier tube 23, and the light intensity is measured by a measuring device (not shown). To do.

  In this measurement method, since the emission intensity is detected with a photomultiplier tube (wavelength 185 to 680 nm) with extremely high sensitivity, high sensitivity measurement is possible. In addition, the number of substances to be measured is amplified according to the concentration ratio between the captured substance to be measured and the released substance to be measured, and the amplification factor is the molar concentration of the contained antigen, the microparticle diameter, and It is related to the antigen concentration to be measured. Therefore, by adjusting these factors, the signal amplification factor can be adjusted in an arbitrary range (for example, 5 to 100 times).

  By the way, many types of cytokines are known. For example, chemokines controlling organ selectivity include MCP-1, MIP-1α, RANTES, MCP-2, TARC, ELC, SLC, MDC, MIP -3α, TECK, Gro-γ, IL-8, IP-10, SDF-1α, CXCL16, and other cytokines include IL-1β, IL-2, IL-4, IL-6, IL- 10, IL-12, IL-18, IL-21, GM-CSF, TNF-α, TFG-β, EGF, PDGF, INF-γ, G-CSF. For this reason, the density | concentration of various cytokine can be measured by selecting the cytokine according to a measuring object.

<Modification of Embodiment>
In the above embodiment, alkaline phosphatase is used as the substance that enables the detection by the light receiving element to be included in the photocleavable microcapsule, but instead of this, a catalyst for converting the dye precursor to the dye or Alternatively, a light-absorbing substance or a fluorescent substance may be used.

<Test example>
(1) Formation of skeletal layer model As a basic technology of the skeleton layer forming step ((a) to (c) in the process diagram of FIG. 3) in the preparation of the photocleavable microcapsule of the present invention, a single crystal quartz crystal A crystal resonator with an Au electrode attached thereto was prepared, and a skeletal layer formation test was performed by an alternate lamination method on the Au electrode. That is,
1) 3μL of piranha solution (concentrated sulfuric acid: 30% hydrogen peroxide solution = 3: 1 mixed solution) is dropped onto the Au electrode of the crystal unit and left for 5 minutes to remove organic substances adhering to the surface. It was. Then, it was washed 3 times with pure water and dried with N ₂ gas.
2) Then, in order to evaluate the stacking amount, a change F (Hz) in resonance frequency was measured using the QCM method (measuring device: manufactured by FINIIX QN Corporation) (hereinafter the same).
3) Furthermore, polyethyleneimine (SigmaAldrich, CAS No. 9002-98-6, average molecular weight ~ 1,800 by GPC, ~ 2,000 by LS, hereinafter referred to as "PEI") was added to 0.5M NaCl solution at a concentration of 1 mg / mL. After immersing the Au electrode in the solution added for a predetermined time, it was washed 3 times with pure water and dried with N ₂ gas, and then the resonance frequency was measured by QCM.
4) Next, poly (sodium 4-styrenesulfonate) (Sigma Aldrich, CAS No. 26062-79-3, average molecular weight 100,000-200,000, hereinafter referred to as “PSS”) in 0.5 M NaCl solution is 1 mg / mL. After immersing the Au electrode in the added solution for a predetermined time, it was washed 3 times with pure water and dried with N2 gas, and then the resonance frequency was measured by QCM.
5) Furthermore, Poly (diallyldimethylammonium chloride) (SigmaAldrich, CAS No.25704-18-1, average molecular weight ~ 70,000), hereinafter referred to as “PDDA”) in the NaCl solution of the prescribed concentration is 1 mg / mL. After immersing the Au electrode in the solution so added for a predetermined time, it was washed three times with pure water and dried with N ₂ gas, and then the resonance frequency was measured by QCM.
6) The steps 4) to 6) were repeated so that the stacking order was 7 layers in the order of PEI (+) → PSS (−) → PDDA (+) → PSS (−).

  As a result of measuring the laminated film, which is a model of the skeleton layer, formed on the Au electrode of the quartz resonator as described above by the QCM method, the amount of decrease in the resonance frequency is 3.89 kHz in total for the seven layers. Was estimated to be 78 nm (see FIG. 6).

