JP2015042958A - Method for detecting microorganism to be detected - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel method for quickly and easily detecting a microorganism with high sensitivity.SOLUTION: A method for detecting a microorganism to be detected contained in a sample includes: an adhesion step for making a metal nano particle adhere to a microorganism to be detected contained in a sample; an evaluation step for irradiating the sample with light and evaluating a scattering characteristic of light corresponding to an adhesion condition of the metal nano particle to the microorganism to be detected; and a detection step for detecting the microorganism to be detected based on an evaluation result of the evaluation step.

Description

  The present invention relates to a detection method for detecting a microorganism to be detected.

  In recent years, interest in microbial detection has increased in the medical industry, food industry, agriculture, livestock, aquaculture, water treatment facilities, and the like. Contaminating microorganisms present in foods, pharmaceuticals, agricultural chemicals, and the like can greatly affect human health even though the amount is very small. Microbial contamination in hospitals and elderly care facilities is becoming a social problem. Furthermore, as seen in the increasing distribution and demand for various antibacterial products, there is an increasing interest in hygiene management in ordinary households. For example, in the case of a food processing factory, a bacteria test is performed by sampling a food to be shipped and a bacteria test in the environment in the factory. In the case of measurement by a culture method, 24 to 48 until a result is obtained. Since it takes about time and becomes a factor that increases the storage cost until shipping, a rapid detection method is required. In the agricultural field, for example, when the number of bacteria in a culture medium of hydroponics increases, the risk of disease increases. Since measures such as sterilization can be taken quickly by grasping the number of bacteria quickly, a rapid detection method is effective.

  Under these circumstances, the need for a technology that can easily detect microbial contamination has increased rapidly in recent years. Moreover, in the medical field, since it is necessary to quickly identify the pathogenic bacteria causing the infectious disease, a technique capable of detecting the pathogenic bacteria quickly and with high sensitivity is required. Examples of methods for detecting and identifying microorganisms include ELISA methods and Western blotting methods. These include, for example, an antigen-antibody reaction between an antibody (primary antibody) and a protein specific to a microorganism, and then a labeled secondary antibody is reacted with an antibody (primary antibody) to cause chemiluminescence of the secondary antibody or ATP. This is a method of detecting by monitoring the hydrolysis reaction.

  International Publication No. 2012/121229 (Patent Document 1) describes a microorganism detection sensor including a template of a microorganism to be detected, and describes that a change in weight according to a supplemented state of the microorganism is detected. ing.

  Hayden, SC et al., J. Amer. Chem. Soc. 2012, 134, 6920-6923 (Non-Patent Document 1) shows the state of aggregation of gold nanoparticles on the surface of Escherichia coli and Bacillus subtilis. It has been reported that it is confirmed by UV-visible spectrum.

International Publication No. 2012/121229

Hayden, S.C. et al., J. Amer. Chem. Soc. 2012, 134, 6920-6923

  However, none of the methods such as the ELISA method and the Western blotting method are methods for detecting the microorganism itself, and the ELISA method is not easy because it is necessary to prepare an antibody against a protein unique to the microorganism.

  An object of the present invention is to provide a novel microorganism detection method that can detect microorganisms quickly, simply, and with high sensitivity.

  The present invention relates to a detection method for detecting a microorganism to be detected contained in a sample, comprising an attachment step of attaching metal nanoparticles to the microorganism to be detected contained in the sample, and irradiating the sample with light to produce the microorganism to be detected. The evaluation process which evaluates the light scattering characteristic corresponding to the adhesion state of the metal nanoparticle, and the detection process which detects a to-be-detected microorganism based on the evaluation result in an evaluation process are provided.

  In the evaluation step, the light scattering property is preferably evaluated by at least one of observation of a dark field microscope image, measurement of transmitted light, and measurement of scattered light. For example, the scattering characteristics of light can be evaluated by measuring the disappearance spectrum based on the intensity of transmitted light or measuring the scattering spectrum based on the intensity of scattered light.

  In the attachment step, the metal nanoparticles may be attached in the form of a composite. Examples of the metal nanoparticles include gold nanoparticles. In the attachment step, probe molecules are bound to the metal nanoparticles, and the metal nanoparticles can be attached to the microorganism to be detected via the probe molecules. As the probe molecule, it is preferable to use a probe molecule that adheres to the microorganism to be detected by chemical interaction or chemical bonding with the surface portion of the microorganism to be detected. For example, i) a probe molecule that adheres to the surface of the microorganism to be detected by electrostatic interaction or hydrophobic interaction with the surface of the microorganism to be detected; and ii) is composed of a protein and binds to a sugar chain on the surface of the microorganism to be detected. A molecule that adheres to the microorganism to be detected, or iii) a probe molecule that consists of an antibody and adheres to the microorganism to be detected by reacting with an antigen on the surface of the microorganism to be detected can be used.

  In the attachment step, the metal nanoparticles are fixed to a polymer layer having a three-dimensional template complementary to the three-dimensional structure of the microorganism to be detected, and the metal nanoparticles adhere to the microorganism to be detected through the template. Form may be sufficient.

  The present invention also provides a detection method for detecting a microorganism to be detected contained in a sample, an attaching step for attaching metal nanoparticles to the microorganism to be detected contained in the sample, and an adding step for adding a fluorescent dye to the sample. , An evaluation process for irradiating the sample with excitation light and evaluating the fluorescence characteristics corresponding to the adhesion state of the metal nanoparticles to the detected microorganism, and a detection process for detecting the detected microorganism based on the evaluation result in the evaluation process And comprising. In the attachment step, the metal nanoparticles may be attached in the form of a composite.

  According to the method of the present invention, it is possible to detect microorganisms quickly and easily with high sensitivity.

