A complex for detecting bacteria, a method for producing the complex, and a method for detecting bacteria using the complex.

Abstract

To provide a bacterial detection complex that can detect bacteria quickly, with high sensitivity, and easily. [Solution] A complex for detecting bacteria, comprising carrier particles of a carbon-based material, a single platinum atom supported on the carrier particles, and an antibody immobilized on the carrier particles that specifically binds to the bacteria to be detected.

Description

[Technical Field]

[0001] The present invention relates to a complex for detecting bacteria, a method for producing the complex, and a method for detecting bacteria using the complex. [Background technology]

[0002] Periodontal disease is a general term for diseases of the periodontal tissues, which are the tissues that support the teeth, and is considered a multifactorial disease caused by various factors. Among these many factors, one of the main ones is infection of the periodontal pockets by periodontal pathogenic bacteria. Such bacterial infection causes the periodontal tissues to break down, eventually leading to tooth loss.

[0003] While numerous periodontal pathogens are known to exist, Porphyromonas gingivalis (also known as "Pg bacteria"), Tannerella forsythia (also known as "Tf bacteria"), and Treponema denticola (also known as "Td bacteria") are particularly likely to be causative agents of periodontal disease, and are also associated with diseases of other organs.

[0004] Many methods have been developed to detect these periodontal disease bacteria. For example, microbiological detection methods include biochemical measurements and hybridization methods using nucleotide probes. Electrochemical methods that measure impedance and molecular biological detection methods utilizing polymerase chain reaction (PCR) have also been reported.

[0005] On the other hand, while Patent Document 1 does not mention bacterial detection, it discloses a resin-platinum complex in which multiple platinum particles are immobilized on resin particles, for use in immunoassays. An antigen or antibody is adsorbed onto the complex to form a ligand, and an analyte that specifically adsorbs to the antigen or antibody is detected.

[0006] Furthermore, one of the inventors of the present invention reported in Non-Patent Literature 1 that hepatitis E virus was detected with high sensitivity by an immunoassay using an antibody labeled with platinum nanoparticles supported on Co3O4 hollow cages (Pt-Co3O4 Hollow cages). This detection method utilizes the fact that Pt-Co3O4 hollow cages catalyze the hydroxylation reaction to electrochemically detect the virus. This catalytically active nanomaterial (Pt-Co3O4 hollow body), i.e., nanozyme, is manufactured by supporting platinum nanoparticles on a zeolite imidazole framework (ZIF-67) and calcining it at 500°C. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2017 / 010391 publication [Non-patent literature]

[0008] [Non-Patent Document 1] ACS Appl. Mater. Interfaces 2020, 12, 50212-50221 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] However, all of the microbiological detection methods described above require bacterial culture. Therefore, they are time-consuming and labor-intensive, and it has been difficult to isolate and identify anaerobic bacterial species such as Pg bacteria and perform regular clinical evaluations. Electrochemical methods measuring impedance did not provide sufficient detection sensitivity. On the other hand, molecular biological detection methods using PCR do not require bacterial culture and have high detection sensitivity. However, they require intensive treatment at specific facilities equipped with special adjustment devices and equipment such as thermal cyclers, and have the drawback of long detection times. Immunological assays can also be considered for bacterial detection, but the method disclosed in Patent Document 1 did not have sufficient detection sensitivity. While a detection method disclosed in Non-Patent Document 1 exists, the development of detection methods using alternative approaches is also desired.

[0010] This invention aims to solve these problems. Specifically, the object of this invention is to provide a complex for bacterial detection that can detect bacteria quickly, with high sensitivity, and in a simple manner. [Means for solving the problem]

[0011] The inventors, after diligently studying to achieve the above objectives, found that the objectives could be achieved with the following configuration.

