JP2014178151A - Microorganism detection method and device using microsphere resonance sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of determining a microorganism from a whispering gallery mode (hereinafter abbreviated as WGM), an inexpensive sensor, and a measuring device.SOLUTION: Provided is an inexpensive microorganism detection device for rapidly and conveniently detecting a target microorganism, that uses a method of preparing a single microparticle in which antibodies are immobilized for specifically detecting a target microorganism existing in a solution and thereby detecting a bonding target microorganism using a split change of a WGM, and that further uses a spectroscope which includes an object lens on which evanescent light is made incident and an object lens capable of detecting scattered light enclosed in the single microparticle and which has resolution power to detect one or more WGM peak signals, and a sensor encapsulated by a glass cover.

Description

  The present invention relates to microbial testing in the field of food testing, and more particularly to a rapid and sensitive detection apparatus and method based on whispering gallery mode (WGM) using a single microparticle.

  Accidents that threaten food safety and security, such as food poisoning, for the food industry will lead to the loss of corporate brand and trust, and so the hygiene management system should be improved.

  In food inspection, microorganisms need to be tested simply, accurately, and quickly, but the conventional culture method, which is a conventional inspection method, requires more than 24 hours to incubate and judge, and a skilled person Work is required. Therefore, it has been desired to develop a sensor capable of simple and rapid high-sensitivity detection, and a detection method and apparatus optimal for them.

  In recent years, research and development using immunochromatography, ELISA, DNA testing, PCR, immunomagnetic bead methods, etc., have been promoted as rapid testing methods. Among them, immunological testing using antigen-antibody reaction is unique. Therefore, it is expected to be developed as a rapid detection method (see, for example, Patent Documents 1 and 2) and is being developed.

  Patent Document 1 discloses a high-sensitivity rapid detection method in which a target microorganism is separated and collected using a target microorganism-specific antibody, ATP of the target microorganism is amplified, and the amplified ATP is measured.

  However, these methods have insufficient detection sensitivity and determination accuracy, so that enrichment culture is required, and it takes 8 hours or more to obtain a general test result. Therefore, for food manufacturers, the shipment waits for the inspection result, or the shipment has a risk of self-collection.

  Patent Document 2 discloses a detection method in which a microorganism is captured using a capturing body on which an antibody that binds to a constituent component of the microorganism is immobilized, and DNA extracted from the captured and washed microorganism is amplified by a PCR method. It has been published.

  However, these methods also require advanced techniques such as extraction and amplification, and it is difficult to obtain test results easily and inexpensively.

  Therefore, a sensor capable of simple and quick high-sensitivity detection without using an expensive device or sensor, and a detection technique and device optimal for them have not yet been provided.

  As a technique for solving such a problem, a biosensor using an optical mode in which probe light is incident and circulated in a single resonator is provided. This optical resonance mode and resonance are called whispering gallery mode (hereinafter abbreviated as WGM) or form-dependent resonance (MDR), and the light incident on the probe light is totally reflected and confined at the interface of the resonator. Occurs when.

  Since WGM is very sensitive to surface conditions, a method has been proposed in which a binding partner that binds to a target analyte is immobilized on the surface of a microspheroid and a profile peak of WGM is detected (for example, a patent) Reference 3-4). Biosensors have also been proposed that increase the proportion of detected signal light by including an optical microresonator coupled to a waveguide before the analyte solution flows too much on the detector surface (eg, patent literature). 5).

  However, in order to detect the wavelength shift of these WGM profile peaks, a detector requiring high resolution is required, and there is also a problem that determination of measurement error and wavelength shift cannot be performed because stray light at the time of measurement is easily picked up. . Therefore, it is desired to develop a quick and simple measurement technique and measurement device without using an expensive measurement device.

JP 2006-81506 A JP 2007-97551 A JP 2012-137490 A Special table 2012-509070 gazette Special table 2008-513776

  An object of the present invention is to solve the above-described conventional problems, and to provide a method for easily determining microorganisms from the split change of the spectrum peak of WGM, and an inexpensive sensor and measurement apparatus without using an expensive measurement apparatus. And

  In order to achieve such an object, the present invention firstly binds and binds a target microorganism to a single microparticle on which an antibody that specifically detects the target microorganism present in a solution is immobilized. A microorganism detection method using a microsphere resonance sensor, which detects a target microorganism by a split change of a peak corresponding to WGM. The single microparticle of the present invention is characterized in that it has a diameter satisfying a condition that resonates at a wavelength that coincides with the immobilization of an antibody and the binding of a target microorganism, and is higher than the refractive index of the medium. By having a diameter of -10 μm, a highly sensitive WGM peak can be detected.

  In addition, the present invention secondly, an excitation objective lens capable of entering evanescent light condensed to a diameter smaller than the diameter of a single microparticle into a single microparticle sealed with a cover glass, and the single microparticle confined. Low cost to detect target microorganisms quickly and easily by using a spectroscope that includes a detection objective lens that can detect scattered light, has dark field illumination, and has a resolution that can detect one or more WGM peak signals It becomes possible to produce a microorganism detection apparatus using a microsphere resonance sensor.