It is a figure which shows typically the adhesion process of 1st Embodiment. It is a figure which shows an example of a metal nanoparticle composite_body | complex typically. It is a figure explaining an example of the adhesion method by the probe molecule | numerator of the adhesion process of 1st Embodiment. It is a figure explaining an example of the adhesion method by the casting_mold | template of the adhesion process of 1st Embodiment. It is a schematic diagram which shows an example of the system used in the evaluation process of 1st Embodiment. It is a figure which shows the TEM image of a gold nanoparticle (a), a copper oxide nanoparticle (b), and a gold nano raspberry (c). In Experimental example 1, it is a figure which shows the electron microscope image (a) at the time of using a 1st metal nanoparticle (negative Au NPs) and a dark field microscope image (b). In Experimental example 1, it is a figure which shows the electron microscope image (a) at the time of using a 2nd metal nanoparticle (positive Au NPs), and a dark field microscope image (b). In Experimental Example 1, a diagram illustrating an electron microscope image in the case of using the third metal nanoparticles (positive Cu ₂ O NPs) and (a) a dark-field microscopic image (b). In Experimental example 1, it is a figure which shows the electron microscope image (a) at the time of using a 1st metal nanoparticle composite_body | complex (positive Au nanoraspberry), and a dark field microscope image (b). In Experimental example 1, it is a figure which shows only a Pseudomonas aeruginosa, and shows a dark-field microscope image when not adding a metal nanoparticle or a metal nanoparticle composite_body | complex. In Experiment 1, showing the scattering spectrum in the case of using the second metal nanoparticles (positive Au NPs) or third metal nanoparticles (positive Cu ₂ O NPs). In Experimental example 1, it is a figure which shows the scattering spectrum of the sample using the 2nd metal nanoparticle (positive Au NPs) or the 1st metal nanoparticle complex (positive Au nanoraspberry), and the sample containing only Pseudomonas aeruginosa. is there. In Experimental example 2, it is a figure which shows the electron microscope image (a) of the samples 2-4, and the dark field microscope image (b). In Experimental Example 2, it is a figure which shows the disappearance spectrum of samples 1-6. It is a figure which shows typically the relationship between the density | concentration of Pseudomonas aeruginosa and the adhesion number of a metal nanoparticle. In Experimental example 3, it is a figure which shows the dark-field microscope image of the samples 8-13. In Experimental Example 3, it is a figure which shows the scattering spectrum of the samples 7-13. In Experimental example 3, it is a figure which shows the relationship of the scattering intensity of wavelength 600nm with respect to the ratio of a metal nanoparticle composite_body | complex. In Experimental example 4, it is a figure which shows the fluorescence spectrum of sample A, B, and C. FIG. In Experimental Example 4, it is a figure which shows the fluorescence spectrum of sample A and 14-21. In Experimental example 4, it is a figure which shows the relationship of the fluorescence intensity in wavelength 512nm with respect to content of a gold nanoparticle. In Experimental Example 4, it is a figure which shows the fluorescence spectrum of sample A2, 22-29. In Experimental example 4, it is a figure which shows the relationship of the fluorescence intensity in wavelength 512nm with respect to content of gold nano raspberry.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
<First Embodiment>
The first embodiment is a detection method for detecting a microorganism to be detected contained in a sample, an attaching step (St1) for attaching metal nanoparticles to the microorganism to be detected contained in the sample, and irradiating the sample with light. Then, an evaluation step (St2) for evaluating the light scattering characteristics corresponding to the adhesion state of the metal nanoparticles to the detection microorganism, and a detection step (St3) for detecting the detection microorganism based on the evaluation result in the evaluation step And comprising.

  In the evaluation step (St2), the light scattering property is preferably evaluated by at least one of observation of a dark field microscope image, measurement of transmitted light, and measurement of scattered light.

[Adhesion process]
FIG. 1 is a diagram schematically showing an adhesion process according to the first embodiment. This is a step of adhering the metal nanoparticles 1 to the microorganism 2 to be detected by mixing a solution containing the metal nanoparticles 1 and a solution containing the microorganism 2 to be detected.

(Detected microorganism)
The microorganism 2 to be detected is not particularly limited. For example, Escherichia genus of Escherichia coli, Pseudomonas genus such as Pseudomonas aeruginosa, Acinetobacter genus such as Acinetobacter calcoaceticus, Serratia genus, Klebsiella genus, Enterobacter genus, Citrobacter genus, Burkholderia genus, Sphingomonadase genus, Chromobacterium genus, Salmonella genus, Salmonella genus, Salmonella genus Bacteria, Legionella, Campylobacter, Yersinia, Proteus, Neisseria, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Corynebacterium, Listeria, Bacillus, Mycobacterium, Chlamydia, Rickettsia, Haemophilus Is exemplified. Examples of viruses include hepatitis A virus, adenovirus, rotavirus, and norovirus, candidia as fungi, and cryptosporidium as protozoa.

(Metal nanoparticles)
The metal nanoparticle 1 is a metal particle having a size on the order of nanometers. The nanometer order includes the range of 1 to several hundred nanometers, and the particle size is typically in the range of 1 nm to 100 nm. The metal nanoparticles are not particularly limited as long as surface plasmons are observed. The metal nanoparticle 1 in which surface plasmon is observed changes in light scattering characteristics in accordance with the state of attachment to the microorganism 2 to be detected. Therefore, the scattering characteristics can be evaluated (evaluation process), the adhesion state to the detected microorganism 2 can be detected based on the evaluation result, and the detected microorganism 2 can be detected (detection process).

  The metal nanoparticle 1 may be a nanoparticle made of a single metal or a nanoparticle made of a metal compound, and examples thereof include gold nanoparticles, silver nanoparticles, copper nanoparticles, and copper oxide nanoparticles. The In addition, as shown in FIG. 1, the metal nanoparticles 1 are attached to the microorganism 2 to be detected. In addition to the form in which each metal nanoparticle 1 is attached to the microorganism 2 to be detected, a plurality of metal nanoparticles are gathered. Thus, a complex (hereinafter also referred to as “metal nanoparticle complex”) is formed, and the metal nanoparticle complex adheres to the microorganism to be detected. The metal nanoparticle composite is not particularly limited as long as it is a form in which a plurality of metal nanoparticles are aggregated, and examples thereof include a form in which a plurality of metal nanoparticles are held on the surface of the polymer layer. .

