JP6034261B2 - Manufacturing method of biomolecule detection chip - Google Patents

Description

The present invention relates to a method for producing a biomolecule detecting chips for detecting biomolecules.

  As a technology to detect multiple chemical components from a very small amount of sample at the same time and to compare similar chemical reactions with high accuracy, biological technologies such as nucleic acid molecules (aptamers) with molecular recognition function, proteins such as antibodies and enzymes, etc. An analysis method using an array chip in which molecules for detecting molecules are arranged in a minute region has attracted attention.

  In such an analysis method using an array chip, analysis is performed based on information indicating which biomolecule detection molecules are arranged in which micro regions. For this reason, a technique for immobilizing molecules for biomolecule detection with a fine pattern with good position controllability is required. In addition, accurate position information must be acquired when performing analysis. As described above, in order to fix the molecule for biomolecule detection in a fine region with good position controllability, there is a problem that a large-scale and complicated apparatus is required and a skilled operation is required.

  Dye that emits light in different colors as an on-chip analysis method that detects multiple chemical components from a very small amount of sample at the same time and accurately compares and measures similar chemical reactions without requiring such complicated processes A photodetection method that independently uses as a probe is conceivable.

  One example is a method for labeling a target substance to be measured. By labeling with different dye molecules for each type of target substance, highly sensitive detection with good selectivity is possible. However, labeling of the target substance has a problem that a complicated pretreatment process is required.

  Another method is more preferred label-free detection. In this method, molecules for detecting biomolecules such as nucleic acid molecules having molecular recognition functions and proteins such as antibodies and enzymes are labeled in advance with dyes that emit light in different colors, and detection is performed by color. . By detecting by color, it is possible to simultaneously detect a plurality of chemical components without fixing the biomolecule detection molecule pattern at different desired locations on the substrate surface.

“OPTICAL INTERFERENCE FILTER”, OMEGA OPTICAL Japanese Catalog 14th Edition, pp. 26-27. E. Treossi et al., "High-Contrast Visualization of Graphene Oxide on Dye-Sensitized Glass, Quartz, and Silicon by Fluorescence Quenching", J. AM. CHEM. SOC., Vol.131, pp.15576-15577, 2009 . J. Kim et al., "Visualizing Graphene Based Sheets by Fluorescence Quenching Microscopy", J. AM. CHEM. SOC., Vol.132, pp.260-267, 2010. S. Husale et al., "SsDNA Binding Reveals the Atomic Structure of Graphene", Langmuir, vol.26, no.23, pp.18078-18082, 2010. D. Li et al., "Processable aqueous dispersions of graphene nanosheets", Nature nanotechnology, vol.3, pp.101-105, 2008.

  Here, the biomolecule detection molecule labeled with a dye emitting light of a different color needs to be in a state in which no luminescence is observed, i.e., quenching, in the absence of a target substance, and exhibits quenching characteristics. It must be used in combination with molecules and materials. Many molecules and materials that exhibit conventional quenching properties often exhibit good quenching properties only for specific dyes. For this reason, a quenching material paired with a dye used for labeling is required for each type of dye, and there is a problem that it is difficult to increase the number of colors on the same platform (Non-Patent Document 1).

  In addition, since many molecules and materials themselves exhibiting extinction characteristics also exhibit light emission, there is a problem that they are obstructed when making multiple colors. Furthermore, in order to show good quenching characteristics, it is necessary to place molecules that exhibit quenching characteristics in the vicinity of biomolecule detection molecules labeled with dyes. There is a problem of being big. As described above, there is a problem in that it is not ready to analyze a plurality of different detection targets using a plurality of different biomolecule detection molecules with a single chip.

  The present invention has been made to solve the above-described problems, and it is possible to easily perform analysis of a plurality of different detection targets using a plurality of different biomolecule detection molecules with one chip. The purpose is to.

Each BIOLOGICAL molecular detection chip includes a plurality of biomolecule detecting molecules that each specifically bind a respective different biomolecule be detected, which is coupled to one end of each of a plurality of biomolecule detecting molecules comprising different and a plurality of dye color, consist formed oxidation graphene reductant on a substrate, and a fixed layer to which the other end of the plurality of biomolecule detecting molecules is fixed.

