JP4853971B2 - Method for producing sensing chip for target molecule - Google Patents

Description

  The present invention relates to a method for producing a sensing chip for a target molecule, and more particularly to a method for producing a sensing chip for a target molecule comprising a molecular imprint polymer on the surface of a chip substrate.

  As a technique used for diagnosing diseases and evaluating biomolecular interactions, array technology is one of the technologies that have been increasing in demand in recent years. By using an array in which many types of molecular recognition bodies are arranged on a chip substrate, it is possible to comprehensively analyze biomolecules in a sample. Conventionally, biomolecules such as proteins, antibodies, and nucleic acids are mainly used as molecular recognition bodies arranged on an array. However, while biomolecules have excellent molecular recognition ability, there is a problem that it takes time and cost to produce and acquire them. Furthermore, there is a problem that long-term storage is difficult and a problem that patterning is difficult when fixing on a substrate.

  Attempts have been made to reproduce molecular recognition in vivo with an artificial system. Molecular imprinting is known as one of the promising techniques. This method is one of the template polymerization methods. By using a molecule for recognition (target molecule) as a template and coexisting in the polymerization reaction, a molecular recognition site that interacts complementarily to the template molecule is polymerized. It is a method of constructing simultaneously at the time of reaction. Recently, it has been reported that a polymer (molecular imprint polymer) obtained by a molecular imprint method is applied to the field of sensor arrays (see Non-Patent Document 1).

  The present inventors have been researching to realize a molecularly imprinted polymer array, and have achieved excellent results. For example, Non-Patent Document 2 synthesizes six types of molecular imprint polymers using three types of template proteins and two types of functional monomers, and uses these six types of imprint polymers to produce three types. It is described that unique fingerprints were obtained for a total of 5 types of proteins, i.e., the template protein and non-template 2 types of proteins.

N. T. Greeneand K. D. Shimizu, J. Am. Chem. Soc., 2005, 127, 5695. T. Takeuchi, D. Goto and H. Shinmori, Analyst, 2007, 132, 101-103.

  However, the binding between the six types of molecularly imprinted polymers described in Non-Patent Document 2 and the target molecule was evaluated in a test tube, and the above six types of molecularly imprinted polymers were placed on the chip substrate. It does not use a placed array. This is because it is very difficult to dispose a plurality of different molecularly imprinted polymers in an arbitrary pattern on the chip substrate, and the technology has not yet been established.

  The present invention has been made in view of the above problems, and an object thereof is to provide a method for producing a sensing chip for a target molecule capable of arranging a molecularly imprinted polymer in an arbitrary pattern on the surface of a chip substrate. It is to be.

  As a result of intensive studies to solve the above problems, the present inventors have arranged nanoparticles in an arbitrary pattern on the surface of the target molecule-immobilized substrate, and immobilized the target molecules on the arranged nanoparticles, We found that by forming a molecularly imprinted polymer on the surface of the chip substrate using the immobilized target molecule as a template, a sensing chip for the target molecule in which the molecularly imprinted polymer is arranged in an arbitrary pattern can be produced, and the present invention is completed. I came to let you.

  That is, the method for producing a sensing chip for a target molecule according to the present invention is a method for producing a sensing chip for a target molecule having a molecular imprint polymer on the surface of the chip substrate. The method is characterized in that a target molecule is immobilized on the arranged nanoparticles and a molecularly imprinted polymer is formed on the chip substrate surface using the immobilized target molecule as a template.

  The method for producing a sensing chip for a target molecule according to the present invention is a method for producing a sensing chip for a target molecule comprising a molecularly imprinted polymer on the surface of the chip substrate, wherein biotinylated nanoparticles are provided on the surface of the target molecule-immobilized substrate. A nanoparticle placement step, avidin binding step for binding avidin to the biotinylated nanoparticles placed in the nanoparticle placement step, biotinylated on avidin bound to biotinylated nanoparticles in the avidin binding step A target molecule immobilization step for binding and immobilizing a target molecule, and a molecular imprint polymer formation step for forming a molecular imprint polymer on the chip substrate surface using the target molecule immobilized in the target molecule immobilization step as a template It is characterized by including.

  In the nanoparticle arranging step, the biotinylated nanoparticles are preferably arranged in an arbitrary pattern on the surface of the target molecule-immobilized substrate.

  In the nanoparticle arranging step, the biotinylated nanoparticles are preferably arranged in a plurality of regions on the surface of the target molecule-immobilized substrate.

  When biotinylated nanoparticles are arranged in a plurality of regions on the target molecule-immobilized substrate surface, a single type of target molecule is immobilized in each of the plurality of regions where the biotinylated nanoparticles are arranged, and the target molecule It is preferable that at least two types of target molecules are immobilized on the surface of the immobilized substrate, and it is more preferable that different types of target molecules are immobilized in each of a plurality of regions where biotinylated nanoparticles are arranged. .

  The target molecule sensing chip produced by the production method of the present invention is preferably a biomolecule target molecule, and more preferably a protein target molecule.

  By using the manufacturing method according to the present invention, the molecular imprint polymer can be easily arranged in an arbitrary pattern on the chip substrate surface. Therefore, it is possible to easily pattern a molecular imprint polymer targeting a plurality of different types of molecules on the surface of the chip substrate, and it is possible to fabricate an array type sensing chip simply and at low cost.

  In addition, since the molecularly imprinted polymer is formed using the immobilized target molecule as a template, a sensing chip including a molecularly imprinted polymer with high selectivity for the target molecule on the surface can be produced.