  FIG. 2 is a diagram schematically illustrating an example of a metal nanoparticle composite. The metal nanoparticle composite 5 includes a plurality of metal nanoparticles 1 and a polymer layer 4 that holds the metal nanoparticles 1. For example, by mixing gold ions and aniline which is a reducing agent for gold ions, the gold ions are reduced to produce gold nanoparticles, and at the same time, the aniline is oxidized to produce aniline oligomers to form the polymer layer 4. A gold nanoparticle composite (hereinafter also referred to as “gold nanoraspberry”) 5 is formed. The diameter of the gold nanoraspberry is usually 50 to 150 nm.

(Adhesion method)
FIG. 3 is a schematic diagram for explaining an example of a method of attaching the metal nanoparticles 1 to the microorganism 2 to be detected in the first embodiment. First, the probe molecules 3 are bonded to the metal nanoparticles 1, and the metal nanoparticles are attached to the surface of the microorganism 2 to be detected via the probe molecules 3. The probe molecule 3 is attached to the detected microorganism 2 by chemical interaction or chemical bond with the surface portion of the detected microorganism 2. Examples of attachment by chemical interaction or chemical bond include attachment by electrostatic interaction, attachment by hydrophobic interaction, and a probe molecule consisting of a sugar chain on the surface of the microorganism 2 to be detected and a protein (lectin or the like). Examples include adhesion by binding, adhesion by antigen-antibody reaction between the antigen on the surface of the microorganism 2 to be detected and a probe molecule composed of an antibody.

  In FIG. 3, a sugar chain or an antigen present on the surface of the microorganism 2 to be detected and a protein such as a lectin or an antibody that specifically binds are used as the probe molecule 3 that binds to the metal nanoparticle 1 via the probe molecule 3. According to the method of attaching the metal nanoparticles 1 to the microorganism 2 to be detected, the microorganism 2 to be detected can be specifically detected even when a plurality of types of microorganisms are contained in the sample.

  FIG. 4 is a schematic diagram for explaining a method for attaching the metal nanoparticles 1 to the microorganism 2 to be detected, which is different from the method shown in FIG. In the method shown in FIG. 4, the metal nanoparticle composite 5 a in which the metal nanoparticles 1 are held by the polymer layer 4 is used. A template 7 having a three-dimensional structure complementary to the three-dimensional structure of the microorganism 2 to be detected is formed on the surface of the polymer layer 4 of the metal nanoparticle complex 5a, and the metal nanoparticle complex 5a is detected via the template 7 This is a method of attaching to 2. The three-dimensional structure of the microorganism 2 to be detected is not limited to the entire three-dimensional structure of the microorganism 2 to be detected, and may be a three-dimensional structure of a specific part of the microorganism 2 to be detected. In FIG. 4, a template 7 having a three-dimensional structure complementary to the three-dimensional structure of lipopolysaccharide (LPS) 8 which is a constituent component of the cell wall outer wall 9 of the microorganism 2 to be detected is formed. When the metal nanoparticle composite 5a attached to the microorganism 2 to be detected is irradiated with light, the scattered light 12 is observed. Therefore, the microorganism to be detected can be detected by measuring the scattering characteristics.

  The template 7 having a three-dimensional structure complementary to the three-dimensional structure of the microorganism 2 to be detected can be formed according to the method described in International Publication WO2012 / 121229. According to the method of attaching the metal nanoparticles 1 to the microorganism 2 to be detected through the template 7 having a three-dimensional structure complementary to the three-dimensional structure of the microorganism 2 to be detected as shown in FIG. Even if it is contained, the microorganism 2 to be detected can be specifically detected, which is preferable.

  In the embodiment of the present invention, a method for attaching the metal nanoparticles 1 to the detected microorganism 2 is selected according to the detected microorganism 2. For example, if the microorganism 2 to be detected is a microorganism having a negative or excessive charge on the entire surface or the surface, an attachment method by electrostatic interaction between the probe molecule 3 and the microorganism 2 to be detected can be selected. Examples of such a combination of metal nanoparticles 1 and probe molecules 3 include a combination of gold nanoparticles and aminoethanethiol. As probe molecules 3 used in combination with gold nanoparticles, molecules having an amino group and a thiol group are preferably used in addition to aminoethanethiol. Such molecules are attached by electrostatic interaction to microorganisms in which the thiol group is coordinated to the gold nanoparticle and the amino group has a positive charge, so that the whole or surface charge is in a negatively charged excess state. Because.

[Evaluation process]
In the evaluation step, light scattering characteristics corresponding to the state of adhesion of the metal nanoparticles to the microorganism to be detected are evaluated. When metal nanoparticles in which surface plasmons are observed adhere to the microorganism to be detected, surface plasmon resonance occurs, and when light is irradiated, scattered light is generated and light scattering characteristics change. Therefore, depending on the presence or absence of the microorganism to be detected or the concentration of the microorganism to be detected, the adhesion state of the metal nanoparticles to the microorganism to be detected changes, the degree of surface plasmon resonance changes, and the intensity of scattered light changes. Or the scattering characteristics of the light change, such as the peak of the scattered light shifting. In the evaluation step, the light scattering characteristics are evaluated. The light scattering property is preferably evaluated by at least one of observation of a dark field microscope image, measurement of an extinction spectrum based on the intensity of transmitted light, and measurement of a scattering spectrum based on the intensity of scattered light. The disappearance spectrum is the sum of the absorption spectrum and the scattering spectrum, and the scattering characteristics can be evaluated by analyzing the disappearance spectrum.