  The biomolecule detection chip may include an adhesion molecule that is bonded to the other end of the biomolecule detection molecule and adheres the other end of the biomolecule detection molecule and the fixing layer. The substrate is preferably transparent.

Method for producing a biomolecule detecting chip according to the present invention includes a fixed layer forming step of forming a fixed layer composed of oxidation graphene reductant on a substrate, each of the respective plurality of different biomolecules be detected A molecule formation process for detecting a dye-coupled biomolecule that binds a dye of a different color to one end of each of a plurality of biomolecule-detecting molecules that specifically bind to the molecule, and a biomolecule detection in which a dye of a different color is bound Dye-forming biomolecule detection that fixes the other end of a plurality of molecules for detecting biomolecules , each of which is bound to a dye of a different color, by bringing a mixed solution in which multiple aqueous solutions containing molecules are mixed into contact with the fixed layer A molecular immobilization step.

  In the method for manufacturing a biomolecule detection chip, the biomolecule detection chip includes an adhesion molecule bonding step of bonding an adhesion molecule that adheres to the fixing layer to the other end of the plurality of biomolecule detection molecules. The other end of the biomolecule detection molecule and the fixing layer may be adhered and fixed by an adhesion molecule bonded to the other end of the biomolecule detection molecule. The substrate is preferably transparent.

  As described above, according to the present invention, it is possible to obtain an excellent effect that analysis of a plurality of different detection targets using a plurality of different biomolecule detection molecules can be easily performed with one chip. .

FIG. 1 is a configuration diagram showing a partial configuration of a biomolecule detection chip according to an embodiment of the present invention. FIG. 2 is a configuration diagram showing a partial configuration of the biomolecule detection chip according to the embodiment of the present invention. FIG. 3 is a flowchart illustrating a method for manufacturing the biomolecule detection chip in the embodiment of the present invention. FIG. 4 is a configuration diagram showing the configuration of the microchannel.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a configuration diagram showing a partial configuration of a biomolecule detection chip according to an embodiment of the present invention. This biomolecule detection chip is formed on a plurality of biomolecule detection molecules 101a and 101b, a plurality of dyes 102a and 102b, and a substrate 103, on which the biomolecule detection molecules 101a and 101b are fixed. 104. Dyes 102a and 102b are bonded to one end of the biomolecule detection molecules 101a and 101b. The other ends of the biomolecule detection molecules 101 a and 101 b are fixed to the fixed layer 104. Note that the pigment 102a is different from the pigment 102b, and these are known.

  The biomolecule detection molecule 101a and the biomolecule detection molecule 101b specifically bind to each of a plurality of different biomolecules to be detected. For example, as shown in FIG. 2, the biomolecule detection molecule 101a is an aptamer that forms a complex with the biomolecule 201a to be detected, and the biomolecule detection molecule 101b is a biomolecule 201b different from the biomolecule 201a. Is an aptamer that forms a complex. The dye 102a and the dye 102b are fluorescent dye molecules that emit fluorescence having different wavelengths.

The fixed layer 104 is made of graphene oxide or a graphene oxide reductant. Graphene has a structure in which carbon atoms composed of carbon sp ² bonds are two-dimensionally bonded, and it can be said that several atomic layers constituting the layered material graphite are extracted from one layer. A material obtained by oxidizing such graphene is graphene oxide. A substance obtained by reducing graphene oxide to some extent is a graphene oxide reductant. The graphene oxide reductant is not in a complete graphene state.

  Graphene oxide or graphene oxide reductant is a quenching material that exhibits extremely efficient quenching characteristics with respect to luminescent dyes in a wide wavelength band (see Non-Patent Documents 2 and 3). Therefore, according to the biomolecule detection chip in the embodiment using the fixed layer 104 having the above-described configuration, multicolor detection can be performed regardless of the type of the dye. In particular, since the graphene oxide reductant is a non-light emitting material, there is an advantage that detection interference due to its own light emission does not occur.