  A method for producing a sensing chip for a target molecule according to the present invention (hereinafter referred to as “manufacturing method according to the present invention”) is a method for producing a sensing chip for a target molecule comprising a molecular imprint polymer on the surface of a chip substrate. Any nanoparticle may be arranged on the surface of the molecule-immobilized substrate, the target molecule may be immobilized on the arranged nanoparticle, and the molecule-imprinted polymer may be formed on the surface of the chip substrate using the immobilized target molecule as a template. .

  In this specification, the “target molecule sensing chip” is a general term for a sensor substrate having a target molecule recognition body on the surface of a chip substrate, and selectively recognizes and binds to a target molecule and its binding information. Those having the function of converting into some signal and measuring the degree of binding of the target molecule are intended. When the sensing chip can detect the signal converted from the binding information (for example, color development or light emission), the sensing chip becomes a measuring device for the degree of binding, and the signal converted from the binding information can be detected by the sensing chip itself. When it is difficult to detect (for example, an electrochemical signal, vibration, etc.), a measurement device for the degree of binding is configured by combining such a signal with a detection device capable of detection. Therefore, the “target molecule sensing chip” includes what is called a sensor element, a sensor array, a sensor substrate, a molecular recognition element, or the like depending on the use mode.

  A “target molecule sensing chip” (hereinafter simply referred to as “sensing chip”) produced by the production method according to the present invention comprises a molecular imprint polymer on a chip substrate surface as a target molecule recognition body.

The molecular imprint polymer is a polymer synthesized using the molecular imprint method (molecular imprint method), which is one of the template polymerization methods. During the polymerization reaction, the molecule (target molecule) for the purpose of recognition is used as a template. By coexisting, a molecular recognition site that interacts complementarily with a template molecule during a polymerization reaction is constructed simultaneously with polymer synthesis. More specifically, first, a functional monomer having both a functional group capable of binding to a target molecule and a polymerizable functional group is bound to the target molecule to form a target molecule / functional monomer complex. This bond needs to be reversible. Next, a crosslinking agent and a polymerization initiator are added to the target molecule / functional monomer complex, and a polymerization reaction is performed. As a result, an organic polymer in which the shape of the target molecule (template molecule) and the arrangement of interaction points are stored is obtained. Finally, by extracting and removing the template molecule from the obtained polymer, a polymer (molecular imprint polymer) having a molecular recognition site that interacts with the template molecule in a substrate-specific manner is obtained. For the method of synthesizing the molecularly imprinted polymer, for example, the reference “Komiyama, M., Takeuchi, T., Mukawa, T., Asanuma, H.“ Molecular
Refer to the description of “Imprinting”, WILEY-VCH, Weinheim, 2002.

  The chip substrate used for the sensing chip manufactured by the manufacturing method according to the present invention may be any substrate that can form a molecular imprint polymer on the surface thereof. Examples of suitable substrates include metal substrates, glass substrates, silicon substrates, optical waveguide substrates, and the like. Moreover, it is preferable to select the material of the substrate as appropriate according to the detection means used for detecting the binding between the molecularly imprinted polymer and the target molecule. For example, when applied to the surface plasmon resonance (SPR) method, a gold substrate for SPR is preferably used, and when applied to the localized plasmon resonance (LSPR) method, an optical waveguide substrate is preferably used. When applied to a sensor, it is preferable to use a quartz crystal as a sensing chip, and when applied to detection by an electrochemical method, it is preferable to use a metal substrate (for example, a gold substrate) serving as an electrode. When applied to detection by light emission or color development, it is preferable to use a glass, quartz substrate, optical fiber, or optical waveguide substrate.

  In the present specification, “nanoparticles” are intended to be fine particles having an average particle diameter of less than 1 μm. The average particle diameter can be measured, for example, with a light scattering photometer. The nanoparticle used in the production method according to the present invention may be any nanoparticle that can bind and immobilize a target molecule directly or indirectly (via another molecule). For example, organic nanoparticles such as polystyrene and polymethyl methacrylate, inorganic nanoparticles such as silica nanoparticles and metal oxide nanoparticles can be suitably used. Moreover, the nanoparticle should just be dispersible in a solvent, The particle diameter has preferable 10 dozen nm-several hundred nm, More preferably, it is 70 nm-800 nm. Nanoparticles may be produced by themselves (see the examples in the subsequent stage), or commercially available products may be used.

  The target molecule-immobilized substrate used in the production method according to the present invention is not particularly limited as long as nanoparticles can be arranged on the surface thereof. For example, a metal substrate, a glass substrate, a silicon substrate, an optical waveguide substrate, or the like can be suitably used. The size is not limited, and may be selected according to the size of the sensing chip to be manufactured.

  Since nanoparticles can be handled in the same manner as a solution, they can be arranged in an arbitrary pattern on a substrate using a known technique such as an arrayer or a spotter. Therefore, the target molecules immobilized on the nanoparticles are also arranged in an arbitrary pattern, and as a result, the molecular imprint polymer created on the chip substrate surface using this as a template is subjected to arbitrary patterning. It will be. Therefore, the production method according to the present invention is particularly suitable for producing an array type sensing chip having a molecular imprint polymer capable of recognizing a plurality of different target molecules on one chip substrate. When an array-type sensing chip is manufactured, a plurality of separated sections may be provided on the surface of the target molecule-immobilized substrate. This makes it easier to arrange the nanoparticles in a plurality of regions in an arbitrary pattern. That is, by using the manufacturing method according to the present invention, an array type sensing chip can be easily manufactured.

  In the production method according to the present invention, the target molecule may be directly immobilized on the nanoparticle disposed on the surface of the target molecule-immobilized substrate, or may be indirectly immobilized on the nanoparticle. “Indirectly immobilized” means a state in which the target molecule is immobilized via a molecule directly immobilized on the nanoparticle, and the target molecule and the nanoparticle are not in direct contact with each other. Also, “immobilization” is intended to prevent molecules from moving to other locations. Note that the number of molecules that mediate between the nanoparticle and the target molecule is not limited to one, and a plurality of molecules may be interposed.