  FIG. 5 is a schematic diagram showing an example of a system used for measuring the disappearance spectrum or the scattering spectrum in the evaluation process. In the system 20, the sample 16 to be detected is irradiated with desired light from the light source 13, and the scattered light 9 from the sample 16 is detected by a detector 18 disposed in a direction of 90 ° with respect to the light irradiation direction. Measure the scattering spectrum. In the detector 19 arranged in the light irradiation direction, the disappearance spectrum can be measured by detecting the transmitted light. The system 20 may be any system in which at least one of the detector 18 that measures scattered light and the detector 19 that measures transmitted light is arranged. As the detectors 18 and 19, for example, a photodiode, a photomultiplier tube (PMT) or the like can be used. In the system 20, a light emitting diode (LED) is preferably used as the light source 13, and light emitted from the light source 13 is preferably visible light having a wavelength of 400 to 800 nm. The light scattering characteristics can be evaluated quickly and easily using a system as shown in FIG.

  The dark-field microscopic image is an image formed by scattered light, and the adhesion state of the metal nanoparticles to the microorganism to be detected can be directly and clearly confirmed from the observation of the image. The observation with the dark field microscope has an advantage that it can be observed quickly and easily as compared with the observation with the electron microscope. In addition, since the scattering intensity increases up to a certain adhesion amount according to the adhesion amount of the metal nanoparticles to each microorganism to be detected in the sample, the observed dark field microscopic image is also clear.

[Detection process]
In the detection step, the microorganism to be detected is detected based on the evaluation result of the evaluation step. Based on the evaluation result of the evaluation process, the presence or absence of the detected microorganism can be detected by checking in advance how the scattering characteristics change depending on the presence or absence of the detected microorganism and the concentration of the detected microorganism. The concentration of the microorganism to be detected can be detected. In addition, according to the observation of the dark field microscopic image, the presence and concentration of the microorganism to be detected can be directly measured.

<Second Embodiment>
The second embodiment is a detection method for detecting a microorganism to be detected contained in a sample, an attaching step (St11) for attaching metal nanoparticles to the microorganism to be detected contained in the sample, and a fluorescent dye on the sample. An addition step (St12) to be added, an evaluation step (St13) in which the sample is irradiated with excitation light and the fluorescence characteristics corresponding to the adhesion state of the metal nanoparticles to the microorganism to be detected are evaluated, and the evaluation results in the evaluation step And a detection step (St14) for detecting the detected microorganism.

  Since the adhesion process (St11) of this embodiment is the same as the adhesion process (St1) of the first embodiment, the description thereof is omitted. In the adding step (St12), a fluorescent dye that emits fluorescence is added. The fluorescence intensity of the fluorescent dye is enhanced by surface plasmons appearing on the metal nanoparticles present in the sample. Differences in fluorescence intensity occur depending on the presence and absence of metal nanoparticles and the amount of metal nanoparticles. Therefore, in the evaluation step (St13), in order to evaluate only the fluorescence characteristics derived from the metal nanoparticles attached to the detected microorganism, the metal nanoparticles not attached to the detected microorganism may be removed in advance. preferable. In the evaluation step (St13), the sample is irradiated with excitation light to evaluate the fluorescence characteristics. Examples of the evaluation of the fluorescence characteristics include measurement of a fluorescence spectrum and observation of a fluorescence microscope image. In the detection step (St14), the microorganism to be detected is detected based on the evaluation of the fluorescence characteristics.

  Examples of fluorescent dyes that can be used in this embodiment include fluorescein, rhodamine, propidium iodide, calcein, acridine orange, and the like. The wavelength of the excitation light is selected according to the fluorescent dye to be used. When fluorescein is used as the fluorescent dye, for example, light having a wavelength of 490 nm can be selected as the excitation light. In the present embodiment, the fluorescence characteristics can be evaluated quickly and easily.

[Experimental Example 1: Selection of metal nanoparticles]
In Experimental Example 1, when Pseudomonas aeruginosa is used as a microorganism to be detected, a metal nanoparticle or a metal nanoparticle complex that can be used for the detection method of the present invention or is suitable for use is used. This was confirmed by observation of an electron microscope image, observation of a dark field microscope image, and measurement of a scattering spectrum.

The metal nanoparticles or metal nanoparticle composites shown below in (1) to (4), to which probe molecules were bound, were prepared.
(1) First metal nanoparticles: gold nanoparticles (sodium citrate bonded as a probe molecule) (negative Au NPs),
(2) Second metal nanoparticle: gold nanoparticle (positive Au NPs) in which aminoethanethiol is bound as a probe molecule,
(3) Third metal nanoparticles: copper oxide nanoparticles (positive Cu ₂ O NPs) bonded with cetyltrimethylammonium bromide as a probe molecule,
(4) First metal nanoparticle complex: gold nanoraspberry (positive Au nanoraspberry) in which aniline is bound as a probe molecule.

  FIG. 6A shows a transmission electron microscope image (TEM image) of gold nanoparticles, FIG. 6B shows a TEM image of copper oxide nanoparticles, and FIG. 6C shows a TEM image of gold nano raspberries. Show. In this experimental example, gold nanoparticles and gold nanoraspberries were prepared as gold nanodispersion and gold nanoraspberry dispersion prepared as follows.

(Preparation of first metal nanoparticle dispersion)
750 μL of 1% tetrachloroauric acid (III) acid tetrahydrate aqueous solution and 563 μL of 2% sodium citrate aqueous solution were added to 25 mL of ultrapure water, and the mixture was stirred for 30 minutes at 80 ° C. Was prepared. The average particle diameter of the first metal nanoparticles in the obtained dispersion was 30 nm, and the zeta potential was −30 mV.

(Preparation of second metal nanoparticle dispersion)
A second metal nanoparticle dispersion was prepared according to the method for preparing the first metal nanoparticle dispersion described above, except that an aminoethanethiol solution was used instead of the 2% aqueous sodium citrate solution. The average particle diameter of the second metal nanoparticles in the obtained dispersion was 30 nm, and the zeta potential was +40 mV.

(Preparation of third metal nanoparticle dispersion)
320 μL of 1.6% aqueous copper sulfate solution was added to 18 mL of ultrapure water, and nitrogen bubbling was performed for 30 minutes. To this solution, 1 mL of 10% cetyltrimethylammonium bromide and 500 μL of 4.2% sodium borohydride were added and stirred for 10 hours. Furthermore, the 3rd metal nanoparticle dispersion liquid was prepared by stirring in air for 10 hours. The average particle diameter of the third metal nanoparticles in the obtained dispersion was 45 nm, and the zeta potential was +30 mV.