  On the other hand, graphene oxide exhibits weak light emission in the red region, but if a dye having a very strong light emission intensity is selected as compared with the light emission of graphene oxide itself, detection interference can be almost ignored. For pigments other than red, the graphene oxide itself does not interfere with detection. Note that the weak red light emission of graphene oxide itself may cause interference when a weak detection signal is observed, such as when the concentration of the target substance is low. In such a case, it is preferable to use a graphene oxide reductant that is a non-light emitting material.

  In addition, graphene oxide or graphene oxide reductant has a strong affinity for aptamers among biomolecule detection molecules. Therefore, aptamers spontaneously approach these surfaces in the absence of a target substance. And adsorb (Non-patent Document 4). As a result, in a steady state, as shown in FIG. 1, the distance between the dyes 102a and 102b, which are dye molecules used for labeling, and the surface of the fixed layer 104 made of graphene oxide or graphene oxide reductant is close, and graphene oxide or graphene oxide The reductant exhibits sufficient quenching properties.

  On the other hand, when a target substance for the aptamer exists, the aptamer and the target substance form a complex, and form a three-dimensional structure on the surface of graphene oxide or the graphene oxide reduced form. As shown in FIG. 2, when the biomolecule detection molecules 101a and 101b form a complex with the biomolecules 201a and 201b, the biomolecule detection molecules 101a and 101b whose other ends are fixed to the fixed layer 104 are , It is not detached from the fixed layer 104. However, by forming a complex with the biomolecules 201a and 201b, one end of the biomolecule detection molecules 101a and 101b is separated from the fixed layer 104 together with the dyes 102a and 102b fixed thereto.

  The quenching efficiency of graphene oxide or graphene oxide reductant depends largely on the distance between the dye and the graphene oxide or graphene oxide reductant surface. For this reason, as the distance increases, the quenching efficiency is remarkably lowered, and the light emission of the dyes 102a and 102b is observed. If aptamers for various target substances are labeled with dyes that emit light of different colors, mixed in advance and immobilized on the surface of graphene oxide or graphene oxide reductant, light emission of a specific color in the presence of a certain target substance Is observed. By performing such color-specific detection, it is possible to detect a plurality of molecules without requiring patterning into a minute region.

  The biomolecule detection molecule 101a and the biomolecule detection molecule 101b may be fixed to the fixing layer 104 via an adhesion molecule (linker molecule). As the adhesion molecule, for example, a molecule containing pyrene in the skeleton such as pyrene butadiene acid may be used. The other end (terminal) amino group of the biomolecule detection molecule 101a and the biomolecule detection molecule 101b is introduced, and the carboxyl group or N-hydroxysuccinimide ester site introduced into the adhesion molecule is reacted therewith to form a chemical bond. You can do it. Such an adhesion molecule having an N-hydroxysuccinimide ester moiety is adhered (adsorbed) to the fixed layer 104, and the other ends of the biomolecule detection molecules 101a and 101b to which the dyes 102a and 102b are bonded at one end. What is necessary is just to couple | bond with an adhesion molecule.

  Next, a manufacturing method will be described. The graphene oxide can form a dispersion solution that is dispersed in water in the form of a sheet of graphene-like carbon atoms. For this reason, by applying an aqueous solution in which graphene oxide (this small piece) is dispersed to a solid substrate by a spin coating method or the like, a layer composed of small pieces of graphene oxide of about 1 to 5 layers is formed on the substrate. Uniform formation is possible.

  The graphene oxide reductant can be obtained by chemically reducing graphene oxide. Note that when the reduction reaction is performed in the state of the graphene oxide dispersion solution, the graphene oxide reductant aggregates, and it becomes difficult to maintain the state of the sheet of one carbon atom. For this reason, when forming a fixed layer from a graphene oxide reduced body, it is good to reduce, after forming a layer in the state of graphene oxide. As a reduction method, exposure to reducing vapor (such as hydrazine) may be performed. Further, a reduction method such as high heat treatment in a gas atmosphere such as hydrogen, nitrogen, argon, or vacuum heating may be used.