(2) Preparation of hollow particles composed of a skeletal layer MF particles composed of melamine formaldehyde condensate with a low degree of crosslinking and soluble in hydrochloric acid (microParticles GmbH, custom-made products (Weakly cross-linked MF particles diam (HCl- soluble)), 2.86 μm, SD = 0.06 μm) were placed in a beaker, an aqueous solution of cationic polymer electrolyte (or anionic polymer electrolyte) was added and stirred, and then incubated for a certain period of time. Then, the operation of centrifuging and removing the supernatant liquid is repeated three times, and then an aqueous solution of anionic polymer electrolyte (or cationic polymer electrolyte) is added and stirred, followed by incubation for a certain period of time, and then the supernatant liquid is centrifuged. The removal operation was repeated 3 times. Thus, the cationic polymer electrolyte and the anionic polymer electrolyte were alternately laminated on the FM particles several times. Finally, the operation of dissolving and removing the MF particles with hydrochloric acid and centrifuging and removing the supernatant was repeated three times. Thus, hollow particles composed of a skeleton layer in which a cationic polymer electrolyte and an anionic polymer electrolyte were alternately laminated were obtained.

The SEM photograph and TEM photograph of the hollow particles obtained as described above are shown in FIG.
(A) is a core (melamine-formaldehyde; MF) particle with a non-laminated skeleton layer, (B) is an MF particle after laminating the skeleton layer, and (C) is a microcapsule (skeleton) after dissolving the MF particle with HCl. (Device name: JSM-7001F, manufactured by JEOL Ltd.). (D) is a TEM image (device name: JEM-2100, manufactured by JEOL Ltd.) of a microcapsule (skeleton layer) after MF particles are dissolved with HCl. In the TEM images of the unstacked (A) and post-laminated (B) MF particles, the particles are blackish. This is because the particles were thick and the electrons could not be transmitted. The outer shape (C) of the laminated MF particles is slightly uneven compared to the unstacked MF particles. On the other hand, it was observed that the hollow microcapsule (D) after dissolving the MF particles with HCl was permeated and hollow. The reason why the hollow microcapsules are collapsed in the TEM image is thought to be due to the observation in a vacuum system.

  The state of lamination of the cationic polymer electrolyte and the anionic polymer electrolyte during the preparation of the hollow particles composed of the skeleton layer was confirmed by measuring the zeta potential at each production step. A zeta potential meter (Zetasizer Nano ZS90, Malvern) was used for the measurement of the zeta potential. The results are shown in FIG. Unstacked MF particles have a positive charge. It is negative after PSS lamination and positive after PDDA lamination. This indicates that when the zeta potential is negative, the PSS layer is formed on the outermost surface, and when the zeta potential is positive, the PDDA layer is formed on the outermost surface, and the PSS layer and the PDDA layer are alternately stacked. .

(3) Formation of shell wall consisting of skeleton layer and TiO ₂ layer on capsule surface and antibody immobilization Formation of shell wall and antibody immobilization on skeleton layer surface in preparation of photocleavable microcapsule of the present invention ( As a basic technique of (f) to (g) in the process diagram of FIG. 3, a TiO ₂ layer is formed on a silicon wafer, and an antibody is immobilized on the surface thereof (see the process diagram shown in FIG. 9). . That is,
1) First, a solution having a mixing ratio of 30% NH ₄ OH: 30% H ₂ O ₂ : water = 1: 1: 5 was kept at 75 ° C., and a silicon wafer was immersed in this solution for 10 minutes. By this operation, silanol groups were formed on the surface of the SiO2 layer present on the surface layer of the silicon wafer to enhance the hydrophilicity (see FIG. 9B). Then, it was immersed in pure water for 15 min to hydrate the SiO ₂ surface and to have a negative charge.
2) A silicon wafer on which silanol groups were formed was immersed in a solution in which PEI was added to a 0.5 M NaCl solution to a concentration of 1 mg / mL for 5 minutes, and then washed three times with pure water.
3) Next, it was immersed in an aqueous sodium chloride solution added to a 0.5 M NaCl solution so that the concentration of PSS was 1 mg / mL for 5 minutes, and then washed three times with pure water.
4) Further, after immersing the silicon wafer in a solution in which PDDA was added to a predetermined concentration of NaCl solution to a concentration of 1 mg / mL for 5 minutes, it was washed three times with pure water.
5) The steps 4) to 6) were repeated so that the total number of layers was 5 in the order of PEI (+) → PSS (−) → PDDA (+) → PSS (−) (FIG. 9C). reference).
6) Further PSS / PDDA dispersion of a silicon wafer formed by laminating TiO ₂ and (TiO _2: diluted 1.99% by weight (Tayca TKS-203 10-fold with pure water), the zeta potential: -49.2 mV) immersion The mixture was allowed to stand for 10 minutes, washed with pure water, and dried at room temperature.
Thus, a multilayer structure in which a polyanion / polycation laminate film and a TiO ₂ layer were laminated on a silicon wafer was prepared (see FIG. 9D). The results of analysis by EDX of this product are shown in Table 1 and FIG. These results show that a layer of TiO ₂ is formed.