(Preparation of gold nano raspberry dispersion)
To 50 mL of ultrapure water, 152 μL of 1% tetrachloroauric (III) acid tetrahydrate aqueous solution was added and stirred at 80 ° C., 1 mL of 0.1 M aniline aqueous solution was added, and the mixture was stirred at 80 ° C. for 30 minutes. 50 mL of the reaction solution was centrifuged at 8500 rpm, 5 ° C. for 30 minutes, and the precipitate was developed in 3 mL of pH 7 phosphate buffer and sonicated for 10 minutes to be uniformly dispersed. This washing operation was repeated three times to prepare a gold nanoraspberry dispersion. The average particle diameter of the gold nanoraspberry in the obtained gold nanoraspberry dispersion was 80 nm, and the zeta potential was +20 mV.

  As shown in FIG. 3, the solution containing the metal nanoparticle or metal nanoparticle complex bound with the probe molecule and the solution containing Pseudomonas aeruginosa are mixed and stirred, and then centrifuged at room temperature (6200 rpm). As a result, the metal nanoparticles not attached to the microorganisms were removed. The adhesion state of metal nanoparticles or metal nanoparticle composites to Pseudomonas aeruginosa was confirmed by an electron microscope image and a dark field microscope image.

FIG. 7 is a diagram showing an electron microscope image (a) and a dark field microscope image (b) when the first metal nanoparticles (negative Au NPs) having a particle size of 30 nm are used. FIG. 8 is a diagram showing an electron microscope image (a) and a dark field microscope image (b) when second metal nanoparticles (positive Au NPs) having a particle size of 30 nm are used. FIG. 9 is a diagram showing an electron microscope image (a) and a dark field microscope image (b) in the case of using third metal nanoparticles (positive Cu ₂ O NPs) having a particle diameter of 45 nm. FIG. 10 is a diagram showing an electron microscope image (a) and a dark field microscope image (b) when the first metal nanoparticle composite (positive Au nanoraspberry) is used. FIG. 11 is a diagram showing a dark field microscopic image in the case of containing only Pseudomonas aeruginosa and not adding metal nanoparticles or metal nanoparticle composites. The dark-field microscope images shown in FIGS. 7 to 11 are all taken under the same conditions such as the exposure time (400 ms).

8-10, the second metal nanoparticles (positive Au NPs), the third metal nanoparticles (positive Cu ₂ O NPs), and the first metal nanoparticle complex (positive Au nanoraspberry) are found in Pseudomonas aeruginosa. It was confirmed that it adheres and can be used for detection of Pseudomonas aeruginosa. This is because the surface of Pseudomonas aeruginosa is negatively charged by phosphate groups, while these probe molecules are positively charged functional groups (amino group of aminoethanethiol, amino group of aniline oligomer, This is because it has an imino group and the like and adheres by electrostatic interaction with Pseudomonas aeruginosa. These probe molecules have functional groups that coordinate and bond to the metal nanoparticles (thiol group of aminoethanethiol, amino group of cetyltrimethylammonium bromide, amino group of aniline oligomer, imino group, etc.). It is coordinate-bonded to the metal nanoparticles via.

FIG. 12 shows a scattering spectrum when the second metal nanoparticles (positive Au NPs) are used and when the third metal nanoparticles (positive Cu ₂ O NPs) are used. As can be seen from FIG. 12, the peak wavelength of the scattering spectrum when the second metal nanoparticles (positive Au NPs) are used is different from that when the third metal nanoparticles (positive Cu ₂ O NPs) are used. It is around 650 nm and around 550 nm. Therefore, the dark field microscope image when the second metal nanoparticles (positive Au NPs) are used is red, and the dark field microscope image when the third metal nanoparticles (positive Cu ₂ O NPs) are used is It is blue. Therefore, by using multiple metal nanoparticles or metal nanoparticle composites with different peak wavelengths in the scattering spectrum, each is designed to adhere to a different type of microorganism to be detected, and the scattering characteristics are evaluated. A plurality of types of microorganisms to be detected can also be specified.

  FIG. 13 shows the case where the second metal nanoparticles (positive Au NPs) are used, the case where the first metal nanoparticle composite (positive Au nanoraspberry) is used, and the metal nanoparticle or metal nanoparticle composite. The scattering spectrum of the sample containing only Pseudomonas aeruginosa without mixing is shown. From the result shown in FIG. 13, when the first metal nanoparticle composite (positive Au nanoraspberry) is used, the intensity of the scattered light is higher than that when the second metal nanoparticle (positive Au NPs) is used. It can be seen that it is much larger. This is clear even when the dark-field microscope images shown in FIGS. 8B and 10B are compared. Therefore, it can be seen that in the embodiment of the present invention, it is preferable to use the first metal nanoparticle composite (positive Au nanoraspberry) from the viewpoint that the intensity of scattered light is large and detection is easy.

  Moreover, it can be seen from the results shown in FIG. 13 that almost no scattered light is observed from a sample containing only Pseudomonas aeruginosa. Therefore, when a sample containing Pseudomonas aeruginosa is mixed with metal nanoparticles or metal nanoparticle complexes that can adhere to Pseudomonas aeruginosa (attachment process), and a scattering spectrum having a peak at a specific wavelength is measured (evaluation Step), it can be confirmed that the target microorganism is present in the sample (detection step). It is also possible to detect the concentration of the microorganism to be detected based on the intensity of the peak of the scattering spectrum.

