JP6682307B2 - Method and apparatus for highly sensitive detection of microsphere resonance sensor - Google Patents

Description

The present invention relates to a microorganism inspection in the field of food inspection, and more particularly to a rapid, highly sensitive and highly accurate detection method and device based on Whispering Gallery Mode (WGM) using a single microsphere.

For the food industry, accidents that threaten food safety and security, such as food poisoning, can damage the brand and credibility of a company, and so it is desirable to improve the hygiene management system.
In food inspection, the inspection of microorganisms needs to be simple, accurate, and rapid, but the conventional inspection method, culture method, requires 24 hours or more before the enrichment culture and determination, and Work is required. Therefore, it has been desired to develop a sensor that is simple and capable of high-sensitivity detection, and a detection method and an apparatus that are optimal for them.

In recent years, research and development by immunochromatography, ELISA, DNA test, PCR, immunomagnetic bead method, etc. have been advanced as rapid test methods, but among them, immunological test method using antigen-antibody reaction is unique. It is expected to be used as a rapid detection method (see, for example, Patent Documents 1 and 2) and is being developed because of its excellent properties, quickness, and simplicity.

Patent Document 1 discloses a highly sensitive rapid detection method in which a target microorganism is isolated and collected using an antibody specific to the target microorganism, ATP of the target microorganism is amplified, and the amplified ATP is measured.

However, in these methods, the detection sensitivity and the determination accuracy are not sufficient, so that the enrichment culture is required, and it takes 8 hours or more to obtain a general test result. Therefore, for food manufacturers, shipment is awaiting inspection results or shipment with the risk of voluntary recall.

Patent Document 2 discloses a detection method in which a microorganism is captured by using a capturing body in which an antibody that binds to a constituent component of the microorganism is immobilized, and further, DNA extracted from the captured and washed microorganism is amplified by a PCR method. Although published, these methods also require advanced techniques such as extraction and amplification, and it is difficult to obtain test results easily and inexpensively.

As a method for solving such a problem, there is provided a biosensor using an optical mode in which a probe light is incident and circulated in a single resonator. This optical resonance mode and resonance are called whispering gallery mode (WGM) or form-dependent resonance (MDR), and occur when the light incident on the probe light is totally reflected and confined at the boundary surface of the resonator. To do.

Since WGM is very sensitive to the state of the surface, a method has been proposed to detect the WGM profile peak by immobilizing the binding partner that binds to the target analyte on the surface of the microspheroid. References 3 to 4). In addition, a biosensor that increases the ratio of detected signal light by including an optical microresonator coupled with a waveguide before the sample solution flows over the surface of the detector (see, for example, Patent Document 5), and , A sensing device in which a plurality of micro optical resonators are arranged on an optical waveguide substrate (for example, refer to Patent Document 6), and a detection method by an attenuated total reflection arrangement using an objective lens (for example, refer to Patent Document 7) are proposed. ing.

However, in order to detect the shift of the resonance peak wavelength of these WGM, a detector that requires high resolution is required, and there is also a problem that the measurement error and the wavelength shift cannot be determined because stray light during measurement is easily picked up. . Furthermore, the WGM of the microspheres placed on the substrate orbits in various directions and is very sensitive to the surface state of the microspheres. And the method of calculating the refractive index (for example, refer to Non-Patent Document 1), it is necessary to improve the S / N ratio of the detected resonance peak wavelength and reduce the influence of the split phenomenon (for example, Non-Patent Document 2). is there.

Therefore, in order to develop a quick and simple measurement method and measurement apparatus without using an expensive measurement apparatus, a detection method and apparatus with improved S / N ratio of resonance peak wavelength and shift accuracy are desired.

JP, 2006-81506, A JP, 2007-97551, A JP2012-137490A Special table 2012-509070 gazette Japanese Patent Publication No. 2008-513776 JP, 2013-96707, A JP, 2014-178151, A

Anal. Sci., Vol. 30, pp. 799-804, 2014 IEEE Sensors 2014 Conf. Proc., Pp. 641-644, 2014

Since the contact area of the WGM of the microspheres that reacted with the antigen-antibody increases with the substrate, light is emitted to the substrate side, and the resonance peak wavelength detected from the scattered light spectrum of the microspheres has an attenuated S / N ratio. Will end up. In addition, since the orbit of the light that circulates around the surface of the microspheres changes, there is a problem that split peaks corresponding to TE polarized light and TM polarized light occur at the same time. Since the split interval and the shift amount of the resonance peak wavelength are approximately the same, the shift amount detected from the resonance peak wavelength becomes less accurate. Further, there has been a problem that the microspheres being detected move due to the temperature change of the irradiation light used for the detection.