  When a target molecule is indirectly immobilized on a nanoparticle, a biotin-modified nanoparticle is used, and avidin is bound and immobilized thereon, and the biotinylated target molecule is bound to the immobilized avidin. Thus, a method of immobilizing them can be suitably used. It is well known that biotin and avidin are biomolecules that bind specifically and strongly, and these bonds can form a state equivalent to immobilization. In the present specification, “avidin” includes streptavidin.

  The inventors of the present invention have developed a molecular imprint polymer formed by using a target molecule immobilized on a target molecule-immobilized substrate as a template, in a polymerization solution (a solution in which a functional monomer, a crosslinking agent, etc. are dissolved in a solvent). It has been found that the selectivity to the target molecule is improved from the molecularly imprinted polymer formed by mixing (see Examples). Therefore, the production method according to the present invention has not only an effect that patterning can be easily performed, but also an effect that a sensing chip including a molecularly imprinted polymer with high selectivity for a target molecule can be provided on the surface. is there.

  The target molecule of the sensing chip produced by the production method according to the present invention is not particularly limited, and a wide range of molecules from low molecular compounds such as drugs to high molecular compounds such as proteins can be applied as target molecules. Preferable target molecules include biomolecules. The biomolecule may be a molecule that exists in an organism. By targeting a biomolecule, it is possible to provide a sensing chip that can be used for disease diagnosis, clinical examination, basic biology research, and the like. Moreover, it is more preferable that protein is a target molecule among biomolecules. This is because protein sensing for protein function evaluation has attracted attention as an essential technique for post-genomic research in recent years. Regarding molecularly imprinted polymers that target proteins, there are few reports of successful examples because the protein itself is sensitive to temperature and pH, which makes it difficult to select an optimal monomer. Furthermore, a sensing chip in which molecularly imprinted polymers that recognize proteins are arranged as an array has not yet been reported. Therefore, the production method according to the present invention capable of realizing a molecularly imprinted polymer array capable of recognizing a plurality of proteins is expected to be a very promising technique in the field of protein analysis.

  Hereinafter, as an embodiment of the present invention, a method for producing a sensing chip using a target molecule immobilized on the surface of a target molecule-immobilized substrate using a biotin-avidin bond will be described in detail with reference to FIG. 1 and FIG. explain. FIG. 1 is a schematic diagram showing a nanoparticle placement step, an avidin binding step, and a target molecule immobilization step in the production method according to the present invention, and FIG. 2 shows a molecular imprint polymer formation step in the production method according to the present invention. It is a schematic diagram which shows a part. Note that the present invention is not limited to this.

[Nanoparticle placement process]
First, biotinylated nanoparticles are placed on the surface of the target molecule-immobilized substrate (see FIG. 1). In this step, it is necessary to prepare biotinylated nanoparticles in advance. The biotinylated nanoparticles, for example, use nanoparticles having a carboxyl group on the surface and biotin into which an amino group is introduced (for example, bitoin- (PEG) ₃ -NH ₂ or the like) as shown in the examples in the subsequent stage. Thus, it can be obtained by condensing the carboxyl group and the amino group. Moreover, it can also acquire by condensing the said amino group and a carboxyl group using the nanoparticle which has an amino group on the surface, and the carboxyl group of the biotin which introduce | transduced the carboxyl group.

  The target molecule-immobilized substrate is preferably washed on the surface in order to form biotinylated nanoparticles on the surface. For example, when a glass substrate, a metal oxide substrate, an optical waveguide substrate, a gold substrate, or the like is used, it may be cleaned with a piranha solution (concentrated sulfuric acid: hydrogen peroxide solution = 3: 1) or UV / ozone. This eliminates the oxide film on the substrate surface and improves the efficiency of surface modification.

  Examples of a method for arranging biotinylated nanoparticles on the surface of a target molecule-immobilized substrate include, for example, a method in which a target molecule-immobilized substrate is immersed in a suspension of biotinylated nanoparticles and dried as it is to evaporate water. A method in which a suspension of nanoparticles is dropped or coated on the surface of a target molecule-immobilized substrate and then dried to evaporate moisture can be suitably used. When biotinylated nanoparticles are arranged in an arbitrary pattern on the target molecule-immobilized substrate surface, it is preferable to use a method of dropping or coating. Also, commercially available spotters, arrayers, and printers can be suitably used. If a spotter, an arrayer or a printer is used, biotinylated nanoparticles can be easily arranged in an arbitrary pattern in a plurality of regions on the surface of the target molecule-immobilized substrate.

[Avidin binding step]
In this step, avidin is bound to the biotinylated nanoparticles placed on the surface of the target molecule-immobilized substrate in the preceding nanoparticle placement step (see FIG. 1). The method for binding avidin to biotinylated nanoparticles is not particularly limited as long as the biotinylated nanoparticles and avidin can be brought into contact with each other. For example, a method of immersing a target molecule-immobilized substrate on which biotinylated nanoparticles are arranged in an avidin solution, a method of dropping or applying an avidin solution on a region on the target molecule-immobilized substrate surface where biotinylated nanoparticles are arranged, etc. Can be mentioned. After the biotinylated nanoparticles and avidin are brought into contact with each other for a certain time, the avidin-bound target molecule-immobilized substrate can be obtained by removing unbound avidin by washing or the like. As mentioned above, since the biotin-avidin bond is very strong, immobilization of avidin is achieved by binding avidin to biotinylated nanoparticles.