[Experimental example 2: Change in mixing ratio of microorganism to be detected and metal nanoparticles, constant concentration of metal nanoparticles]
In Experimental Example 2, Pseudomonas aeruginosa was used as the microorganism to be detected, and the second metal nanoparticles (positive Au NPs) used in Experimental Example 1 were used as the metal nanoparticles. Were changed to observe an electron microscope image, a dark field microscope image, and a disappearance spectrum.
The following samples 1 to 6 were prepared. The content of the second metal nanoparticles (positive Au NPs) in Samples 2 to 6 was 0.1 mg.
(1) Sample 1: a sample containing Pseudomonas aeruginosa at a concentration of 3.1 × 10 ⁸ CFUmL ⁻¹ and without adding metal nanoparticles,
(2) Sample 2: a sample in which second metal nanoparticles (positive Au NPs) are added to a solution containing Pseudomonas aeruginosa at a concentration of 4.6 × 10 ⁷ CFUmL ⁻¹ ,
(3) Sample 3: Sample obtained by adding second metal nanoparticles (positive Au NPs) to a solution containing Pseudomonas aeruginosa at a concentration of 9.2 × 10 ⁷ CFUmL ⁻¹ ,
(4) Sample 4: a sample in which second metal nanoparticles (positive Au NPs) are added to a solution containing Pseudomonas aeruginosa at a concentration of 1.5 × 10 ⁸ CFUmL ⁻¹ ,
(5) Sample 5: a sample obtained by adding second metal nanoparticles (positive Au NPs) to a solution containing Pseudomonas aeruginosa at a concentration of 3.1 × 10 ⁸ CFUmL ⁻¹ ,
(6) Sample 6: A sample that contains only the second metal nanoparticles (positive Au NPs) and does not contain the microorganism to be detected.

14A shows electron microscope images of Samples 2 to 4, and FIG. 14B shows dark field microscope images of Samples 2 to 4. FIG. 15 shows the disappearance spectra of Samples 1-6. From the dark field microscopic image shown in FIG. 14 (b), it can be seen that the brighter the dark field microscopic image is observed, the lower the concentration of Pseudomonas aeruginosa. In sample 5, there was almost no difference in comparison with the dark field microscopic image (FIG. 11) of sample 1 (only Pseudomonas aeruginosa). Since the concentration of the metal nanoparticles is constant, as shown in FIG. 16, the number of metal nanoparticles 1 attached to Pseudomonas aeruginosa 2 increases as the concentration of Pseudomonas aeruginosa 2 decreases. It is understood that it is due to.
From the disappearance spectrum shown in FIG. 15, samples 1 (Pseudomonas aeruginosa only) and sample 6 (metal nanoparticles only) have almost no disappearance light in the vicinity of a wavelength of 600 to 700 nm. It was observed that the intensity of the lost light increased with increasing concentration of Pseudomonas aeruginosa. This is considered to be caused by the fact that scattered light is generated in the vicinity of a wavelength of 600 to 700 nm due to adhesion of metal nanoparticles to the surface of Pseudomonas aeruginosa. From the above, it can be seen that the presence or absence of the microorganism to be detected can be detected by measuring the disappearance spectrum, and the concentration of the microorganism to be detected can be detected based on the intensity of the specific wavelength of the disappearance spectrum. .

[Experimental Example 3: Change in mixing ratio of microorganism to be detected and metal nanoparticle composite]
In Experimental Example 3, Pseudomonas aeruginosa was used as the microorganism to be detected, and the first metal nanoparticle complex (positive Au nanoraspberry) used in Experimental Example 1 was used as the metal nanoparticle. The density of the nanoparticle complex (positive Au nanoraspberry) was varied to observe a dark field microscope image and to measure the scattering spectrum.

  As shown in FIG. 3, a solution containing a metal nanoparticle complex (positive Au nanoraspberry) and a solution containing Pseudomonas aeruginosa are mixed and stirred, and then centrifuged at room temperature (6200 rpm) to give microorganisms. Unattached metal nanoparticles were removed. The adhesion state of metal nanoparticles or metal nanoparticle composites to Pseudomonas aeruginosa was confirmed by an electron microscope image and a dark field microscope image.

The following samples 7 to 13 were prepared. Samples 7 to 13 contain Pseudomonas aeruginosa at a concentration of 1.5 × 10 ⁸ CFUmL ^{−1 and} the concentration of the first metal nanoparticle complex (positive Au nanoraspberry) is 76 μg. It is described in terms of this ratio assuming that it is contained at a ratio of 1.0 to Pseudomonas aeruginosa.
(1) Sample 7: a sample containing Pseudomonas aeruginosa at a concentration of 7.5 × 10 ⁸ CFUmL ⁻¹ and containing a first metal nanoparticle complex (positive Au nanoraspberry) at a ratio of 0.2,
(2) Sample 8: a sample containing Pseudomonas aeruginosa at a concentration of 3.8 × 10 ⁸ CFUmL ⁻¹ and containing a first metal nanoparticle complex (positive Au nanoraspberry) at a ratio of 0.4,
(3) Sample 9: a sample containing Pseudomonas aeruginosa at a concentration of 1.5 × 10 ⁸ CFUmL ⁻¹ and containing a first metal nanoparticle complex (positive Au nanoraspberry) at a ratio of 1.0,
(4) Sample 10: a sample containing Pseudomonas aeruginosa at a concentration of 6.0 × 10 ⁸ CFUmL ⁻¹ and containing a first metal nanoparticle complex (positive Au nanoraspberry) at a ratio of 1.25,
(5) Sample 11: a sample containing Pseudomonas aeruginosa at a concentration of 3.8 × 10 ⁸ CFUmL ⁻¹ and containing a first metal nanoparticle complex (positive Au nanoraspberry) at a ratio of 2.0,
(6) Sample 12: a sample containing Pseudomonas aeruginosa at a concentration of 3.0 × 10 ⁸ CFUmL ⁻¹ and containing a first metal nanoparticle complex (positive Au nanoraspberry) at a ratio of 2.5,
(7) Sample 13: A sample containing Pseudomonas aeruginosa at a concentration of 1.5 × 10 ⁸ CFUmL ⁻¹ and containing a first metal nanoparticle complex (positive Au nanoraspberry) at a ratio of 5.0.