Therefore, the present invention, in order to solve the problems that occur in the case of the conventional detection method described above, eliminates the attenuation and split phenomenon of the resonance peak wavelength, further, by suppressing the movement of the microspheres, the resonance peak wavelength of An object of the present invention is to provide a method and a device for detecting changes with high sensitivity and high accuracy.

In order to achieve such an object, the present invention firstly arranges microspheres on a substrate, resonates the whispering gallery mode excited on the surface of the microspheres by the evanescent light exuding from the substrate, A method and apparatus for detecting scattered light of a sphere, wherein the fine sphere is arranged on the substrate so that the substrate and the orbit of the whispering gallery mode of the microsphere do not come into contact with each other, and Provide a device.

A method and apparatus for detecting scattered light is characterized by arranging microspheres on a substrate on which grooves are formed, and by forming the grooves to be narrower than the diameter of the microspheres, the microspheres arranged on the grooves are formed. The deviation can be suppressed. Since the orbit of the whispering gallery mode on the surface of the microsphere is not in contact with the substrate, it is possible to manufacture a sensor chip that is not affected by the increase of deposits on the surface of the microsphere.

The scattered light detection method and apparatus described above are an excitation objective lens capable of converging irradiation light from a light source into a microsphere diameter or less to generate evanescent light, and a detection objective lens capable of detecting a scattered light spectrum from the microsphere. It is desirable to use a microsphere resonance sensor equipped with a spectroscope having a resolution capable of detecting the resonance peak wavelength of the whispering gallery mode.

In addition, the present invention is secondly a method and a device for detecting microbial contamination in a target solution using the above-mentioned scattered light detection method, wherein the antibody is immobilized on microspheres, and the antibody is immobilized. A method and apparatus for detecting microbial contamination in a target solution, which is characterized by detecting a resonance peak wavelength of a whispering gallery mode of a microsphere brought into contact with a solution and detecting a change in the resonance peak wavelength. provide.

(EN) A method and an apparatus for detecting a change in resonance peak wavelength with high sensitivity and accuracy, and a method for detecting a microbial contamination using an inexpensive microsphere resonance sensor for detecting a desired microbial contamination in a quick and easy manner. It will be possible.

The method and apparatus for detecting microbial contamination using the microsphere resonance sensor according to the present invention does not require the use of a culture method in the inspection process because the sensitivity is amplified by a single microsphere. In addition, since it is possible to suppress the attenuation of the resonance peak wavelength and the split phenomenon simply by causing the evanescent light to act on the surface of the microsphere in the cutting direction from the cut concave groove, the S / N of the resonance peak wavelength due to the antigen-antibody reaction The ratio and shift accuracy are improved, making it possible to evaluate trace amounts of target substances. That is, by utilizing the method of the present invention, it becomes possible to determine the target microbial contamination with high sensitivity and high accuracy, and it is also possible to develop an inexpensive disposable sensor chip or an inexpensive small-sized device.

Further, the present invention can be used as a simple, quick, inexpensive test determination for biosensors, chemical sensors, microchannels, etc., in the field of foods, environment, and in fields requiring microbial testing such as medical care. Become.

(A) is a schematic diagram of WGM that excites microspheres on a planar substrate with evanescent light and orbits the surface, and (b) is a schematic diagram showing WGM and re-emitted light after antigen-antibody reaction. . 6 is a graph showing the resonance peak wavelength of WGM in the surface state of microspheres calculated from Mie scattering theory. (A) is a schematic diagram showing a WGM of a microsphere fixed on a concave groove of a flat substrate, and (b) shows an antigen-antibody reaction on the surface of a microsphere fixed on a concave groove of a flat substrate. FIG. It is a schematic diagram showing excitation and observation of a microsphere of the present invention, and detection of scattered light. 7 is a graph showing the resonance peak wavelength of WGM after an antigen-antibody reaction in the case where there is a concave groove on the planar substrate. It is a figure which shows the Example of the detection method and apparatus of microbial contamination.