[Target molecule immobilization process]
In this step, the target molecule is immobilized on the surface of the target molecule-immobilized substrate by binding the biotinylated target molecule to the avidin bound to the biotinylated nanoparticle in the previous avidin binding step (see FIG. 1). In this step, it is necessary to prepare a biotinylated target molecule in advance. The biotinylated target molecule can be obtained, for example, by reacting a biotinylated reagent activated with NHS (N-hydroxysuccinimide) with a primary amine of the target molecule. As the biotinylation reagent (for example, vitamin- (PEG) ₃ -NH ₂ etc.), a commercially available one can be suitably used. In addition, a biotinylated target molecule can be obtained using the carboxyl group of biotin itself. In this case, the carboxyl group of biotin itself may be reacted with the target molecule, and an amino group, thiol group, maleimide group, active ester group, photo-bonded directly or via an appropriate spacer to this carboxyl group. It may be a reactive group. When biotin is reacted with the target molecule, an appropriate coupling reagent such as a rubodiimide compound can be used as necessary.

  The method for binding the biotinylated target molecule to avidin is not particularly limited as long as it is a method capable of contacting avidin and the biotinylated target molecule. For example, a method in which a target molecule-immobilized substrate on which avidin is immobilized is immersed in a biotinylated target molecule solution, a method in which a biotinylated target molecule solution is dropped on a region where avidin is immobilized on the surface of a target molecule-immobilized substrate, etc. Is mentioned. After the avidin and the biotinylated target molecule are brought into contact with each other for a certain period of time, the target molecule-immobilized substrate to which the target molecule is bound can be obtained by removing the biotinylated target molecule that is not bound by washing or the like. As described above, since the biotin-avidin bond is very strong, immobilization of the target molecule can be achieved by binding a biotinylated target molecule to avidin. A mixture of two or more types of target molecules can be contacted with avidin as a target molecule solution, but usually a single type of target molecule is immobilized in one region where avidin is immobilized. The

  In the nanoparticle arranging step, biotinylated nanoparticles are arranged in a plurality of regions on the target molecule-immobilized substrate surface, and when at least two kinds of target molecules are immobilized on the target molecule-immobilized substrate surface, A biotinylated target molecule different from the first biotinylated target molecule may be bound to avidin and immobilized in a region different from the region where the biotinylated target molecule is bound to avidin and immobilized. When the number of regions where biotinylated nanoparticles are arranged is larger than the number of types of target molecules, the same type of target molecule may be immobilized in a plurality of regions. Preferably, different types of target molecules are immobilized in each of the plurality of regions where the biotinylated nanoparticles are arranged. Thereby, the number of target molecules to be immobilized can be increased to the same number as the number of regions where biotinylated nanoparticles are arranged. When using a plurality of target molecules, it is necessary to biotinylate all target molecules to be used in advance.

[Molecular imprint polymer formation process]
In this step, a molecular imprint polymer is formed on the chip substrate surface using the target molecule immobilized in the target molecule immobilization step as a template.

(a) Dropping of the polymerization solution First, a polymerization solution is prepared, and this is dropped onto the target molecule-immobilized substrate surface where the target molecule is immobilized, and the target molecule is brought into contact with the polymerization solution (see FIG. 2). . The polymerization solution is prepared by dissolving a functional monomer and a crosslinking agent in a polymerization solvent. Examples of the solvent used for the polymerization solution include organic solvents such as chloroform, toluene, acetone, acetonitrile, tetrahydrofuran, and various alcohols, and aqueous solvents such as buffer solutions. It is preferable to select appropriately according to the target molecule used as a template. For example, when the target molecule is a protein, an aqueous solvent is preferable because it may be denatured when it is exposed to an organic solvent.

  The functional monomer only needs to have both a functional group capable of binding to the target molecule and a polymerizable functional group. For example, acrylic acid, methacrylic acid, acrylamide, 2- (dimethylamino) ethyl methacrylate, hydroxyethyl methacrylate and the like can be mentioned. The functional monomer is also preferably selected as appropriate according to the target molecule used as a template.

  As the crosslinking agent, it is preferable to use a molecule having at least two polymerizable functional groups (such as vinyl groups) in the molecule. For example, ethylene glycol dimethyl acrylate, N, N′-methylenebisacrylamide, divinylbenzene and the like can be mentioned.

  A polymerization initiator may be added to the polymerization. As the polymerization initiator, for example, a peroxide such as ammonium persulfate or potassium persulfate, or an azo polymerization initiator such as azobisisobutyronitrile can be used. Further, a polymerization accelerator such as N, N, N ′, N′-tetramethylethylenediamine may be added.

  When multiple types of target molecules are immobilized on the target molecule-immobilized substrate surface, if a polymerization solution with the same composition can be used for all target molecules, the entire structure where multiple types of target molecules are immobilized The polymerization solution can be dropped into the region. On the other hand, when it is necessary to change the composition of the polymerization solution according to the type of the target molecule, a polymerization solution having a different composition is dropped for each target molecule region. In such a case, if the polymerization solution of the same composition is grouped for each target molecule that can be used and is patterned so that the target molecules of the same group are arranged in close proximity, the dropping operation of the polymerization solution can be performed efficiently. be able to. If a target molecule-immobilized substrate having a plurality of separated compartments on the surface is used, a polymerization solution having a different composition for each compartment can be easily dropped.

(b) Place the chip substrate Next, the chip substrate is placed on the droplets of the polymerization solution, and the polymerization solution is sandwiched between the target molecule-immobilized substrate and the chip substrate (see FIG. 2). At this time, it is preferable to introduce a polymerizable functional group (such as a vinyl group) into the surface of the chip substrate. Thereby, the functional group on the surface of the chip substrate and the crosslinking agent in the polymerization solution are polymerized, and a film of molecular imprint polymer can be formed on the surface of the chip substrate.