A self-assembled monomolecule on the TiO ₂ film, which is the uppermost layer of a multilayer structure in which a polyanion / polycation laminate film and a TiO ₂ layer are laminated on the silicon wafer shown in FIG. A film (SAM film) was formed (see FIG. 9E).
That is, the silicon wafer having the multilayer structure shown in FIG. 9D was immersed in an ethanol solution of carboxydecylphosphonic acid (manufactured by Dojin Chemical Laboratories) for 12 hours. Thereafter, it was washed with ethanol, dried at room temperature while blowing nitrogen gas, and further heated at 50 ° C. for 30 minutes for drying. As a result, a hydroxyl group on the surface of TiO ₂ and carboxydecylphosphonic acid were dehydrated and condensed to form a phosphonate ester bond, thereby forming a self-assembled monolayer (SAM film) (see FIG. 9 (e)). ).

Furthermore, the carboxyl group of the SAM membrane was activated ester by the amine coupling method and immobilized by binding with cort-3-BSA. (Refer FIG.9 (f)). Details are shown below.
1) 200μL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (WSC) solution with a concentration of 100 mmol / L in a microtube and N-hydroxysuccinimide (NHS) solution with a concentration of 100 mmol / L 200 μl was pipetted and mixed.
2) The mixed solution prepared in 1) was dropped on the surface of the SAM membrane, allowed to stand at room temperature for 10 minutes, and then washed three times with phosphate buffered saline.
3) The sample solution was dropped on the pad surface and left at room temperature for 30 minutes. The sample solution used was a solution obtained by dissolving cort-3-BSA at 10 μg / mL in the attached Activation Buffer.
5) Washed 3 times with phosphate buffered saline.
6) Blocking solution was dropped onto the self-assembled monolayer (SAM film) and left at room temperature for 30 minutes.
7) After washing with phosphate buffered saline three times, drying at room temperature, water repellent treatment with a water repellent, and further blocking treatment (Milk protein), so that cortisol is mediated by BSA A cortisol-immobilized silicon wafer immobilized on the SAM film was obtained.

Using the cortisol-immobilized silicon wafer obtained as described above, the cortisol concentration of the cortisol-containing sample was measured. For the measurement, a centrifugal immunosensor shown in FIG. 11 was used (see Non-Patent Documents 1 and 2 below). This centrifugal immunosensor includes a disk chip for analysis, a stepping motor for rotating the disk chip at an arbitrary rotation speed, and a high electron multiplier for measurement (left of FIG. 11). The analytical disc chip is composed of each tank and a fine channel connecting them, and the solution dropped into the conjugate tank and the substrate tank is sent to the reaction tank by centrifugal force, and the luminescence intensity is measured. (Right side of Fig. 11).
1) Masaki Yamaguchi, Hiroki Katagata, Yuki Tezuka, Daisuke Niwa, Vivek Shettyc. “Automated-immunosensor with centrifugal fluid valves for salivary cortisol measurement”. Sensing and Bio-Sensing Research 1, 15-20. (2014).
2) Masaki Yamaguchi, Yohei Matsuda, Shohei Sasaki, Makoto Sasaki, Yoshihiro Kadoma, Yoshikatsu Imai, Daisuke Niwa, Vivek Shetty: Immunosensor with fluid control mechanism for salivary cortisol analysis, Biosensors and Bioelectronics 41: 186-191, (2012).