In the present invention, the target microbial contamination is coliforms, and β-galactosidase, an enzyme produced by the coliforms, is detected. A method for detecting scattered light with high sensitivity and high accuracy by arranging microspheres on a concave shape formed on a flat substrate will be described. Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

As shown in FIG. 1A, a spherical polystyrene microsphere 1 or a fluorescent polystyrene microsphere 1 is used as a microcavity resonator. The microsphere surface is excited by the evanescent light 2 generated when the incident light 3 enters from the back surface of the planar substrate 7 and is totally reflected by the front surface, and WGM that confines and circulates the light inside the microsphere surface is generated. At this time, a spectrum having a periodic resonance peak wavelength due to WGM is detected from the scattered light emitted from the surface of the microsphere. This resonance peak wavelength is very sensitive to the diameter and refractive index of the microspheres, the state of the periphery of the microspheres, etc., so that a highly sensitive sensor using the surface of the microspheres 1 can be manufactured. Since the microspheres 1 without surface modification have few contact portions 4 with the flat substrate 7, when the evanescent light 2 having momentum in the -X direction acts, the excitation direction WGM5 that orbits clockwise in the XZ plane is mainly generated. Be excited. At the same time, the oblique direction WGM6 is also excited, but since the component of the excitation direction WGM5 is large, the resonance peak wavelength due to the excitation direction WGM5 is acquired from the scattered light.

FIG. 1 (b) shows the state of WGM circulating on the surface of the microsphere after the antigen-antibody reaction. By immobilizing the antibody 8 that reacts with the target enzyme 9 on the microspheres 1, it becomes possible to determine microbial contamination. The WGM that is excited by evanescent light or the like and confines and circulates the light within the surface of the microsphere 1 emits scattered light from the surface of the microsphere 1 to form a periodic wavelength spectrum. Since the resonance condition changes due to the antigen-antibody reaction, the target detection substance can be evaluated from the shift change in the resonance wavelength of WGM. In this evaluation method, since a single microsphere is used as a bioprobe, a sensor chip can be manufactured at low cost and can be disposable. In addition, since the contaminated sensor is not reused, it can be provided as a safe and secure inspection method for food inspection.

However, in FIG. 1B, since the contact portion 4 between the surface of the microsphere and the planar substrate 7 increases due to the antigen-antibody reaction, the excitation direction WGM5 orbiting the XZ plane has a large interaction with the substrate. Therefore, the re-emitted light 10 from the planar substrate 7 and the contact portion 4 becomes larger than when the WGM is scattered above the microspheres. On the other hand, since the oblique direction WGM6 is not affected by the planar substrate 7 and the contact portion 4, the scattered light from the microsphere 1 is mainly obtained at the resonance peak wavelength due to the oblique direction WGM6 on the surface of the microsphere. Resonant peak wavelengths corresponding to TE and TM polarization are likely to occur at the same time.

FIG. 2 shows a scattering cross-section spectrum in the surface state of polystyrene microspheres (hereinafter abbreviated as PS microspheres). The diameter and refractive index of unmodified PS microspheres are 10.04 μm and 1.59, and the refractive index of pure water is 1.33. Antibodies (anti-β-Galactosidase) and antigens are adsorbed on the surface of PS microspheres. It can be seen that the resonance peak wavelength of the WGM that orbits the surface of the microsphere shifts to the long wavelength side. Here, it is assumed that the antibody "Y" is immobilized as a single layer on the surface of PS microspheres, the refractive index of the antigen-antibody layer is 1.50, the antibody thickness is 14 nm, and the antigen thickness is 16 nm. . When an antibody or antigen is adsorbed on the surface of PS microspheres, the resonance peak wavelength shifts to the long wavelength side by about 1 to 4 nm. It is desirable to evaluate the corresponding resonance peak wavelength with high sensitivity and accuracy.

Therefore, in the present invention, as shown in FIG. 3A, a sensor chip in which the microsphere 1 can be arranged by cutting the concave groove 11 on the flat substrate 7 with a probe having a tip diameter of 5 μm was devised. Even if the evanescent light 2 exuding from the concave surface due to the attenuated total reflection arrangement excites the excitation direction WGM5 that orbits the XZ plane, as shown in FIG. 3 (b), the antibody 8 adsorbed on the surface of the microsphere Since the target enzyme 9 is in the concave groove 11, it is not affected by the contact portion 4 with the flat substrate 7. Therefore, the re-emitted light 10 from the contact portion 4 is reduced, the S / N ratio is improved, and at the same time the split of the resonance peak wavelength is suppressed, so that the evaluation can be performed with high sensitivity and high accuracy. Furthermore, since the microspheres 1 can be fixed in the concave grooves 11, it is possible to suppress the movement of the microspheres 1 caused by the temperature rise of the incident light 3.