  According to the reduction method described above, a graphene oxide reductant can be obtained without peeling off while maintaining the shape fixed to the substrate surface as graphene oxide. As a result, it is possible to uniformly form a layer composed of a plurality of small pieces of graphene oxide reduced body having a single layer to 5 layers or less on the substrate. By using a substrate in which graphene oxide or graphene oxide reductant is fixed to form a fixed layer by such a formation method, dye labeling is performed more easily than fixing a molecule exhibiting extinction characteristics at a predetermined position on the substrate. An elevating means can be installed in the vicinity of the biomolecule detection molecule.

  By the way, there is no particular limitation on the region where various aptamers (molecules for detecting biomolecules) labeled with dyes that emit light of different colors are mixed and fixed. The pinned layer made of graphene oxide or graphene oxide reductant can be patterned with aptamer into a microregion using a conventional representative method, so an array of aptamers may be prepared. Alternatively, a fixed layer formed by previously forming a graphene oxide or graphene oxide reduced body into a film shape and patterning it may be used.

  This will be described in more detail below. First, a method for manufacturing a biomolecule detection chip will be described with reference to the flowchart of FIG. First, in step S301, a fixed layer is formed on a substrate. The substrate may be made of silicon or glass, but there are no particular restrictions on the material. The fixed layer can be obtained by first applying an aqueous solution in which graphene oxide is dispersed to a substrate by a spin coating method or the like. The graphene oxide dispersion solution can be prepared by a reported synthesis method (see Non-Patent Document 5). By applying this graphene oxide dispersion solution, a fixed layer made of small pieces of graphene oxide of about 1 to 5 layers can be uniformly formed on the substrate.

  Next, when the fixed layer is composed of a graphene oxide reductant, the fixed layer made of graphene oxide prepared as described above may be exposed to a reducing atmosphere (step S302). For example, the substrate is placed in a hermetically sealed container in which a small amount of an aqueous solution containing about 5% of hydrazine is sealed, and the treatment is performed at 60 to 95 ° C. for 30 to 60 minutes. The graphene oxide constituting the fixed layer formed on the substrate surface undergoes a reduction reaction when exposed to hydrazine vapor, and changes to a graphene oxide reduced form. The graphene oxide reductant generated in this manner maintains a shape fixed to the substrate surface as graphene oxide, and thus does not cause aggregation as seen in a reduction reaction in a solution. Thus, by reducing after forming a fixed layer, the fixed layer which consists of a small piece of a graphene oxide reduced body of a single layer-about 5 layers can be formed uniformly.

  On the other hand, a molecule for biomolecule detection combined with a dye is prepared (step S303). As the biomolecule detection molecule, a nucleic acid (aptamer) that specifically binds to the target substance, an antibody that specifically binds to the target substance, or an enzyme that performs a specific chemical reaction with the target substance can be used. This biomolecule detection molecule may be labeled with a different dye for each type, or may be labeled with the same type of dye even if the types are different. In addition, you may produce the molecule | numerator for biomolecule detection which couple | bonded the pigment | dye previously.

  In addition, a linker molecule having a property of adsorbing well to the fixed layer may be bound to a previously designed site of the dye-binding biomolecule detection molecule as described above. When a linker molecule is used, for example, pyrene butyric acid or the like can be used. In this case, in step S304, a solution containing pyrenebutyric acid succinimide ester is immersed in the surface of the fixed layer, and this solution and the surface of the fixed layer are kept in contact with each other, for example, at a constant temperature of room temperature to 37 ° C. for a predetermined time. (Normally 30 minutes to 60 minutes) Let stand. Thereafter, excess linker molecules that have not been immobilized are removed from the substrate surface by washing.

  Next, in step S305, a solution containing (mixed) a dye-binding biomolecule detection molecule having an amino group introduced into a predetermined site is brought into contact with the fixed layer. For example, a mixed solution containing the above-described dye-binding biomolecule detection molecule may be dropped on the fixed layer. This state is allowed to stand for a predetermined time (usually 30 minutes to 60 minutes), for example, in a constant temperature state of room temperature to 37 ° C. In these treatments, the solution may be allowed to stand under saturated vapor pressure so as not to dry. By doing so, pyrenebutyric acid (linker molecule) and a dye-binding biomolecule detection molecule can be covalently bonded. Next, the (mixed) solution containing the dye-binding biomolecule detection molecules is removed, and excess biomolecule detection molecules that have not been immobilized are removed from the substrate surface by washing. In addition, when the molecule | numerator for biomolecule detection itself has a function of adsorb | sucking to a fixed layer, it is not necessary to use a linker molecule.