  The vinyl group can be introduced into the chip substrate surface by a known method according to the material of the chip substrate. For example, when the chip substrate is a gold substrate, as shown in FIG. 3, using the gold-thiol bond, the gold substrate is immersed in a methanol solution of N, N′-Bis (acryloyl) cystamine, and then washed. Thus, a vinyl group-modified gold substrate can be obtained. When the chip substrate is a glass substrate, a vinyl group-modified glass substrate can be obtained using a silane coupling agent such as 3-methacryloyloxypropyltrimethoxysilane (see Example 3).

(c) Polymerization Next, the polymerization solution sandwiched between the gaps between the target molecule-immobilized substrate and the chip substrate is polymerized. The polymerization method is not particularly limited, but photopolymerization or thermal polymerization is preferable. When photopolymerization is performed, ultraviolet light (UV) may be radiated from 0 to 4 ° C. for about 10 minutes to 4 hours using a UV light or the like (see FIG. 2). In the case of thermal polymerization, for example, polymerization may be performed at 40 ° C. for about 10 minutes to 3 hours.

(d) Separation of target molecule-immobilized substrate and chip substrate Next, the target molecule-immobilized substrate and the chip substrate are separated. The two substrates may be physically peeled off so that the molecular imprint polymer formed by the previous polymerization is not peeled off from the chip substrate surface.

(e) Removal of target molecule Finally, a template molecule is removed from the formed molecular imprint polymer, thereby completing a sensing chip for the target molecule. As a method for removing the template molecule from the molecularly imprinted polymer, for example, when the template molecule is bound to the molecularly imprinted polymer by electrostatic interaction (for example, when the template molecule is a protein), it is added to a 1M NaCl solution. It can be removed by dipping. Further, when the template molecule is bonded to the molecular imprint polymer by a hydrogen bond, it can be removed by washing with a polar solvent such as methanol.

[Use of sensing chip manufactured by the manufacturing method according to the present invention]
In order to detect a target molecule, a sensing chip manufactured by the manufacturing method according to the present invention uses a surface plasmon resonance (SPR) method, a localized plasmon resonance (LSPR) method, a crystal resonator sensor, an electrochemical method, light emission This method can be applied to a method, a coloring method, an optical waveguide spectroscopy, and the like.

  Here, surface plasmon resonance (SPR) is a phenomenon in which, when light of a specific wavelength is applied from a specific angle, the energy of photons is absorbed by electrons on the surface of the molecularly imprinted polymer and is not reflected. It is used to measure the interaction of biopolymers. For example, it can be measured using an SPR measuring device manufactured by BIACORE.

  Localized plasmon resonance (LSPR) is a resonance phenomenon that an electric field generated to cancel polarization caused by vibration of free electrons in a molecularly imprinted polymer caused by incident light (electromagnetic wave) on a sample surface is caused by incident light. Since incident light is remarkably absorbed, even a small amount of material in a minute region can be detected.

  A quartz oscillator sensor measures the binding of molecules by utilizing the fact that the frequency changes when a substance binds to the surface of a quartz oscillator.

  The electrochemical method measures changes in current, voltage, impedance, conductance, and the like when a target molecule is bound to the molecularly imprinted polymer.

  Luminescence and color development measure light (fluorescence) generated when a target molecule is bound to a molecularly imprinted polymer and changes in color tone.

  In optical waveguide spectroscopy, laser light is incident on an optical waveguide having a structure in which a material layer (core layer) having a high refractive index is sandwiched between layers (cladding layer) having a small refractive index, and is repeatedly reflected. This is a method for measuring the absorption spectrum information of the evanescent wave generated at the optical waveguide interface with high sensitivity. Since the evanescent wave is generated only at the interface, a substance existing near the waveguide interface can be selectively measured.

  As described above, the production method according to the present invention can realize a molecularly imprinted polymer array capable of recognizing a plurality of target molecules, and is expected to be a very promising technique particularly in the field of biomolecular analysis. Is done.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.

[Example 1: Production and evaluation of sensing chip provided with imprint polymer using immobilized avidin as template]
(1-1) Synthesis of nanoparticles In order to introduce biotin onto the surface, nanoparticles having a carboxyl group were synthesized by the following procedure.

  To 27 g of water, 230 mg of methacrylic acid (1 eq, manufactured by Wako Pure Chemical Industries), 2770 mg of styrene (10 eq, manufactured by Wako Pure Chemical Industries), and 240 mg of potassium persulfate (made by Wako Pure Chemical Industries) as a polymerization initiator were added, and nitrogen was added. The mixture was stirred at 80 ° C. for 24 hours under an atmosphere (200 rpm). Then, centrifugation was performed 3 times and the emulsion was wash | cleaned. The polymerization rate of the obtained emulsion was 99.5%. As a result of observing the obtained nanoparticles (hereinafter referred to as “NP”) using a scanning electron microscope (Keyence VE9800, hereinafter referred to as “SEM”), it was a uniform spherical particle having a particle size of about 250 nm. It was confirmed.

(1-2) Introduction of biotin into nanoparticles First, the carboxyl group on the nanoparticle surface was activated. NP 10 mg, 1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide 14 mg (72 μmol, manufactured by Watanabe Chemical Industries), N-hydroxysuccinimide 8.3 mg (72 μmol, manufactured by Aldrich) were suspended in water, The reaction was allowed to stir at room temperature for 5 hours. After completion of stirring, the mixture was centrifuged three times and purified. Next, the obtained NP having activated carboxylic acid was suspended in 10 mM PBS (pH 8.5), and 10 mg of vitamin- (PEG) ₃ —NH ₂ (24 μmol, manufactured by Funakoshi) was added to perform a condensation reaction. After stirring for 6 hours, the mixture was centrifuged 3 times and purified.