  The microorganism detection method using the microsphere resonance sensor according to the present invention amplifies the sensitivity with a single microparticle, so there is no need to use a culture method in the inspection process, and further, the split change of the peak signal of WGM Therefore, microorganisms can be easily determined. That is, it becomes possible to determine the target microorganism with only a single fine particle, and by using a light source having a wavelength corresponding to the peak signal, it becomes possible to develop a disposable and inexpensive sensor and a small device.

  In addition, it is possible to perform simple, quick, and inexpensive test determination in the food field and other fields that require microbiological testing such as the environment and medical treatment.

Schematic which shows the single microparticle which fix | immobilized the antibody which detects the target microorganism. The figure which shows the observation of the polystyrene fine particle in a solution. The figure which shows the split signal of WGM at the time of couple | bonding with the enzyme which the target microorganism produced. Schematic which shows the WGM light detection part of this invention. It is a figure which shows the Example of a microorganisms detection apparatus.

  In the present invention, the microorganism to be detected is the coliform group, and a microorganism detection method for rapidly and accurately detecting β-galactosidase, an enzyme produced by the coliform group, will be described.

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

  As shown in FIG. 1, as a biosensor for a microcavity resonator, a single microparticle 1 of polystyrene having a sphericity or an antibody 2 that reacts with a target to be detected on a single microparticle 1 of fluorescent polystyrene. The microorganism can be determined by immobilization. Scattered light is emitted from the surface of the single microparticle 1 by the WGM excited by evanescent light and confined and circulated within the surface of the single microparticle 1 to form a periodic wavelength spectrum. Since this wavelength spectrum is very sensitive to the diameter and refractive index of the microparticles, the state around the microparticles, etc., by immobilizing the antibody 2 against the target β-galactosidase on the surface of the single microparticle 1, The resonance condition changes due to the antibody reaction. Therefore, it is possible to determine the target detection object from the shift of the wavelength at which the WGM resonates and the signal change of the spectrum peak. In this measurement method, since a single microparticle is used as a biosensor, the sensor can be produced at low cost and can be disposable. We could provide a safe and secure inspection method for food inspection because the contaminated sensor was not reused.

  In order to confine light in a single microparticle 1 in a solution and generate WGM, it is necessary to have a high refractive index with respect to a solution containing β-galactosidase. In order to generate WGM, if the refractive index of the single microparticle 1 is 1.50 or less, a periodic wavelength spectrum is not formed in the solution, so it is desirable that it be 1.50 or more. Depends on the diameter of the single microparticle 1, and the larger the diameter, the narrower the spectral peak interval, so the diameter of the single microparticle 1 is reduced in stray light and peak detection accuracy as shown in Table 1. From the observation / operability of the inspection, it is desirable that the thickness is 1 to 10 μm.

  Since the single microparticle 1 in the solution of the present invention has a smaller refractive index difference from the substrate glass 3 than the single microparticle 1 in the air, as shown in FIG. In order to confirm the above, by using the dark field illumination in which the observation illumination light 5 is incident on the substrate glass 3 and using the sealing glass 6, the single microparticle 1 can be obtained even for the solution containing the target enzyme 4. It became possible to observe.

  As shown in FIG. 3, an antibody against β-galactosidase, which is the target enzyme, is immobilized on the surface of the microparticle, and an antigen-antibody reaction occurs, whereby it is possible to confirm a split change in the spectrum peak. When the antibody against β-galactosidase is not immobilized, only the shift of the spectrum peak can be confirmed, but the split change cannot be confirmed. By using this test method in which the optical resonance condition circulating through a single microparticle changes due to the binding of β-galactosidase and a new resonance mode is generated, microorganisms can be easily determined.

  As shown in FIG. 4, the WGM light detection unit 16 of the present invention drops and fixes fine particles on the substrate glass 3. Since the incident light 8 is incident by the excitation objective lens 7 having a large numerical aperture (NA), the angle of the incident light 8 on the substrate glass 3 becomes equal to or greater than the total reflection angle, and is localized on the surface of the substrate glass 3. Some evanescent light 11 is generated. The thickness of the substrate glass 3 is 170 μm or less so that an immersion oil 9 is used so that the excitation objective lens 7 is focused on fine particles, and the incident light 8 from the excitation objective lens 7 is condensed. Is desirable. By using an excitation objective lens 7 having a large numerical aperture (NA) and an XYZ moving stage 10 for moving the substrate glass 3, the spot diameter of incident light can be made smaller than the diameter of the single microparticle 1, so that a single microparticle Stray light from other sources can be suppressed, and detection with a high S / N ratio is possible.

  The state of scattered light from the microparticles 1 varies depending on the amount of the dropped solution. Since the inspection part after dropping is covered with the sealing glass 6, it is possible to suppress evaporation and unevenness of the solution, so that it is possible to detect the spectrum with high accuracy. Scattered light from a single microparticle is collected by a detection objective lens 13 attached to the upper part, guided by an optical fiber 14 and detected by a spectrometer 15.