[0012] [1] A complex for bacterial detection, Carbon-based material support particles, Platinum single atoms supported on the aforementioned carrier particles, A complex comprising an antibody immobilized on the carrier particle and specifically binding to the bacteria to be detected. [2] The composite according to [1], wherein the carrier particles are hollow. [3] The composite according to [1] or [2], wherein the carrier particles include a carbide of a zeolite-like imidazole structure. [4] The complex according to [3], wherein the zeolite-like imidazole structure is ZIF-67. [5] The composite according to any one of [1] to [4], wherein the carrier particles contain nitrogen atoms. [6] The composite according to any one of [1] to [5], wherein the average particle diameter of the carrier particles is 200 nm to 300 nm. [7] The composite according to any one of [1] to [6], wherein the antibody is an anti-Porphyromonas gingivalis antibody. [8] A method for producing the composite according to any one of [1] to [7], comprising: Loading platinum nanoparticles onto particulate zeolite-like imidazole structures; Covering the surface of the zeolite-like imidazole structure loaded with the platinum nanoparticles with a layer containing poly(cyclotriphosphazene-co-4,4'-sulfonyldiphenol) to obtain a nanozyme precursor; Calcining the nanozyme precursor to obtain a nanozyme; Fixing the antibody to the nanozyme, a method for producing a composite comprising the above steps. [9] The method for producing a composite according to [8], wherein the platinum single atoms are formed from the platinum nanoparticles by calcining the nanozyme precursor.

[10] The method for producing a composite according to [8] or [9], wherein the carrier particles, which are hollow bodies, are formed from the zeolite-like imidazole structure and the layer by calcining the nanozyme precursor.

[11] A method for detecting bacteria, comprising performing an immunological measurement method using the composite according to any one of [1] to [7]. [Advantages of the Invention]

[0013] The present invention provides a composite for detecting bacteria, which can detect bacteria with high sensitivity and simplicity in a short time. [Brief Description of the Drawings]

[0014] [Figure 1] A diagram for explaining the method for producing a composite in an embodiment. [Figure 2] A diagram for explaining the method for detecting bacteria using a composite in an embodiment. [Figure 3]This is a flowchart illustrating the method for manufacturing the composite in the embodiment. [Figure 4] The following are the observation results of the nanozymes synthesized in the examples using a transmission electron microscope (TEM). Figure 4(a) is a TEM image of the Pt-SACs precursor, Figure 4(b) is a TEM image of Pt-SACs, and Figure 4(c) is a HAADF-STEM (High-Angle Annular Dark Field Scanning TEM) image of Pt-SACs. Figures 4(d) and (e) show the results of analysis by energy-dispersive X-ray spectroscopy (EDS), with Figure 4(d) showing platinum (Pt) mapping and Figure 4(e) showing carbon (C) mapping. [Figure 5] This figure shows the calibration curve (a straight line showing the relationship between PG cell concentration and absorbance) obtained in the example. [Figure 6] This figure shows the results of bacterial detection using nanozymes (Pt-SACs) and platinum nanoparticles (Pt-NPs) in the examples. [Figure 7] This figure shows the selectivity of the complex (AbPG-Pt-SACs) for bacterial detection in the examples. [Modes for carrying out the invention]

[0015] The present invention will now be described in detail. The following descriptions of constituent elements may be based on representative embodiments of the present invention, but the present invention is not limited to such embodiments. In this specification, numerical ranges represented by "~" mean a range that includes the numbers written before and after "~" as the lower and upper limits.

[0016] [Complex for bacterial detection] As shown in Figure 1, the bacterial detection complex 100 of this embodiment includes carrier particles 10 made of a carbon-based material, a single platinum atom 20 supported on the carrier particles 10, and an antibody 30 immobilized on the carrier particles 10 that specifically binds to the bacteria 50 to be detected (see Figure 2). The complex 100 can be used, for example, to detect bacteria 50 in an immunoassay (immunological measurement method). An immunoassay is an analytical method that utilizes a reaction in which bacteria 50, which are antigens (Ag), and antibodies (Ab) 30 react to produce an antigen-antibody conjugate (Ag-Ab), and has extremely high specificity and sensitivity. In the complex 100 of this embodiment, the carrier particles 10 and the single platinum atom 20 supported thereon constitute a nanozyme 40. A nanozyme refers to a nanomaterial that has enzyme-like properties. In the complex 100 of this embodiment, the nanozyme 40 having the single platinum atom 20 can further enhance the detection sensitivity of bacteria 50.