  FIG. 17 shows dark field microscopic images of Samples 8 to 13, FIG. 18 shows scattering spectra of Samples 7 to 13, and FIG. 19 shows the ratio of the metal nanoparticle composite prepared based on the scattering spectrum of FIG. It is a figure which shows the relationship of the scattering intensity of wavelength 600nm. In the dark field microscope image shown in FIG. 17, the exposure time was 100 ms, and all other imaging conditions were the same. From the dark field microscopic image shown in FIG. 17, in the samples (samples 8 to 11) in which the ratio of the metal nanoparticle composite is 2.0, the darker dark field microscopic image is observed as the metal nanoparticle composite concentration is higher. I found out that It is understood that this is because the number of metal nanoparticle complexes attached to Pseudomonas aeruginosa increases as the concentration of the metal nanoparticle complex increases with respect to Pseudomonas aeruginosa.

  From the scattering spectrum shown in FIG. 18, in the case of sample 7 (only Pseudomonas aeruginosa), the scattered light is not measured, and in samples 8 to 13, the higher the concentration of the metal nanoparticle complex, the higher the peak intensity of the scattering spectrum. Was observed, but the peak intensities of Samples 12 and 13 were almost the same. Further, from the results shown in FIG. 19, it was found that the intensity of the scattered light does not increase even when the ratio of the metal nanoparticle composite is 3.0 or more. Therefore, when the detection is performed using the first metal nanoparticle composite (positive Au nanoraspberry), the first metal nanoparticle composite (positive Au nanoraspberry) is compared to the predicted concentration of the microorganism to be detected. It has been found that it is preferable to add in the range where the ratio is 1.25 to 2.5 and to evaluate the scattering characteristics.

[Experimental Example 4]
In Experimental Example 4, measurement was performed to confirm the fluorescence enhancement effect of gold nanoparticles and gold nanoraspberries. First, fluorescence measurement was performed at an excitation wavelength of 490 nm for a 1 μM fluorescein aqueous solution. Next, a gold nanoparticle dispersion prepared as follows was dropped into a 1 μM fluorescein aqueous solution, and fluorescence was measured at an excitation wavelength of 490 nm. Further, the gold nanoraspberry dispersion prepared in Experimental Example 1 was dropped into a 1 μM fluorescein aqueous solution, and fluorescence measurement was performed at an excitation wavelength of 490 nm.

(Preparation of gold nanoparticle dispersion)
750 μL of 1% tetrachloroauric (III) acid tetrahydrate aqueous solution and 0.1 M aqueous potassium carbonate solution (0.5 mL) were added to 25 mL of ultrapure water, and the mixture was stirred at 4 ° C. Thereafter, 0.5 mL of 13 mM sodium borohydride aqueous solution was added 5 times (total amount 2.5 mL) and stirred for 20 minutes to prepare a gold nanoparticle dispersion. The average particle diameter of the gold nanoparticles in the obtained gold nanoparticle dispersion was 5 nm, and the zeta potential was −15 mV.

  FIG. 20 shows the fluorescence spectrum of a 1 μM fluorescein aqueous solution (sample A), the fluorescence spectrum of an aqueous solution (sample B) containing 0.970 μg of gold nanoparticles by dropping a gold nanoparticle dispersion into a 1 μM fluorescein aqueous solution, and 1 μM. Shows a fluorescence spectrum of an aqueous solution (sample C) containing 0.727 μg of gold nanoraspberry by dropping an aqueous solution of gold nanoraspberry into an aqueous solution of fluorescein.

  As shown in FIG. 20, the 1 μM fluorescein aqueous solution (sample A) exhibited a spectrum having a maximum value at 512 nm. The intensity of this fluorescent peak was 95. The peak intensity of the aqueous solution (sample B) containing 0.970 μg of gold nanoparticles was 154, which was 1.5 times higher than the peak intensity of sample A. The peak intensity of the aqueous solution (sample C) containing 0.727 μg of gold nanoraspberry was 1000 or more, which was enhanced by 10 times or more compared to the peak intensity of sample A.

FIG. 21 shows a fluorescence spectrum of a 1 μM fluorescein aqueous solution (sample A) and a fluorescence spectrum of samples 14 to 21 in which a gold nanoparticle dispersion is dropped into a 1 μM fluorescein aqueous solution and contains gold nanoparticles in the following amounts.
(1) Sample 14: content of gold nanoparticles 0.194 μg,
(2) Sample 15: Gold nanoparticle content of 0.582 μg,
(3) Sample 16: content of gold nanoparticles 0.970 μg,
(4) Sample 17: Gold nanoparticle content 1.94 μg,
(5) Sample 18: Gold nanoparticle content 5.82 μg,
(6) Sample 19: Content of gold nanoparticles 9.70 μg,
(7) Sample 20: Gold nanoparticle content 19.4 μg,
(8) Sample 21: Gold nanoparticle content 32.2 μg.

  FIG. 22 is a diagram showing the relationship of the fluorescence intensity at a wavelength of 512 nm with respect to the content of gold nanoparticles, created based on the fluorescence spectrum of FIG. 21 and 22, the fluorescence intensity at a wavelength of 512 nm increased corresponding to the increase in the gold nanoparticle content until the gold nanoparticle content reached 9.70 μg (sample 19). The peak intensity decreased abruptly when the value was increased (9.70 to 32.2 μg), and the sample 21 was smaller than the fluorescein alone. This is understood to be due to quenching of the electron transfer of the fluorescent dye adjacent to the gold nanoparticle while the fluorescence enhancement by the enhanced electric field on the gold nanoparticle surface. Therefore, when detecting the microorganism to be detected according to this experimental example, it is preferable to select the concentration of the gold nanoparticle and the concentration of the fluorescent dye according to the expected concentration of the microorganism to be detected.