  An avidin adsorption experiment was performed on the obtained biotinylated NP, and the rate of biotin introduction into the NP was calculated. Specifically, 3 mg each of biotinylated NP and unmodified NP was added to each concentration of avidin solution and incubated at 25 ° C. for 3 hours. Thereafter, the NP was removed by centrifugation, the supernatant was diluted 4-fold, and the UV / vis spectrum was measured to calculate the amount of avidin adsorbed on the NP. A UV / vis spectrum measurement device V-560 manufactured by Jasco was used for the measurement of the UV / vis spectrum.

  The results are shown in FIG. Since biotinylated NP adsorbed more avidin than unmodified NP, it was confirmed that biotin was introduced into biotinylated NP. In addition, since the amount of avidin adsorbed by biotinylated NP increased linearly, the amount of unmodified NP adsorbed was saturated, indicating that biotin was introduced into biotinylated NP. It could be confirmed.

(1-3) Immobilization of Avidin on Biotinylated Nanoparticles FIG. 5 shows a schematic diagram of the procedure from the placement of biotinylated NP on the target molecule-immobilized substrate to the completion of the sensing chip. First, in order to form biotinylated NP on the cover glass, the cover glass was washed with a Piranha solution (concentrated sulfuric acid: hydrogen peroxide solution = 3: 1) and dried. On top of that, 20 μL of biotinylated NP was dropped and dried slowly overnight at room temperature. Next, the obtained cover glass was immersed in a 10 nM avidin solution for 1 hour, and then washed with water to immobilize the avidin on the cover glass (see FIG. 5).

(1-4) Production of sensing chip provided with imprint polymer The cover glass on which avidin obtained in 1-3 above was immobilized was placed on a slide glass, and a polymerization solution (acrylic acid 1.75 μL (manufactured by Wako Pure Chemical Industries, Ltd.) ), N, N′-methylenebisacrylamide 38.5 mg (manufactured by Sigma), 3-methacryloyloxypropyltrimethoxysilane 21 mg (MPC, manufactured by Shin-Etsu Chemical Co., Ltd.), 2,2′-azobis [2-methyl-N- [ 1,1-bis (hydroxymethyl) -2-hydroxyethyl] propionamide] 1 mg (VA-080, manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 3 mL of PBS (see Table 1)) was dropped 5 μL (see FIG. 5). ).

  An SPR sensor chip (gold substrate) modified with a vinyl group is placed on the droplet of this solution, and UV-LED light (manufactured by OMRON, control unit: ZUV-C10, head unit: ZUV-H10) at 4 ° C. for 10 minutes. And photopolymerization was carried out. After completion of the polymerization, the gold substrate and the cover glass were peeled off, and the obtained polymer-coated gold substrate was washed successively with water and methanol. Thereafter, the avidin as a template was removed by immersion in a 1M NaCl aqueous solution for 24 hours to obtain a sensing chip having the target imprint polymer “IP” (see FIG. 5).

  A gold-thiol bond was used to introduce a vinyl group into the gold substrate. Specifically, a gold substrate is immersed in a solution in which 3.6 mg of N, N′-Bis (acryloyl) cystamine is dissolved in 5 mL of methanol, and then the substrate is sequentially washed with water and methanol to obtain a vinyl group-modified gold substrate. (See FIG. 3).

  As a control, a sensing chip including a reference polymer “RP”, a blank polymer “BP”, and a bulk imprint polymer “bulkIP” was similarly prepared. “RP” is an imprint polymer using NP without biotin modification as a template, “BP” is a polymer without template, and “bulkIP” is not immobilized avidin but dispersed in the polymerization solution. It is an imprint polymer polymerized using avidin as a template.

(1-5) Adsorption experiment by surface plasmon resonance (SPR) measurement BiacoreQ manufactured by Biacore was used as the surface plasmon resonance measurement apparatus. The measurement was performed at 25 ° C. As the running buffer, 10 mM PBS pH 7.4 (80 mM NaCl) was used at a flow rate of 20 μL / min. As the regeneration solution, a 1M NaCl aqueous solution and 100 mM glycine buffer pH 10 (1M NaCl) were used, and 20 μL each was added in this order. The amount of the analyte protein solution added was 40 μL. The SPR signal 60 seconds after the completion of addition was defined as the amount of binding.

  First, the affinity of avidin for each polymer of IP, RP, and BP was evaluated. The results are shown in FIG. As is clear from FIG. 6, it was shown that IP using immobilized avidin as a template has a significantly larger amount of avidin binding than the other two types of polymers.

  Next, the selectivity of each polymer of IP, RP, BP, and bulkIP was evaluated using three types of protein solutions (4 μM) of avidin, lysozyme, and ribonuclease A. The results are shown in FIG. As is apparent from FIG. 7, the affinity for avidin was significantly higher in IP among the four types of polymers, indicating that IP has a high avidin selectivity. In bulk IP, the affinity between avidin and lysozyme is almost the same, and the imprint effect using avidin as a template is not observed. This demonstrates the effectiveness of using immobilized avidin as a template.