[0017] <Nanozyme> The nanozyme 40 of this embodiment includes carrier particles 10 and platinum single atoms 20 supported thereon. The carrier particles 10 are not particularly limited as long as they are particles of a carbon-based material (a material mainly composed of carbon), but preferably include, for example, carbides of a zeolitic imidazolate framework (ZIF). As will be described in detail later, as shown in Figure 1, by calcining a precursor 41 in which platinum nanoparticles 21 are supported on a zeolitic imidazolate framework (hereinafter referred to as "framework (ZIF)") 11, the platinum nanoparticles 21 can be thermally decomposed to form platinum single atoms 20, and the nanozyme 40 can be efficiently produced.

[0018] The structure (ZIF) 11 is a type of material with a metal-organic framework (MOF) as its backbone, where the metal is cobalt (Co) or zinc (Zn) and the organic bridging ligands consist of imidazole substituents, resulting in a three-dimensional microporous material similar to a zeolite. Examples of such structures (ZIF) 11 include ZIF-7, ZIF-22, ZIF-8, ZIF-67, ZIF-69, ZIF-71, ZIF-78, ZIF-90, and ZIF-95, depending on differences in pore size and the type of imidazole substituent. Among these, ZIF-67, i.e., a sodalite-type crystalline structure in which Co ions are bridged by 2-methylimidazole, is preferred from the viewpoint of efficiently synthesizing nanozyme 40. The presence of ZIF-67 carbides in the carrier particles 10 can be simply determined from the fact that the shape of the carrier particles 10 is dodecahedral. Furthermore, one type of structure (ZIF)11 may be used, or two or more types may be used.

[0019] The carrier particles 10 are preferably hollow bodies having a hollow structure. Of the platinum single atoms 20 contained in the carrier particles 10, those that function as catalysts are the atoms located on and near the surface of the carrier particles 10. By making the carrier particles 10 hollow bodies, more platinum single elements 20 can be placed on and near the surface of the carrier particles 10, and as a result, catalytic activity can be further enhanced. Hollow carrier particles can be efficiently produced, for example, by covering the surface of a structure (ZIF) 11 with a layer containing poly(cyclotriphosphazene-co-4,4′-sulfonyldiphenol) (PZS layer) to create a core-shell structure (ZIF-PZS structure) nanozyme precursor 41, and then calcining it. During the calcination process of the precursor, gas is released, and as a result, the carrier particles 10 obtain a hollow void and a porous structure (e.g., a mesoporous structure). In this case, the carrier particles contain the structure (ZIF) 11 and the carbide of the PZS layer.

[0020] The carbon-based material of the carrier particles 10 is preferably mainly composed of carbon (C), and may consist only of carbon (C), or may contain other elements as long as they do not produce the effects of the present invention. For example, the carrier particles 10 may contain nitrogen atoms (N). Including nitrogen atoms (N) may further improve the catalytic activity of the nanozyme.

[0021] From the viewpoint of improving the sensitivity of bacterial detection, the average particle diameter of Nanozyme 40 (carrier particles 10 on which platinum single atoms 20 are supported) is preferably 200 nm to 300 nm. In this specification, the average particle diameter may be calculated, for example, from TEM image analysis.

[0022] Nanozyme 40 exhibits extremely high catalytic activity because the catalytically active platinum (Pt) exists not as particles, but as single atoms (single platinum atoms 20). When platinum exists as particles, only the atoms exposed on the surface of the particles can contribute to catalytic activity. However, in this embodiment, since it exists as single platinum atoms 20, more atoms can contribute to catalytic activity without waste.

[0023] <Antibody> The antibody 30 is not particularly limited as long as it is an antibody that specifically binds to the target bacteria (antigen) 50. For example, polyclonal antibodies, monoclonal antibodies, antibodies obtained by genetic engineering, and antibody fragments that can bind to the antigen [e.g., H chain, L chain, Fab, F(ab′)2, etc.] can be used. In addition, any of IgG, IgM, IgA, IgE, or IgD can be used as the immunoglobulin. The animal species that produces the antibody can be humans, as well as other animals (e.g., mice, rats, rabbits, goats, horses, etc.).

[0024] The bacteria (antigen) 50 to be detected in this embodiment are not particularly limited, and examples include Porphyromonas gingivalis (Pg bacteria), Tannerella forsythia (Tf bacteria), and Treponema denticola (Td bacteria), which are causative agents of periodontal disease. In addition, Staphylococcus aureus, Clostridium tetani, etc., may be the bacteria to be detected.