  By repeating the step of fixing the dye-binding biomolecule detection molecule described above, the biomolecule detection molecule may be fixed in several steps. During the repetition, the type of (mixed) solution containing the biomolecule detection molecule may be changed. Thereby, a substrate having a plurality of different types of biomolecule detection molecules immobilized on the surface of the immobilization layer is obtained. Thereafter, in step S306, a measurement microchannel for introducing the sample solution may be mounted at the formation position of the fixed layer of the obtained substrate. By using the micro flow channel, it becomes possible to observe the response of a very small amount of sample. In addition, by providing a plurality of flow paths, it is possible to simultaneously compare responses for different solutions. Through the above steps, the biomolecule detection chip in the embodiment of the present invention is completed.

[Example 1]
Hereinafter, description will be made using examples. First, Example 1 will be described. First, a cover glass was prepared as a substrate. Next, an aqueous solution in which graphene oxide is dispersed is applied to the substrate by a spin coating method to form a coating layer. The graphene oxide dispersion solution can be prepared by a reported synthesis method (see Non-Patent Document 5). Note that the size of the graphene oxide can be adjusted to small pieces of about several hundred nm by ultrasonicating the dispersion solution. By this coating, a coating layer composed of small pieces of graphene oxide of about 1 to 5 layers can be uniformly formed on the surface of the substrate. Further, the concentration of graphene oxide in the dispersion solution may be about 0.01 to 1 wt%.

  Next, the substrate on which the coating layer has been formed as described above is sealed in a sealed container together with a solution in which a 35% hydrazine aqueous solution and a 28% ammonia aqueous solution are mixed at a volume ratio of 1: 7. As a result, the graphene oxide constituting the coating layer is exposed to hydrazine / ammonia vapor. By allowing this state to stand at 95 ° C. for 1 hour, graphene oxide can be reduced, and a fixed layer made of a graphene oxide reductant can be formed. The graphene oxide reductant produced at this time retains the shape fixed to the substrate surface as graphene oxide, and thus does not cause aggregation as seen in the reduction reaction in the solution. As described above, a fixed layer in which small pieces of graphene oxide reduced body of about 1 to 5 layers are uniformly dispersed can be formed.

  Next, a 0.5 mM dimethylformamide solution of pyrenebutyric acid succinimide ester in which the COOH group of pyrenebutyric acid is activated with succinimide is dropped on a fixed layer on the substrate, and left at room temperature (about 25 ° C.) for 1 hour. Adsorbed on the surface of the fixed layer. This is washed with dimethylformamide and air dried. Pyrene functions as a linker molecule for firmly fixing a graphene oxide reductant and an aptamer that is a biomolecule detection molecule used in a later step.

  Next, a biomolecule detection molecule to which a dye is bound is prepared. A molecule having an aptamer structure was used as a biomolecule detection molecule. As the DNA sequence of the aptamer part, 5'-AATTAAGCTCGCCATCAAAATAGC-3 'was prepared as one that specifically binds to prostate specific antigen (PSA). Moreover, 5'-AGTCCGTGGGTAGGGCAGGTTGGGGTGACT-3 'was prepared as what specifically binds to thrombin. “A” is adenine, “G” is guanine, “C” is cytosine, and “T” is thymine.

  In addition, for aptamers that specifically bind to PSA, a green fluorescent molecule (6-carbofluorescein; FAM) is bound to three ends as a dye that causes a detectable signal change, and a biomolecule detection molecule to which the dye is bound, and To do. In addition, the aptamer that specifically binds to thrombin is a biomolecule detection molecule in which a red fluorescent molecule (TAMRA) is bound to three ends as a signal material that causes a detectable signal change, and a dye is bound. In addition, about 10 thymines may be bound between the aptamer sequence and the dye molecule as a spacer sequence having no specific binding function.