FIG. 4 is a schematic diagram showing excitation and observation of the microsphere 1 of the present invention and detection of scattered light. A white light source is used as the excitation light source 12, and the polarizer 13 switches the excitation directions of TE polarization and TM polarization, and the reflection mirror 14 irradiates the inverted excitation objective lens 15 with the incident light 3. The microspheres 1 are dropped and placed on a planar substrate 7 attached to the bottom surface of the dish. Since the incident light 3 enters through the excitation objective lens 15 having a large numerical aperture (NA), the angle of the incident light 3 on the planar substrate 7 becomes equal to or greater than the total reflection angle, and is localized on the surface of the planar substrate 7. Evanescent light 2, which is a wave, is generated. In order to fill the space between the objective lens 15 for excitation and the planar substrate 7 with the immersion oil 16 so that the focal point matches the microsphere 1, the thickness of the planar substrate 7 is preferably 170 μm or less. By using the excitation objective lens 15 having a large numerical aperture (NA), the spot diameter of the incident light 3 becomes equal to or smaller than the diameter of the microsphere 1, stray light from other than the microsphere 1 is not detected, and a high S / N ratio is obtained. It became possible to detect. Further, by covering the detection part onto which the target enzyme 9 was dropped with the sealing glass 17, it was possible to suppress the evaporation of the solution and the thickness unevenness, and it was possible to detect the spectrum with high accuracy. The scattered light from the microspheres 1 is condensed by the detection lens 18 attached to the upper part, and is evaluated by switching the observation of the CCD camera 20 and the spectrum detection of the spectroscope 21 by the X-axis moving mirror 19.

As shown in FIG. 5, when the scattered light of the microsphere 1 that has undergone the antigen-antibody reaction on the flat substrate 7 without the concave groove 11 is detected, the resonance corresponding to the TM polarized light is generated despite the TE polarized light excitation. The peak wavelength is easy to detect. However, if the microsphere 1 is excited after processing the concave groove 11, only the resonance peak wavelength corresponding to the polarization direction is detected, and the shift amount can be accurately estimated. Moreover, since the re-emitted light 10 from the contact portion 4 of the planar substrate 7 is reduced, the intensity of the resonance peak wavelength is approximately doubled, and the S / N ratio can be improved. Since the resonance peak wavelength of the scattered light detected by the spectroscope 21 is shifted to the long wavelength side by about 1 to 4 nm, the resolution of the spectroscope is preferably 1 nm or less.

FIG. 6 shows an embodiment of a method and apparatus for detecting microbial contamination. The scattered light acquired from the WGM light detector 22 is guided by the optical fiber 23 and detected by the spectroscope 21. Regarding the electric signal converted from the optical signal by the spectroscope 21, the change amount of the resonance peak wavelength is calculated by the wavelength change detection unit 24, and the target contaminant is bound to the surface of the microsphere 1 by the microbial contamination determination unit 25. Can be determined.

The situation of the demonstration experiment will be described below.

(1) The target enzyme coliforms is widely used as a contamination indicator bacterium in the field of food hygiene. Β-D-galactosidase (manufactured by Wako Pure Chemical Industries, Ltd.), which is one of the enzymes produced when coliform bacteria decompose sugar, was used.

(2) Antibody A polyclonal antibody of Rabbit IgG (manufactured by Institute of Medical Biology) was used to react with the target β-galactosidase.

(3) Irradiation light used The excitation light source 12 used was a white light source (170-2100 nm) manufactured by Technology. A polarization direction of TE or TM was selected from random polarization by rotating a polarizer 13 (Glan-Thompson prism) manufactured by Sigma Koki Co., Ltd. by 90 °. In addition, for ultraviolet light that damages proteins and infrared light that raises the temperature of the irradiation part, use the sharp cut filter and cold filter manufactured by Sigma Koki Co., Ltd., and evaluate mainly in the wavelength range of 500 to 600 nm. Carried out.

(4) Cutting Method of Planar Substrate To cut the concave groove 11 on the planar substrate 7, a hard metal probe (tip diameter 5 μm) manufactured by Micro Support Co. was used. A concave groove 11 having a line width of 5 μm or less was produced by performing a combination operation with a three-dimensional hydraulic micromanipulator (minimum driving distance 1 μm) made by Narishige.

(5) Preparation of microsphere probe and evaluation method PS microspheres with a diameter of 10 μm and modified with a carboxyl group (manufactured by micromod) are washed twice, and then the carboxyl group is activated by a water-soluble carbodiimide (manufactured by Polysciences). Then, the antibody was immobilized on the surface of the microsphere. After reacting for about 60 minutes, the supernatant was removed with a centrifuge to prepare a solution containing antibody-immobilized microspheres.