[Example 2: Production and evaluation of sensing chip including imprint polymer using immobilized glucose oxidase as template]
(2-1) Immobilization of glucose oxidase By the same method as in Example 1, avidin was immobilized on a cover glass on which biotinylated NP was formed. Next, glucose oxidase (hereinafter referred to as “GOx”) was immobilized on the substrate. Specifically, a 10 nM solution of biotinylated glucose oxidase (manufactured by Funakoshi) was prepared, and a cover glass on which avidin was immobilized was immersed in the solution for 1 hour. Thereafter, the cover glass was washed with water to obtain a cover glass on which glucose oxidase was immobilized (see FIG. 1).

(2-2) Production of sensing chip provided with imprint polymer In the same manner as in Example 1, the cover glass on which GOx obtained in 2-1 above was immobilized was placed on a slide glass, and 5 μL of a polymerization solution was dropped and polymerized. An SPR sensor chip (gold substrate) modified with a vinyl group is placed on the solution droplet, and a UV-LED light (manufactured by OMRON, control unit: ZUV-C10, head unit: ZUV-H10) at 4 ° C. for 10 minutes. Photopolymerization was performed by irradiation (see FIG. 2). Since GOx is an acidic protein, it is negatively charged under the buffer conditions (pH 7.4) during polymer polymerization. Therefore, the functional monomer is cationic 2- (N, N-dimethylamino) ethyl acrylate. Rat (DMAEA, manufactured by Wako Pure Chemical Industries, Ltd.) was used. That is, 1.75 μL of acrylic acid in the composition of the polymerization solution of Example 1 (see Table 1) was changed to 2 μL of DMAEA in this example.

  After completion of the polymerization, the gold substrate and the cover glass were peeled off, and the obtained polymer-coated gold substrate was washed successively with water and methanol. Then, the sensing chip provided with the target imprint polymer “GOx-IP” was obtained by immersing in 1M NaCl aqueous solution for 24 hours to remove the GOx of the template. In addition, the sensing chip provided with blank polymer "BP" (polymer without a template) as a control was produced similarly.

(2-3) Adsorption experiment by SPR measurement BiacoreQ manufactured by Biacore was used for the surface plasmon resonance measurement apparatus. The measurement was performed at 25 ° C. As the running buffer, 10 mM PBS pH 7.4 (80 mM NaCl) was used at a flow rate of 20 μL / min. As the regeneration solution, 1 M NaCl aqueous solution and 100 mM Glycine buffer pH 10 (1 M NaCl) were used, and 10 μL each was added in this order. The amount of the analyte protein solution added was 40 μL. A value obtained by performing an equilibrium value analysis near the end of the coupled portion was defined as the coupled amount.

  First, the affinity of GOx to GOx-IP and BP was evaluated. The results are shown in FIG. From FIG. 8, it was confirmed that the amount of GOx adsorbed on GOx-IP tends to be larger than the amount of GOx adsorbed on BP.

  Next, the selectivity of GOx-IP and BP was evaluated using three protein solutions (0.5 μM) of GOx, BSA (bovine serum albumin) and myoglobin. The results are shown in FIG. As can be seen from FIG. 9, BP did not show any selectivity for the three proteins, but GOx-IP showed high selectivity for GOx. In addition, although data is not shown, in the low concentration region, it corresponds to GOx-IP and GOx-bulkIP (imprinted polymer obtained by polymerizing GOx dispersed in the polymerization solution as a template, not immobilized GOx). A clear difference in selectivity was confirmed, which showed the effectiveness of using immobilized GOx as a template.

[Example 3: Examination of patterning]
(3-1) Immobilization of biotinylated NP spots and streptavidin Place a metal with holes in only three places on the cover glass and wash with UV ozone cleaner (Meiwaforsys TC003) for 30 minutes. Only was hydrophilized. 5 μL of biotinylated NP was dropped at three locations subjected to hydrophilic treatment and dried to obtain a cover glass spotted with biotinylated NP at three locations. This cover glass was immersed in a FITC-modified streptavidin (manufactured by Wako Pure Chemical Industries) solution and then washed with water to immobilize the FITC-modified streptavidin.

(3-2) Production of sensing chip provided with patterned imprint polymer As a glass substrate, a slide glass into which a vinyl group was introduced was produced. That is, 50 μL of acetic acid was added to 5 mL of water and stirred. While stirring, 50 μL of a silane coupling agent (3-methacryloyloxypropyltrimethoxysilane, manufactured by Shin-Etsu Chemical Co., Ltd.) was slowly dropped and stirred for 30 minutes. 10 μL of the obtained solution was dropped on a slide glass, and a cover glass was placed from above and allowed to stand overnight. The cover glass was peeled off and washed sequentially with water and methanol to obtain a vinyl group-modified slide glass.

  The cover glass on which the FITC-modified streptavidin obtained in 3-1 was immobilized was placed on a slide glass, and a polymerization solution (acrylic acid 1.75 μL (manufactured by Wako Pure Chemical Industries), N, N′-methylenebisacrylamide 38 0.5 mg (manufactured by Sigma), 3-methacryloyloxypropyltrimethoxysilane 21 mg (MPC, manufactured by Shin-Etsu Chemical Co., Ltd.), 2,2′-azobis [2-methyl-N- [1,1-bis (hydroxymethyl) -2 -Hydroxyethyl] propionamide] 1 mg (VA-080, manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 3 mL of PBS was added dropwise. A vinyl group-modified slide glass was placed thereon, and photopolymerization was performed by irradiating with UV-LED light (manufactured by OMRON, control unit: ZUV-C10, head unit: ZUV-H10) at 4 ° C. for 10 minutes. After completion of the polymerization, the glass substrate (slide glass) and the cover glass were peeled off, and the resulting polymer-coated glass substrate was washed successively with water and methanol. Then, the sensing chip provided with the target imprint polymer was obtained by immersing in 1M NaCl aqueous solution for 24 hours to remove the FITC-modified streptavidin of the template.