[0025] In this embodiment, an antibody 30 that specifically binds to bacteria 50 is selected according to the type of bacteria 50 to be detected. As for the antibody 30, one type of antibody may be used for detecting one type of bacteria, or two or more types of antibodies may be used for detecting two or more types of bacteria, but from the viewpoint of increasing detection sensitivity, it is preferable to use only one type of antibody.

[0026] The antibody 30 is immobilized on the carrier particle 10 by chemical or physical binding or adsorption. The antibody 30 may be directly bound to the carrier particle 10, or it may be bound via any linker molecule. From the viewpoint of stable immobilization and ease of controlling the amount of immobilized antibody 30, it is preferable that the antibody 30 is immobilized on the carrier particle 10 directly or indirectly by chemical bonding (e.g., amide bonding).

[0027] [Method for producing the composite] The method for manufacturing the composite 100 is not particularly limited, but one example will be described according to the flowcharts in Figures 1 and 3. The manufacturing method of this embodiment includes the following steps S1 to S4.

[0028] (Step S1) A step of supporting platinum nanoparticles 21 on particulate zeolite-like imidazole structures (ZIF) 11, (Step S2) A step to obtain a nanozyme precursor 41 by covering the surface of a structure (ZIF) 11 supporting platinum nanoparticles 21 with a layer (PZS layer, not shown) containing poly(cyclotriphosphazene-co-4,4′-sulfonyldiphenol) (PZS), (Step S3) A step of calcining the nanozyme precursor 41 to obtain nanozyme 40, and (Step S4) A step in which the antibody 30 is immobilized on the nanozyme 40.

[0029] <Process S1> First, platinum nanoparticles 21 are supported on the structure (ZIF) 11 (step S1 in Figure 3). The structure (ZIF) 11 may be a commercially available product or may be synthesized by a known method (see, for example, Non-Patent Document 1). The preferred embodiment (type, etc.) of the structure 11 is as described above. The average particle size of the structure 11 is preferably 200 nm to 300 nm. If the average particle size of the structure 11 is within the above range, it is easy to obtain a composite 100 of a size that provides sufficient bacterial detection sensitivity.

[0030] The platinum nanoparticles 21 may be commercially available or synthesized by known methods (see, for example, Non-Patent Document 1). The average particle size of the platinum nanoparticles 21 is not particularly limited, but is preferably, for example, 10 nm to 15 nm. If the average particle size of the platinum nanoparticles 21 is within the above range, single platinum atoms 20 can be easily obtained by thermal decomposition.

[0031] The method for supporting the platinum nanoparticles 21 on the structure 11 is not particularly limited, and known methods can be used. For example, the structure 11 and the platinum nanoparticles 21 may be mixed in a solvent. This impregnates and supports the porous structure 11 with the platinum nanoparticles 21.

[0032] <Process S2> Next, the surface of the structure (ZIF) 11 supporting the platinum nanoparticles 21 is covered with a PZS layer (step S2 in Figure 3). This yields a nanozyme precursor 41 having a core-shell structure (ZIF-PZS structure). The method for covering the surface of the structure (ZIF) 11 with the PZS layer is not particularly limited, and known methods can be used. For example, as disclosed in the above-mentioned reference (1), the structure (ZIF) 11 supporting the platinum nanoparticles 21 may be dispersed in a solvent, and a hexachlorocyclophosphazene solution and a 4,4′-sulfonyldiphenol solution may be added and reacted to form a PZS layer on the surface of the structure (ZIF) 11.

[0033] The thickness of the PZS layer forming the shell may be, for example, 10 nm to 20 nm. By setting the thickness of the PZS layer within the above range, carrier particles 10 having a hollow structure can be obtained more efficiently.

[0034] <Process S3> The obtained nanozyme precursor 41 is calcined (step S3 in Figure 3). During calcination, the platinum nanoparticles 21 are thermally decomposed to form single platinum atoms 20, and at the same time, carrier particles 10 made of a carbon-based material having a hollow structure are formed from the core-shell structure (ZIF-PZS structure). This yields nanozyme 40, which is the carrier particle 10 supporting the single platinum atoms 20.

[0035] The firing temperature and firing time may be adjusted as appropriate. For example, the firing temperature and firing time may be 1000°C for 6 hours. It is preferable to carry out the firing in an inert gas atmosphere such as nitrogen.