A calibration curve for cortisol was prepared by the following method.
1) A cortisol-immobilized silicon wafer is attached to the reaction tank on the right side of FIG.
2) Mix 30 µL of the sample (cortisol standard solution 1.0, 3.0, 10 ng / mL) and 30 µL of the conjugate solution, and let the antibody react for 1 minute at a constant room temperature of 25 ° C. (See FIG. 12 (a)). Here, the conjugate is an anti-cortisol antibody obtained by amide-bonding alkaline phosphatase (ALP) and can be easily prepared using a commercially available alkaline phosphatase labeling kit.
3) Dispense 20 μL of the reaction solution that has undergone the antigen-antibody reaction into the conjugation tank of the analytical disk chip, flow it by centrifugal force, move it to the reaction tank, and cortisol on the cortisol-immobilized silicon wafer for 1 minute at 25 ° C. And an antigen-antibody reaction (FIGS. 12B and 12C).
4) After introducing 30 μL of buffer solution from the buffer solution tank by centrifugal force and washing the reaction vessel, 30 μL of the luminescent substrate that emits light by alkaline phosphatase (ALP) was dispensed, and the luminescence intensity characteristics were evaluated (FIG. 12). (D)). The measurement conditions are as follows.
Cortisol standard solution concentration: 1 ng / mL, 3 ng / mL, 10 ng / mL
・ Measurement temperature 25 ℃ ・ Number of measurements: n = 3,
・ At the start of emission intensity measurement: Value after 300 seconds from the reaction

As a result, as shown in FIG. 13, a calibration curve in which the output (luminescence intensity) was inversely proportional to the cortisol concentration was obtained, and the calibration curve determination coefficient R ² = 0.99 was obtained. This measurement method uses a reaction in which cortisol in a measurement solution and cortisol immobilized on a silicon wafer competitively bind to an alkaline phosphatase (ALP) -modified anti-cortisol antibody, and the measurement is performed on a silicon wafer. It is an alkaline phosphatase (ALP) modified anti-cortisol antibody bound to cortisol immobilized on (see FIG. 12). For this reason, the higher the concentration of cortisol in the measurement solution, the fewer alkaline phosphatase (ALP) -modified anti-cortisol antibodies bound to cortisol immobilized on the silicon wafer, and as a result the output (luminescence intensity) becomes the cortisol concentration. It was an inversely proportional calibration curve.

DESCRIPTION OF SYMBOLS 1 ... Skeletal layer, 2 ... Lipid bilayer, 3 ... Outer shell layer, 4 ... Antibody modification layer, 5 ... Antibody 6 ... Alkaline phosphatase (6a ... Epitope), 7 ... Aqueous solvent, 16 ... Photocleavable microcapsule, 22 ... sensor recess (22a ... mixing part, 22b ... measuring part, 22c ... groove)

Claims (7)

A microcapsule whose surface is modified with a molecular recognition probe capable of specifically binding to a substance to be measured,
A substance for enabling detection by a light receiving element is contained inside the microcapsule, and the shell wall of the microcapsule contains a photocatalyst and a material that is decomposed by light irradiation to the photocatalyst. Features photocleavable microcapsules for sensor signal amplification.   The substance for enabling detection by the light receiving element is any one of a catalyst for emitting a light emitting precursor, a catalyst for converting a dye precursor into a dye, a light absorbing substance, and a fluorescent substance. The photocleavable microcapsule according to claim 1.   The photocleavable microcapsule according to claim 1 or 2, wherein the molecular recognition probe is an antibody.   The photocleavable microcapsule according to claim 3, wherein the antibody is an antibody having an antigen as a cytokine.   The microcapsule includes a skeleton layer, a lipid bilayer membrane covering the skeleton tank, and an outer shell layer covering the lipid bilayer membrane and containing a photocatalyst. The photocleavable microcapsule according to claim 1. The photocleavable microcapsule according to any one of claims 1 to 5,
An immobilization part in which a molecular recognition probe capable of specifically binding to the substance to be measured is immobilized on the surface;
A light irradiator for irradiating the fixed part or the liquid to be measured with light;
A light receiving part for sensing light generated by the action of a substance for enabling detection by the light receiving element;
A sensor comprising: A first step in which a measurement liquid containing a substance to be measured is brought into contact with a fixing portion where a molecular recognition probe capable of specifically binding to the substance to be measured is fixed on the surface;
A second step of removing the liquid to be measured on the fixed part;
A third step in which the capsule-containing liquid in which the photocleavable microcapsules of any one of claims 1 to 5 are dispersed is brought into contact with the immobilization part in which the substance to be measured is captured by the molecular recognition probe;
A fourth step of removing the capsule-containing liquid on the fixing part;
A fifth step of irradiating the fixing part or the removed capsule-containing liquid with light to cleave the photocleavable capsule to release a substance enabling detection by a light receiving element;
A sixth step of sensing the intensity of light generated by the action of the substance;
A method for measuring a substance to be measured.