(3-3) Adsorption of target molecule The sensing chip obtained in 3-2 above was immersed in a 1 μM solution of FITC-modified streptavidin for 30 minutes and washed with water.

(3-4) Evaluation of patterning At each time point below, patterning was evaluated by observing fluorescence emitted when the cover glass or the sensing chip was irradiated with visible light.
(A) Cover glass on which FITC-modified streptavidin is immobilized (see (3-1))
(B) Sensing chip after polymer polymerization and cleaning (see (3-2))
(C) Sensing chip after target molecule adsorption (see (3-3))

  The results are shown in FIG. In (a), three places of fluorescence were observed on the cover glass, and it was confirmed that the biotinylated NP was immobilized only at the spot spot. In (b), no fluorescence was observed on the sensing chip, and it was confirmed that FITC-modified streptavidin as a template was removed from the imprint polymer. In (c), fluorescence was observed at three locations on the sensing chip, and it was confirmed that three binding sites selective for FITC-labeled streptavidin were constructed in the obtained sensing chip.

  From the above results, it was shown that imprinted polymer patterning can be easily performed by using a method of immobilizing an arbitrary protein on biotinylated NP.

  The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  The present invention can be widely used in manufacturing industries such as medical equipment and analytical equipment.

It is a schematic diagram which shows the nanoparticle arrangement | positioning process, avidin coupling | bonding process, and target molecule immobilization process of the preparation methods which concern on this invention. It is a schematic diagram which shows a part of molecular imprint polymer formation process of the preparation methods which concern on this invention. It is a schematic diagram which shows the preparation methods of the gold substrate modified with the vinyl group. It is a graph which shows the result of the adsorption experiment of avidin to a biotinylated nanoparticle. It is a schematic diagram which shows the procedure from arrangement | positioning of the biotinylated nanoparticle to the target molecule fixed substrate surface to completion of a sensing chip. It is a graph which shows the adsorption isotherm with respect to avidin of each polymer (IP, RP, and BP). It is a graph which shows the result of the selectivity evaluation with respect to three types of protein of each polymer (IP, RP, BP, and bulkIP). It is a graph which shows the adsorption isotherm with respect to glucose oxidase (GOx) of each polymer (GOx-IP and BP). It is a graph which shows the result of selectivity evaluation with respect to three types of proteins of each polymer (GOx-IP and BP). (A) is a figure which shows the patterning in the cover glass which fix | immobilized FITC modification streptavidin, (b) is a figure which shows that the template molecule | numerator is removed in the sensing chip after polymer polymerization and washing | cleaning, c) is a diagram showing that patterning of template molecules is reflected in the fabricated sensing chip.

Claims (8)

This is a method for producing a sensing chip for a target molecule that has a molecularly imprinted polymer as a target molecule recognition body on the chip substrate surface, and has both the function of selectively binding to the target molecule and the function of converting the binding information into a signal. And
Place nanoparticles on the target molecule-immobilized substrate surface, immobilize the target molecules on the arranged nanoparticles, and target the polymerization solution containing functional monomer and cross-linking agent between the target molecule-immobilized substrate and the chip substrate The molecular imprint formed by separating the target molecule-immobilized substrate and the chip substrate so that the formed molecular imprint polymer is not peeled off the chip substrate surface by sandwiching it in contact with the molecules and polymerizing the polymerization solution. A method for producing a sensing chip for a target molecule, comprising removing the target molecule from a polymer . This is a method for producing a sensing chip for a target molecule that has a molecularly imprinted polymer as a target molecule recognition body on the chip substrate surface, and has both the function of selectively binding to the target molecule and the function of converting the binding information into a signal. And
A nanoparticle placement step of placing biotinylated nanoparticles on the surface of the target molecule-immobilized substrate;
An avidin binding step of binding avidin to the biotinylated nanoparticles placed in the nanoparticle placement step;
A target molecule immobilization step of immobilizing a biotinylated target molecule by immobilizing the biotinylated target molecule to avidin bound to the biotinylated nanoparticle in the avidin binding step;
Using the target molecule immobilized in the target molecule immobilization step as a template, and forming a molecular imprint polymer on the chip substrate surface ,
In the molecular imprint polymer forming step, a polymerization solution containing a functional monomer and a cross-linking agent is sandwiched between the target molecule-immobilized substrate and the chip substrate in contact with the target molecule, and the polymerization solution is polymerized to form. The target molecule sensing chip is characterized in that the target molecule-immobilized substrate and the chip substrate are separated so that the molecular imprint polymer does not peel off from the chip substrate surface, and the target molecule is removed from the formed molecular imprint polymer. Manufacturing method. 3. The method for producing a target molecule sensing chip according to claim 2, wherein in the nanoparticle arranging step, the biotinylated nanoparticles are arranged by patterning on the surface of the target molecule-immobilized substrate.   The method for producing a target molecule sensing chip according to claim 3, wherein in the nanoparticle arrangement step, biotinylated nanoparticles are arranged in a plurality of regions on the surface of the target molecule-immobilized substrate.   A single type of target molecule is immobilized on each of a plurality of regions where biotinylated nanoparticles are arranged, and at least two types of target molecules are immobilized on the surface of the target molecule-immobilized substrate. A method for producing a sensing chip for a target molecule according to claim 4.   6. The method for producing a sensing chip for target molecules according to claim 5, wherein different types of target molecules are immobilized in each of the plurality of regions where the biotinylated nanoparticles are arranged.   The method for producing a target molecule sensing chip according to any one of claims 1 to 6, wherein the target molecule is a biomolecule.   The method for producing a target molecule sensing chip according to claim 7, wherein the biomolecule is a protein.

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