[0036] <Process S4> The antibody 30 is immobilized on the nanozyme 40 (step S4 in Figure 3). As described above, the antibody 30 can be appropriately selected to specifically bind to the bacteria 50 to be detected. The antibody 30 may be a commercially available product or it may be prepared in the process.

[0037] The method for immobilizing the antibody 30 onto the carrier particles 10 of the nanozyme 40 is not particularly limited, and known methods may be used. The antibody 30 may be immobilized on the carrier particles 10 by chemical or physical binding or adsorption. The antibody 30 may be directly bound to the carrier particles 10, or it may be bound via any linker molecule. From the viewpoint of stable immobilization and ease of controlling the amount of immobilized antibody 30, it is preferable to immobilize the antibody 30 on the carrier particles 10 by chemical bonding, either directly or indirectly. For example, as will be explained in the examples described later, the antibody 30 may be immobilized on the carrier particles 10 by covalent bonding (amide bonding) between carboxyl groups introduced on the carrier particles 10 and free amino groups contained in the antibody 30.

[0038] The manufacturing method for the composite 100 of this embodiment, as described above, allows for the simultaneous monatomicization of platinum nanoparticles 21 and the generation of hollow carrier particles 10 by calcining the nanozyme precursor 41, thereby enabling the efficient production of the composite 100.

[0039] [Methods for detecting bacteria] The bacterial detection method of this embodiment includes performing an immunological assay using the complex 100 described above. More specifically, the immunological assay method is an enzyme immunoassay (enzyme-linked immunosorbent assay, EIA, or ELISA), which is a type of labeling method. Examples of enzyme immunoassays include the direct method and the sandwich method. In these methods, the complex 100 of this embodiment is used as an enzyme-labeled antibody that specifically binds to the target bacterium 50 (antigen). The bacterial detection method of this embodiment can use the same procedure as conventional immunological assays, except for the use of the complex 100. The detection method of this embodiment can not only determine the presence or absence of bacterium 50 but also quantify bacterium 50.

[0040] As an example, the detection method for bacterium 50 using the EIA sandwich method, as shown in Figure 2, will be described. First, antibody 70 of bacterium 50, the target of detection, is adsorbed or otherwise immobilized on the surface of well 60 of the microplate. It is preferable to use antibody 70 that has a different antigen recognition site (epitope) from antibody 30 of complex 100. Next, the sample containing bacterium 50 is added to well 60 to bind the bacterium 50 to antibody 70. After washing, complex 100 is added to bind antibody 30 to bacterium 50. That is, complex 100 functions as the second antibody (secondary antibody) in the sandwich method. After washing away the excess complex 100, a solution of chromogenic substrate (a substance that develops color by an enzymatic reaction) is added. The chromogenic substrate develops color due to the complex 100 present in well 60, and by detecting this color, bacterium 50 can be detected. The chromogenic substrate is not particularly limited and any known substrate can be used, for example, 3,3′,5,5′-tetramethylbenzidine (TMB). The TMB solution contains, for example, hydrogen peroxide, and the hydrogen peroxide is decomposed by complex 100, and the resulting reactive oxygen species oxidize the TMB. The resulting blue-violet pigment turns yellow (absorption at 450 nm) under sulfuric acid acid, which is added to stop the reaction, and this absorbance is measured. For example, quantitative analysis of bacteria 50 is possible by determining a calibration curve of absorbance.

[0041] The bacterial detection method of this embodiment uses an immunological measurement method, eliminating the need for bacterial culture or large-scale, specialized equipment, thus enabling simple and rapid bacterial detection. Furthermore, the sensitivity of bacterial detection can be further enhanced by using the complex 100. This is presumed to be because the nanozyme 40 contains a single platinum atom 20, which strongly catalyzes the color reaction of the chromogenic substrate. Because the bacterial detection method of this embodiment has high detection sensitivity, quantitative analysis of bacteria 50 is possible over a wide range from low to high concentrations. With the highly sensitive bacterial detection method of this embodiment, it becomes possible to diagnose early periodontal disease, which was difficult with conventional detection methods, and to detect even small amounts of bacteria remaining in the root area of ​​teeth that have undergone caries treatment. [Examples]

[0042] The present invention will be described in more detail below based on examples. The materials, amounts used, proportions, processing content, and processing procedures shown in the following examples can be modified as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the examples shown below.