Next, since the fluorescence intensity exceeded the limit of the apparatus when gold nano raspberry was added in FIG. 20 (intensity 1000 or more), a 0.1 μM fluorescein aqueous solution (sample A2) in which the concentration of fluorescein was diluted 10-fold from sample A Was prepared, and the gold nanoraspberry dispersion prepared in Experimental Example 1 was added dropwise thereto, and fluorescence was measured at an excitation wavelength of 490 nm. FIG. 23 shows the fluorescence spectrum of sample A2 and the fluorescence spectra of samples 22 to 29 in which a gold nanoraspberry dispersion was dropped into a 0.1 μM fluorescein aqueous solution and the following amount of gold nanoraspberry was contained.
(1) Sample 22: Gold nano raspberry content 0.0727 μg,
(2) Sample 23: content of gold nano raspberry 0.218 μg,
(3) Sample 24: Gold nano raspberry content 0.364 μg,
(4) Sample 25: content of gold nano raspberry 0.727 μg,
(5) Sample 26: Gold nano raspberry content 2.18 μg,
(6) Sample 27: Gold nano raspberry content 3.64 μg,
(7) Sample 28: Gold nano raspberry content 7.27 μg,
(8) Sample 29: Gold nano raspberry content 12.1 μg.

  FIG. 24 is a diagram showing the relationship of the fluorescence intensity at a wavelength of 512 nm with respect to the content of gold nanoraspberry created based on the fluorescence spectrum of FIG. According to FIGS. 23 and 24, the fluorescence intensity at a wavelength of 512 nm increased corresponding to the increase in the content of gold nanoraspberry until the content of gold nanoraspberry reached 3.64 μg (sample 27). The peak intensity showed a substantially constant intensity even when the value was increased (3.64 to 12.1 μg). This is understood to be based on the fact that gold nanoparticles in gold nano raspberry have a structure assembled with aniline oligomers as linkers. That is, it is understood that quenching is suppressed because fluorescein is captured by the aniline oligomer and does not directly contact the gold nanoparticles. When detecting the microorganism to be detected according to this experimental example, it is preferable to select the concentration of the gold nanoraspberry and the concentration of the fluorescent dye according to the expected concentration of the microorganism to be detected.

  Considering the above results, it can be understood that the peak intensity in sample B was enhanced because the enhanced electric field (surface plasmon) on the surface of the gold nanoparticles enhanced the fluorescence of the nearby fluorescent dye. . In Sample C, the peak intensity was enhanced in the gold nanoraspberry having a structure in which gold nanoparticles are aggregated to form a hot spot by coupling the surface-enhanced electric field in adjacent gold nanoparticles, This hot spot is understood to be due to the further enhancement effect.

  From the result of Experimental Example 4, first, metal nanoparticles or metal nanoparticle composites are attached to the microorganisms to be detected included in the sample in the attachment step, and then metal nanoparticles or metal nanoparticle composites that are not attached to the microorganisms to be detected. It was found that the microorganism to be detected can be detected based on the evaluation result by removing the body, adding a fluorescent dye, and evaluating the fluorescence characteristics of the sample in the evaluation step. Alternatively, the metal nanoparticle complex holding the fluorescent dye in advance is attached to the microorganism to be detected contained in the sample, and the fluorescence characteristics of the sample are evaluated in the evaluation process, so that the microorganism to be detected is based on the evaluation result. It was found that can be detected. This is because the peak intensity of the fluorescence spectrum is enhanced when the metal nanoparticle complex attached to the microorganism to be detected exists.

  1 metal nanoparticle, 2 microorganism to be detected, 3 probe molecule, 4 polymer layer, 5 metal nanoparticle complex (gold nanoraspberry), 7 template, 8 lipopolysaccharide, 9,12 scattered light, 13 light source, 16 sample, 18, 19 Detector.

Claims (12)

A detection method for detecting a microorganism to be detected contained in a sample,
An attachment step of attaching metal nanoparticles to the microorganism to be detected contained in the sample;
An evaluation step of irradiating the sample with light to evaluate the light scattering characteristics corresponding to the state of attachment of the metal nanoparticles to the microorganism to be detected;
A detection step of detecting the microorganism to be detected based on an evaluation result in the evaluation step.   2. The detection method according to claim 1, wherein in the evaluation step, the light scattering characteristic is evaluated by at least one of observation of a dark field microscope image, measurement of transmitted light, and measurement of scattered light.   The detection method according to claim 2, wherein in the evaluation step, the light scattering characteristic is evaluated by measuring a disappearance spectrum based on an intensity of the transmitted light.   The detection method according to claim 2, wherein in the evaluation step, the light scattering characteristic is evaluated by measuring a scattering spectrum based on an intensity of the scattered light.   5. The detection method according to claim 1, wherein in the attaching step, the metal nanoparticles are attached in the form of a complex.   The detection method according to claim 1, wherein the metal nanoparticles are gold nanoparticles.   The said adhesion | attachment process WHEREIN: The probe molecule is couple | bonded with the said metal nanoparticle, and the said metal nanoparticle adheres to the said to-be-detected microorganisms via the said probe molecule | numerator. Detection method.   The detection method according to claim 7, wherein the probe molecule is attached to the detected microorganism by chemical interaction or chemical bonding with a surface site of the detected microorganism.   The probe molecule is made of a protein that adheres to the detected microorganism by electrostatic interaction or hydrophobic interaction with a surface site of the detected microorganism, and binds to a sugar chain on the surface of the detected microorganism. The detection method according to claim 8, wherein the detection method adheres to the microorganism to be detected, or consists of an antibody and adheres to the microorganism to be detected by an antigen-antibody reaction with an antigen on the surface of the microorganism to be detected.   In the attaching step, the metal nanoparticles are fixed to a polymer layer having a template having a three-dimensional structure complementary to the three-dimensional structure of the microorganism to be detected. The detection method according to claim 1, which adheres to a detection microorganism. A detection method for detecting a microorganism to be detected contained in a sample,
An attachment step of attaching metal nanoparticles to the microorganism to be detected contained in the sample;
An addition step of adding a fluorescent dye to the sample;
An evaluation step of irradiating the sample with excitation light and evaluating fluorescence characteristics corresponding to the state of attachment of the metal nanoparticles to the microorganism to be detected;
A detection step of detecting the microorganism to be detected based on an evaluation result in the evaluation step.   The detection method according to claim 11, wherein in the attaching step, the metal nanoparticles are attached in the form of a complex.