The prepared microspheres are placed on the flat substrate 7 attached to the bottom surface of the glass base dish (manufactured by Iwaki) shown in FIG. 4, and about 100 μl of the same amount of β-D-galactosidase is dropped. The detection part was covered with the sealing glass 17 to excite the microspheres with a white light source, and the scattered light spectrum was detected in the wavelength region of 570 to 600 nm.

After β-D-galactosidase was added dropwise and reacted for 15 minutes, the resonance peak wavelength of WGM showing an optical resonance reaction reflecting the surface state of the microspheres could be detected in the scattered light spectrum. Regarding the position of the resonance peak wavelength, the resonance peak wavelength of the TE polarized light can be acquired on the longer wavelength side of 2 to 3 nm than the resonance peak wavelength of the TM polarized light.

The detected resonance peak wavelength was fitted to the scattering cross section according to the Mie theory shown in the graph of FIG. 2 to estimate the refractive index and the thickness of the adherent, which was in agreement with the antibody and antigen specifications. As shown in the graph of FIG. 5, when the microspheres 1 that have undergone an antigen-antibody reaction on the concave groove 11 are excited to detect scattered light, a resonance peak wavelength that suppresses the split phenomenon can be detected. Furthermore, since the re-emitted light 10 from the contact portion 4 of the planar substrate 7 can be suppressed, the intensity of the resonance peak wavelength detected from the scattered light is approximately doubled, and the S / N ratio can be improved. did it.

The resonance peak wavelength is shifted to the long wavelength side by about 1 to 4 nm before and after the antigen-antibody reaction by the wavelength change detecting unit 24 in FIG. When the wavelength resolution of the spectroscope 21 is set to 1 nm and the microorganism detection determination unit 18 determines the contamination of microorganisms, the determination time is 8 minutes and the lower detection limit concentration is 5 μg / mL.

As described above, according to the present invention, it is possible to provide an apparatus capable of rapidly detecting a microorganism by using a highly accurate and highly sensitive detection method of a microsphere sensor.

The present invention relates to an inspection method in the field of food that requires inspection of microorganisms, but can also be used in the fields of environmental hygiene, the field of pharmaceuticals, etc., in which biosensors, chemical sensors, microchannels, etc. It can be used as a simple, quick, and inexpensive inspection judgment. Further, it is possible to provide a microbe test using a microsphere resonance sensor that can realize a highly sensitive and highly accurate and low-cost test chip without using an expensive detection device or the like.

1 Microsphere 2 Evanescent light 3 Incident light 4 Contact part 5 Excitation direction WGM
6 Diagonal WGM
7 Planar substrate 8 Antibody 9 Target enzyme 10 Re-emitted light 11 Concave groove 12 Excitation light source 13 Polarizer 14 Reflection mirror 15 Excitation objective lens 16 Immersion oil 17 Sealing glass 18 Detection lens 19 X-axis moving mirror 20 CCD Camera 21 spectroscope 22 WGM light detector 23 optical fiber 24 wavelength change detector 25 microbial contamination determiner

Claims (5)

The microspheres were placed on a substrate, resonates the whispering gallery modes to be excited in microspheres surface by evanescent light oozed from the substrate, a way to detect the scattered light of the microspheres,
Detection how the scattered light, characterized in that the trajectory of the whispering gallery mode of the substrate and the microsphere is placed microspheres to the substrate not in contact. Detection how the scattered light according to claim 1, wherein placing the microspheres on a substrate in which grooves are formed. The resonance objective wavelength of the whispering gallery mode is provided with an objective lens for excitation that can collect the emitted light from the light source to a microsphere diameter or less to generate evanescent light and an objective lens for detection that can detect the scattered light spectrum from the microsphere. detection how the scattered light according to claim 1, wherein the use of microsphere resonance sensor with a spectrometer having a resolution that can be detected. A detecting how the microbial contamination of the target solution using a method of detecting scattered light according to any one of claims 1 to 3,
Detecting the resonance peak wavelength of whispering gallery mode of antibody-immobilized microspheres and microspheres in which a solution is contacted with antibody-immobilized microspheres, and detecting the change in the resonance peak wavelength. detection how the microbial contamination of the target solution and said. A substrate having a groove formed therein, and a microsphere that can be arranged on the groove of the substrate,
The microspheres are arranged on the substrate so that the substrate and the orbits of the whispering gallery mode of the microspheres do not contact with each other, and the whispering gallery mode excited by the evanescent light exuding from the substrate resonates the whispering gallery mode. , Detect scattered light from microspheres
A chip for a biosensor or a chemical sensor, which is characterized in that

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