[0043] [Nanozyme] <Synthesis of platinum nanoparticles> A precursor was synthesized by mixing an aqueous solution of hydrogen hexachloroplatinate (H2PtCl6, 6.0 mM, 4 mL) and polyvinylpyrrolidone (PVP, molecular weight 29,000, 106.6 mg) in 36 mL of methanol. The mixture containing the precursor was heated to 75°C and stirred for 3 hours. The methanol in the mixture was then removed using a rotary evaporator. Acetone was added to the mixture to precipitate the platinum nanoparticles, which were recovered by centrifugation. The recovered platinum nanoparticles were redispersed in methanol and stored in methanol until use.

[0044] <Synthesis of zeolite-like imidazolate structure (ZIF-67)> ZIF-67 was synthesized by mixing 4 mmol of cobalt chloride (CoCl2-6H2O) and 32 mmol of 2-methylimidazole in 80 mL of methanol with stirring for 12 hours. After drying overnight in a vacuum, the mixture was further dried at 60°C for 12 hours to obtain ZIF-67 powder.

[0045] <Synthesis of nanozymes> Dispersion (I) was prepared by ultrasonically dispersing 200 μL of platinum nanoparticle solution (1 mg / mL) and 400 mg of ZIF-67 in 30 mL of methanol and stirring for 30 minutes. Solution (II) was prepared by dissolving 320 mg of bis(4-hydroxyphenyl) sulfone and 150 mg of phosphonitrile chloride trimer in 100 mL of methanol.

[0046] Solution (II) was added to dispersion (I), and the mixture was stirred for 30 minutes to obtain dispersion (III). Furthermore, 2 mL of triethylamine was slowly introduced into dispersion (III) while stirring at room temperature for 15 hours to obtain a precipitate. The precipitate was collected, washed, and dried under vacuum conditions at 60°C for 12 hours. The obtained precipitate is a nanozyme precursor in which ZIF-67 supported by platinum nanoparticles is coated with a poly(cyclotriphosphazene-co-4,4′-sulfonyldiphenol) layer (PZS layer).

[0047] The nanozyme precursor was heated (calcined) in an N2 atmosphere at 1000°C for 6 hours to obtain the nanozyme. During calcination, ZIF-67 was carbonized, and the platinum nanoparticles underwent thermal decomposition to become single platinum atoms.

[0048] <TEM observation of nanozymes (Pt-SACs)> Hereafter, the nanozymes synthesized in this example may be referred to as "Pt-SACs" (Pt-Single Aton Catalyst). Using a transmission electron microscope (TEM), we observed the Pt-SACs precursor before calcination and the Pt-SACs after calcination. Figure 4(a) shows the TEM image of the Pt-SACs precursor, and Figure 4(b) shows the TEM image of the Pt-SACs. Furthermore, Figure 4(c) shows the HAADF-STEM (High-Angle Annular Dark Field Scanning TEM) image of the Pt-SACs, and the corresponding platinum (Pt) mapping by energy dispersive X-ray spectroscopy (EDS) is shown in Figure 4(d), and the carbon (C) mapping is shown in Figure 4(e).

[0049] Figures 4(a) to 4(e) show that, upon firing, Pt-SACs formed a hollow carbon polyhedral structure, with platinum (Pt) uniformly present on its surface. Furthermore, surface analysis of Pt-SACs was performed using X-ray photoelectron spectroscopy (XPS). The results showed that nitrogen (N) was also uniformly present on the surface of the Pt-SACs.

[0050] [Synthesis of complexes] A carboxyl group was introduced into nanozymes (Pt-SACs), and a complex (AbPG-Pt-SACs) was synthesized by amide bonding of the carboxyl group to a free amino group contained in the antibody (detection antibody) using an EDC / NHS chemical reaction. Details are described below.

[0051] 50 mg of nanozyme (powder) was ground in a mortar and pestle, then dispersed in 20 mL of 0.1% mercaptosuccinic acid (MSA) aqueous solution and subjected to sonication for 20 minutes. This introduced carboxyl groups into the nanozyme carrier particles. To remove excess MSA, pure water was added to the carboxylated nanozyme and centrifugation (10,000 G for 10 minutes x 3 times) was performed. After centrifugation, the precipitated carboxylated nanozyme was redispersed in 5 mL of phosphate-buffered saline (PBS) solution and stored at 4°C.