  In addition, an amino group is introduced as a binding site at the 5 terminus of each of the two aptamers described above. The 5-terminal amino group reacts with the COOH group of pyrenebutyric acid to form a peptide bond, and through this, a molecule containing an aptamer structure can be fixed to the surface of the fixed layer.

  The aqueous solutions of the two types of dye-binding biomolecule detection molecules described above are mixed and adjusted so that each concentration becomes 50 μM, and then the mixed solution is brought into contact with the surface of the fixed layer. This contact state is placed in a saturated steam atmosphere so that the mixed solution does not dry, and is maintained at room temperature for 60 minutes. As a result, two types of dye-binding biomolecule detection molecules are fixed to the surface of the fixing layer formed on the glass substrate by binding of the 5-terminal amino group and pyrene, which is a linker molecule. Next, the substrate is washed several times with ultrapure water, and excess dye-binding biomolecule detection molecules that have not been immobilized are removed from the substrate surface.

  The microchannel for detection in which the channels are arranged in parallel is mounted on the biomolecule detection chip of Example 1 manufactured by the above process. The microchannel may be made of a polymer material that is transparent and has good adhesion to the substrate, such as polydimethylsiloxane (PDMS). This microchannel will be described with reference to FIG. 4A is a plan view, and FIG. 4B is a cross-sectional view.

  This microchannel is composed of a support substrate 401 and a channel substrate 403. A fixed layer (not shown) is formed on the surface of the support substrate 401. In the flow path substrate 403, three flow path forming grooves 404, a flow path forming groove 405, and a flow path forming groove 406 are formed. The flow path forming groove 404, the flow path forming groove 405, and the flow path forming groove 406 include measurement regions arranged in parallel to each other. The flow path forming groove 404, the flow path forming groove 405, and the flow path forming groove 406 are formed to have a width of 50 μm and a depth of 100 μm, for example. Moreover, the length of the parallel location which passes a measurement area | region was 5 mm, and the space | interval of each groove | channel in a measurement area | region was 100 micrometers.

  Further, the flow path forming groove 404, the flow path forming groove 405, and the flow path forming groove 406 are formed with introduction ports 404a, 405a, and 406a each having a hole penetrating the flow path substrate 403 at one end and the other end. Discharge ports 404b, 405b, and 406b, which are holes that penetrate the flow path substrate 403, are formed. The inlets 404a, 405a, 406a and the outlets 404b, 405b, 406b are formed in succession to the channel forming groove 404, the channel forming groove 405, and the channel forming groove 406, respectively.

  In addition, a surfactant is introduced into the PDMS so that liquid can be easily fed using the capillary phenomenon in the flow path forming groove 404, the flow path forming groove 405, and the flow path forming groove 406. Thus, the flow path substrate 403 is molded to improve the hydrophilicity of the surfaces of the flow path forming groove 404, the flow path forming groove 405, and the flow path forming groove 406.

  The main surface of the support substrate 401 is brought into close contact with the surface on which the flow path forming groove 404, the flow path forming groove 405, and the flow path forming groove 406 of the flow path substrate 403 are configured as described above. By doing so, it is set as the state provided with the flow path 1, the flow path 2, and the flow path 3 by each groove | channel. As described above, the fixed layer is formed on the surface of the support substrate 401, and the surface of each flow path on the support substrate 401 side is configured by the fixed layer. For example, each surface may be washed with ethanol and then subjected to oxygen plasma treatment to be in close contact, and allowed to stand at room temperature for 30 minutes, and then bonded by chemical bonding.

  The biomolecule detection chip of Example 1 described above is observed using a confocal laser fluorescence microscope. First, 1 μL of a 100 μg / ml PSA aqueous solution is introduced into the channel 1. In addition, 1 μL of 100 μg / ml thrombin is introduced into the flow path 2. Further, 1 μL of ultrapure water is introduced into the flow path 3.