  As shown in FIG. 5, the scattered light detected by the spectroscope 15 is recognized by the split change detection unit 17 as a split change in the WGM peak signal. It can be determined that β-galactosidase, which is the target enzyme, is bound to the surface. In addition, since the split interval of the wavelength peak of the scattered light detected is 3 nm or more, the resolution of the spectrometer is desirably 3 nm or less.

The status of the demonstration experiment is described below.
(1) Target enzyme The coliform group is widely used as a contamination indicator in the field of food hygiene. Β-D-galactosidase (manufactured by Wako Pure Chemical Industries, Ltd.), a kind of enzyme produced when the coliform group decomposes sugar, was used.
(2) Antibody In order to react with the target β-galactosidase, a polyclonal antibody of Rabbit IgG (manufactured by Institute of Medical Biology) was used.
(3) Selection of single fine particle The diameter of the single fine particle is selected from 1 to 10 μm by comparing the S / N ratio of the peak signal to be detected and the observation / operability of the inspection. As shown in Table 1, stray light from the substrate glass 3 could be reduced by making the spot diameter of the excitation light smaller than the diameter of the single microparticle. Furthermore, since the interval between the peaks of scattered light from a single microparticle is 4 nm or more apart, it can be easily detected with a spectrometer. Moreover, since the observation with an optical microscope becomes possible, so that the diameter of a single microparticle becomes large, confirmation of the detection part became easy.

(4) Microorganism determination using single microparticles After washing the fluorescent polystyrene microparticles (micromod) modified with a carboxyl group having a diameter of 10 μm twice, the carboxyl groups were dissolved in water-soluble carbodiimide (Polysciences). And the antibody was immobilized on the surface of the microparticles. After reacting for about 60 minutes, the supernatant was removed with a centrifuge to prepare a solution containing microparticles with immobilized antibodies. About 20 to 50 μl of the produced fine particles are dropped on a glass base dish (manufactured by Iwaki) installed on the XYZ moving stage 10 in FIG. 4, and the same amount of β-D-galactosidase is further dropped. When microparticles were excited with 532 nm light, a fluorescence spectrum was detected in the wavelength region of 560 to 620 nm. After dropping β-D-galactosidase and reacting for 15 minutes, the spectrum of the wavelength peak of WGM showing the optical resonance reaction confined in the fine particles can be detected in the fluorescence spectrum, and is shown in the broken line graph of FIG. Thus, it was confirmed that the peak spectrum was split into two. When β-D-galactosidase is not added dropwise, or because no split is detected in the wavelength peak of WGM of fine particles to which no antibody is immobilized, it can be seen that this mode is caused by the binding of β-D-galactosidase. It is shown that the microorganism detection determination unit 18 can quickly and easily determine the microorganism by detecting whether the split change appears in the peak signal of the WGM having the highest detection sensitivity.

  As described above, according to the present invention, it is possible to provide an apparatus capable of detecting microorganisms quickly by using a highly accurate and inexpensive microparticle sensor.

  The present invention relates to a test method in the food field that requires microbiological tests, but can also be used in the environmental health field, the pharmaceutical field, and the like. It is possible to provide a microorganism test using a microsphere resonance sensor capable of realizing a high-sensitivity, rapid, and low-cost test chip without using an expensive detection device or the like.

DESCRIPTION OF SYMBOLS 1 Single microparticle 2 Antibody 3 Substrate glass 4 Target enzyme 5 Observation illumination light 6 Sealing glass 7 Excitation objective lens 8 Incident light 9 Immersion oil 10 XYZ moving stage 11 Evanescent light 12 Observation light source 13 Detection objective lens 14 Optical fiber 15 Spectrometer 16 WGM light detection unit 17 Split change detection unit 18 Microorganism detection determination unit

Claims (5)

  An antibody that specifically detects a target microorganism present in a solution is immobilized on a single microparticle, and the target target microorganism is detected by splitting a peak corresponding to the whispering gallery mode. A method for detecting microorganisms using a spherical resonance sensor.   2. The microsphere according to claim 1, wherein the single microparticle has a diameter satisfying a condition of resonating at a wavelength corresponding to the immobilization of an antibody and binding of a target microorganism, and a refractive index of the medium is higher. A method for detecting microorganisms using a resonance sensor.   The microorganism detection method using the microsphere resonance sensor according to claim 1, wherein the single fine particle has a diameter of 1 to 10 μm.   An excitation objective lens capable of condensing less than the diameter of the single microparticle according to claim 2 or 3 and receiving evanescent light, and a detection objective lens capable of detecting scattered light confined in the single microparticle. A microorganism detection apparatus using a microsphere resonance sensor, comprising a spectroscope including dark field illumination and having a resolution capable of detecting one or more whispering gallery mode peak signals.   The microorganism detecting apparatus using the microsphere resonance sensor according to claim 4, wherein the single microparticle is sealed with a cover glass.