[0052] The nanozyme in PBS was mixed with 0.1 M EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) to activate the carboxyl group introduced into the nanozyme into an ester intermediate. In PBS, the ester intermediate was reacted with an excess of 0.4 M NHS (N-hydroxysuccinimide) and 5.1 μg of detection antibody. The detection antibody used was an anti-Porphyromonas gingivalis (PG) antibody (SAB4200832) prepared in rabbits.

[0053] Finally, the mixture was stirred overnight at 7°C, then centrifuged to remove unreacted chemicals, and the resulting complexes (AbPG-Pt-SACs) were stored at 4°C.

[0054] [Bacterial detection test] The synthesized complex (AbPG-Pt-SACs) was used for bacterial detection testing using the sandwich ELISA method described below.

[0055] <Creating a calibration curve> (a) Immobilization of anti-PG antibody As a capture antibody, an anti-PG antibody (Sigma-Aldrich, SAB4200832) produced in rabbits was prepared and diluted to 1 μg / mL in 0.1 M PBS (pH 6.5). 100 μL of this solution was added dropwise to the wells of a microtiter plate and incubated at room temperature for 3 hours.

[0056] (b) Blocking of microtiter plates After removing the antibody solution remaining in the microtiter plate, 200 μL of blocking solution containing a blocking agent (5% bovine serum albumin in 0.1 M PBS (pH 6.5)) was added, and the mixture was incubated at room temperature for a further 3 hours.

[0057] (c) Detection of PG bacteria For the measurement samples, PG bacteria solutions of different concentrations were prepared. After removing the remaining blocking solution, 100 μL of the measurement sample was added dropwise to the wells and incubated for 20 minutes. After incubation, the remaining solution was removed and the wells were washed three times with 300 μL of 0.1 M PBS (pH 6.5). Next, 100 μL of the previously prepared PBS solution of the complex (AbPG-Pt-SACs) was added to the wells and incubated for another 20 minutes. As a result, the PG bacteria captured by the capture antibody further bound to the detection antibody of the complex, forming an immune complex sandwich. After incubation, the remaining solution was removed and the wells were washed three times with 300 μL of 0.1 M PBS (pH 6.5).

[0058] A 0.4 M sodium acetate buffer (pH 4.0) solution containing 4 M H2O2 and 40 mM 3,3′,5,5′-tetramethylbenzidine (TMB) was prepared as the substrate solution. When 100 μL of the prepared substrate solution was added to the well, it rapidly developed a blue color. To stop the reaction, 10% H2SO4 was added after 5 minutes, and the blue color changed to yellow. Finally, the absorbance at 450 nm and 655 nm was recorded using a microplate reader. The obtained standard curve is shown in Figure 5.

[0059] As shown in Figure 5, the correlation coefficient (R) is expressed as a function of PG cell concentration.2 ) 0.991, linear range 10 0 ~10 6 cells mL -1 A linear calibration curve was obtained. That is, in the detection method using the complex (AbPG-Pt-SACs), in the range of 10 0 ~10 6 cells mL -1 bacteria can be detected over a wide concentration range.

[0060] <Comparison between Nanozyme (Pt-SACs) and Platinum Nanoparticles (Pt-NPs)> In the above calibration curve preparation, the detection results of 5 samples with a PG bacteria solution concentration of 10 1 ~10 5 cells mL -1 are shown in Fig. 6 as "Pt-SACs".

[0061] For comparison, instead of the complex (AbPG-Pt-SACs), a detection antibody labeled with platinum nanoparticles (AbPG-Pt-NPs) was used, and bacteria detection was performed by the same method otherwise. The detection results are shown in Fig. 6 as "Pt-NPs". The AbPG-Pt-NPs used for measurement were synthesized by a conventionally known method (see Non-Cited Reference 1). The platinum nanoparticles (Pt-NPs) used had a circular particle size of 5 nm to 10 nm.

[0062] As shown in Fig. 6, in all samples (PG bacteria solution) with different concentrations, Pt-SACs showed higher absorbance than Pt-NPs, indicating higher catalytic activity. Especially in samples with low concentrations, the difference in absorbance between the two was large. From these results, it was confirmed that bacteria can be detected with high sensitivity even from low-concentration samples by using the complex (AbPG-Pt-SACs).