  A fluorescence image on the surface of the fixed layer is measured at a position where the straight portions of the three flow paths are observed within the same field of view of the confocal laser fluorescence microscope. Green fluorescence is observed inside the channel 1 and red fluorescence is observed inside the channel 2. These results are as follows. First, PSA and thrombin selectively bind to aptamers that specifically bind to each other, whereby the three-dimensional structure of each aptamer changes. Due to this structural change, FAM and TAMRA modified to the aptamer are separated from the fixed layer to such an extent that energy transfer to the fixed layer does not occur. As a result, FAM and TAMRA emit light.

  On the other hand, almost no fluorescence is observed inside the flow path 3 into which ultrapure water is introduced. This is because there is no biomolecule that binds to each aptamer, and the structural change of each aptamer does not occur, so the distance between FAM and TAMRA and the fixed layer is short. In the state where the fluorescence of these dye molecules is arranged near the fixed layer, it is quenched by the graphene oxide reductant constituting the fixed layer. Moreover, it has shown that the graphene oxide reductant itself which fixes the aptamer does not emit light. The optical observation as described above can be easily performed if, for example, the support substrate 401 and the flow path substrate 403 are transparent.

  As described above, according to Example 1, PSA and thrombin can be detected simultaneously with one chip without patterning the aptamer that is a molecule for detecting biomolecules. According to Example 1, it is shown that it is possible to provide a method capable of simultaneously detecting a plurality of chemical components from a very small amount of sample without requiring patterning into a minute region based on a light detection method for each color. .

[Example 2]
Next, Example 2 will be described. In Example 2, the fixed layer is formed without reducing graphene oxide. Other conditions regarding the substrate are the same as those in the first embodiment.

  As a biomolecule detection molecule, 5′-GUCGGCCAUGCGGUA-3 ′ having an RNA sequence that specifically binds to hemagglutinin (HA), which is an influenza virus antigenic glycoprotein, is used. “U” is uracil. Moreover, 5'-GGTTGGGTGGGTTGG-3 'is used as a DNA sequence that specifically binds to thrombin.

  In addition, an aptamer that binds to HA is bound to a green fluorescent molecule (6-carbofluorescein; FAM) at the three ends as a dye, thereby obtaining a biomolecule detection molecule to which the dye is bound. On the other hand, a red fluorescent molecule (TAMRA) is bound as a dye to the aptamer to be thrombin, and a biomolecule detection molecule having a dye bound thereto is obtained. Using the two types of dye-binding biomolecule detection molecules described above, a biomolecule detection chip having a microchannel was prepared in the same manner as in Example 1 described above.

  The biomolecule detection chip of Example 2 described above is observed using a confocal laser fluorescence microscope. First, 1 μL of a 100 μg / ml HA aqueous solution is introduced into the channel 1. In addition, 1 μL of 100 μg / ml thrombin is introduced into the flow path 2. Further, 1 μL of ultrapure water is introduced into the flow path 3.

  The fluorescence image on the surface of the fixed layer is measured at a position where the straight portions of these three flow paths are observed within the same field of view of the confocal laser fluorescence microscope. Green fluorescence is observed inside the channel 1 and red fluorescence is observed inside the channel 2. These results are as follows. First, HA and thrombin selectively bind to aptamers that specifically bind to each other, whereby the three-dimensional structure of each aptamer changes. Due to this structural change, FAM and TAMRA modified to the aptamer are separated from the fixed layer to such an extent that energy transfer to the fixed layer does not occur. As a result, FAM and TAMRA emit light.

  On the other hand, first, green fluorescence is not observed in the flow path 3 into which ultrapure water is introduced. However, very weak red fluorescence due to graphene oxide is observed. In addition, it was distinguishable from the red fluorescence in the channel 2 described above by a clear intensity difference. This is because there is no biomolecule that binds to each aptamer, and the structural change of each aptamer does not occur, so the distance between FAM and TAMRA and the fixed layer is short. In the state where the fluorescence of these dye molecules is arranged near the fixed layer, it is quenched by the graphene oxide constituting the fixed layer.

  From the above results, it was first shown that the green fluorescent dye did not interfere with detection by the graphene oxide itself. On the other hand, graphene oxide shows weak light emission in the red region, but the emission intensity of the dye is very strong compared to the light emission of graphene oxide itself, indicating that the interference in detection is almost negligible. From the above lack, an analysis method that can simultaneously detect HA and thrombin with one chip without patterning an aptamer that is a molecule for detecting biomolecules has been shown. Even in Example 2 using a fixed layer of graphene oxide, there is a method capable of simultaneously detecting a plurality of chemical components from a very small amount of sample without the need for patterning into a minute region based on a light detection method for each color. It is shown that it can be provided.

  As described above, according to the present invention, a plurality of biomolecule detection molecules to which different dyes are bonded are fixed to a fixed layer composed of graphene oxide or a graphene oxide reductant. Analysis of a plurality of different detection targets using a plurality of biomolecule detection molecules can be easily performed. According to the present invention, a quenching material paired with a dye used for labeling is not required for each type of dye, and multicoloring using green or red fluorescence is possible. In addition, when graphene oxide reductant is used, it is shown that multicolorization is not hindered by its non-light emission characteristics.

  In general, the absolute value of fluorescence intensity includes the effects of deterioration of dye molecules and changes over time, so it is difficult to make a quantitative comparison. In addition, it is possible to accurately compare the difference between the response signals (channels 1 and 2) and the reference signal (channel 3) for the same aptamer. Therefore, by using microchannels, it is possible to cancel different chemical reactions from a very small amount of sample of about 1 μL, cancel the influence of deterioration of dye molecules and changes over time, and compare and measure the difference in taste response simultaneously with high accuracy. Can be obtained.

  The present invention is not limited to the embodiment described above, and many modifications and combinations can be implemented by those having ordinary knowledge in the art within the technical idea of the present invention. It is obvious. For example, in the above description, the case where two types of biomolecule detection molecules are mainly fixed to the fixed layer has been described as an example. However, the present invention is not limited to this, and each of a plurality of different biomolecules to be detected It goes without saying that three (three types) or more molecules for biomolecule detection that specifically bind may be used.

  In addition, the aptamer is not limited to a nucleic acid but may be a peptide. Further, a molecule in which a nucleic acid or a peptide is arranged may be further bonded to the end of the aptamer to which a signal molecule or an adhesion molecule is bonded.

  Adhesion molecules are not limited to those containing pyrene in the skeleton such as pyrene butadiene acid, and examples include benzene and polycyclic aromatic molecules (naphthalene, anthracene, tetracene, pentacene, phenanthrene, pyrene, perylene, coronene), etc. It may be. Further, the functional group (molecule) for bonding the adhesion molecule to the aptamer is not limited to butadiene acid, and may be “—COOH” and “— (CH 2) n COOH”. Note that n is preferably in the range of 2 to 10.

  101a ... biomolecule detection molecule, 101b ... biomolecule detection molecule, 102a ... dye, 102b ... dye, 103 ... substrate, 104 ... fixed layer.

Claims (3)

A pinned layer forming step of forming a fixed layer composed of oxidation graphene reductant on a substrate,
A dye-binding biomolecule detection molecule forming step of binding a dye of a different color to one end of each of a plurality of biomolecule detection molecules that specifically bind to each of a plurality of different biomolecules to be detected;
Detection of a plurality of biomolecules to which dyes of different colors are bonded by bringing a mixed solution in which a plurality of aqueous solutions containing the molecules for detection of biomolecules to which the dyes of different colors are combined are brought into contact with the fixed layer And a molecule-fixing step for detecting a dye-forming biomolecule that fixes the other end of the molecule to the fixing layer. In the manufacturing method of the biomolecule detection chip according to claim 1,
An adhesive molecule binding step of binding an adhesive molecule that adheres to the fixed layer to the other end of the plurality of biomolecule detection molecules,
In the molecule-fixing step for detecting a dye-forming biomolecule, the other end of the biomolecule detecting molecule and the fixing layer are bonded and fixed by the adhesive molecule bonded to the other end of the biomolecule detecting molecule. A method for producing a biomolecule detection chip as a feature. In the manufacturing method of the biomolecule detection chip according to claim 1 or 2,
A method for producing a biomolecule detection chip, wherein the substrate is transparent.

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