[0063] [Selectivity Evaluation of Complex] Samples containing two types of bacteria (PG bacteria and other bacteria) were prepared, and bacteria detection was performed by the same method as in the above calibration curve preparation. As the other bacteria, the following 7 types of bacteria were used. In each sample, the PG concentration was 10 4cells mL -1 as, and the other bacterial concentration was 10 6 cells mL -1 as. The results are shown in Fig. 7. In Fig. 7, the other bacteria are shown as "Interference". <Other bacteria other than PG bacteria> E.coli: Escherichia coli SM: Streptococcus mutans CO: Corynebacterium matruchotii AA: Aggregatibacter actinomycetemcomitans CM: Corynebacterium matruchotii FH: fusobacterium hwasookii FN: fusobacterium nucleatum

[0064] In addition, measurement samples containing each bacterium (PG bacteria and other bacteria) one by one were also prepared, and bacteria detection was performed by the same method. The PG concentration was 10 4 cells mL -1 as, and the other bacterial concentration was 10 6 cells mL -1 as. The results are shown together in Fig. 7.

[0065] As shown in Fig. 7, all the samples containing PG bacteria showed a high absorbance of about 0.4. The samples in which PG bacteria and other bacteria coexisted (Interference + PG) also had a high absorbance equivalent to that of the samples containing PG bacteria alone. On the other hand, the absorbance of the samples containing other bacteria alone was very low, less than 0.1. From these results, it was confirmed that the complex (AbPG-Pt-SACs) did not bind to other bacteria and showed high selectivity due to the specificity of the antibody and the blocking process.

Industrial Applicability

[0066] The complex of the present invention can be used in bacterial detection methods. More specifically, enzyme immunoassay using the complex enables rapid, highly sensitive, and convenient detection of bacteria. By using the complex of the present invention, it becomes possible to diagnose early periodontal disease, which was difficult with conventional detection methods, and to detect even the small amount of bacteria remaining in the root area of ​​teeth that have been treated for cavities. [Explanation of Symbols]

[0067] 10 Carrier particles 11. Zeolite-like imidazolate structure (ZIF) 20 Platinum Monoatoms 21 Platinum Nanoparticles 30,70 antibodies 40 Nanozymes 41 Precursors 50 Bacteria 60 microplate wells 100 complex

Claims

1. A complex for detecting bacteria, Carbon-based material support particles, Platinum single atoms supported on the aforementioned carrier particles, A complex comprising an antibody immobilized on the carrier particle and specifically binding to the bacteria to be detected.

2. The composite according to claim 1, wherein the carrier particles are hollow bodies.

3. The composite according to claim 1 or 2, wherein the carrier particles include a carbide of a zeolite-like imidazole structure.

4. The composite according to claim 3, wherein the zeolite-like imidazole structure is ZIF-67.

5. The composite according to any one of claims 1 to 4, wherein the carrier particles contain nitrogen atoms.

6. The composite according to any one of claims 1 to 5, wherein the average particle size of the carrier particles is 200 nm to 300 nm.

7. The conjugate according to any one of claims 1 to 6, wherein the antibody is an anti-porphyromonas gingivalis antibody.

8. A method for producing a composite according to any one of claims 1 to 7, Platinum nanoparticles are supported on a particulate zeolite-like imidazole structure, The surface of the zeolite-like imidazole structure supporting the aforementioned platinum nanoparticles is covered with a layer containing poly(cyclotriphosphazene-co-4,4'-sulfonyldiphenol) to obtain a nanozyme precursor. The nanozyme precursor is calcined to obtain the nanozyme, A method for producing a complex, comprising immobilizing the antibody on the nanozyme.

9. A method for producing the composite according to claim 8, wherein the platinum single atom is formed from the platinum nanoparticles by calcining the nanozyme precursor.

10. A method for producing the composite according to claim 8 or 9, wherein the zeolite-like imidazole structure and the hollow carrier particles are formed from the nanozyme precursor by calcining the nanozyme precursor.

11. A method for detecting bacteria, comprising performing an immunological measurement method using the complex described in any one of claims 1 to 7.

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