JP2017166825A - High sensitivity detecting method and device for microsphere resonance sensors - Google Patents

High sensitivity detecting method and device for microsphere resonance sensors Download PDF

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JP2017166825A
JP2017166825A JP2016049128A JP2016049128A JP2017166825A JP 2017166825 A JP2017166825 A JP 2017166825A JP 2016049128 A JP2016049128 A JP 2016049128A JP 2016049128 A JP2016049128 A JP 2016049128A JP 2017166825 A JP2017166825 A JP 2017166825A
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健志 田尻
Takeshi Tajiri
健志 田尻
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Abstract

PROBLEM TO BE SOLVED: To provide a high-sensitivity and high-precision method and device for variations of resonance wavelength by restraining radiation in a whispering gallery mode (hereinafter abbreviated to WGM) toward the substrate to eliminate attenuation and/or split phenomenon and further by restraining shifts of microspheres.SOLUTION: Microspheres are arranged over a concave groove 11 cut in a flat substrate 7, WGM is resonated by evanescent light 2 oozing out of the concave groove, and variations in peak resonant wavelength are detected from the scattered light spectrum of microspheres. By using a spectroscope equipped with an objective lens for exciting use that can collect radiation light from a light source into the diameter of microspheres or a smaller size to generate evanescent light and an objective lens for detecting use having a resolving capability to detect the peak WGM resonance wavelength, any microorganic contamination in the object solution is detected.SELECTED DRAWING: Figure 3

Description

本発明は、食品検査分野での微生物検査に係り、特に単一の微小球を使用したウィスパリングギャラリーモード（WGM）に基づく迅速および高感度で高精度な検出方法および装置に関するものである。 The present invention relates to microorganism testing in the field of food inspection, and more particularly to a rapid, highly sensitive and highly accurate detection method and apparatus based on whispering gallery mode (WGM) using a single microsphere.

食品業界にとって食中毒などの食の安全・安心を脅かす事故は、企業のブランドと信用を失墜させることにつながるため、衛生管理体制の高度化が望まれている。
食品検査において微生物の検査は、簡便、正確、迅速に行なわれる必要があるが、従来の検査手法である培養法は、増菌培養し判定するまでに２４時間以上は必要で、かつ、熟練者による作業が必要となる。そのため、簡便で迅速に高感度検出が可能なセンサー、並びに、それらに最適な検出方法と装置の開発が望まれていた。 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.

近年、迅速検査手法としてイムノクロマト法、ELISA法、DNA検査法、PCR法、免疫磁気ビーズ法などによる研究や開発が進められているが、中でも抗原‐抗体反応を用いた免疫学的検査法は特異性、迅速性、簡便性等の点で優れているため、迅速検出方法（例えば、特許文献１〜２参照）として期待され開発が行われている。 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 and developed as a rapid detection method (for example, see Patent Documents 1 and 2).

特許文献１には、標的微生物特異的抗体を用いて標的微生物を分離・回収した後、標的微生物のATPを増幅し、増幅されたATPを測定する高感度迅速検出法が公開されている。 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.

しかし、これらの方法は検出感度や判定精度が十分でないため増菌培養が必要となり、一般的な検査結果を得るまでは８時間以上を要している。そのため、食品製造業者にとっては、検査結果を待った出荷、あるいは、自主回収のリスクを抱えた出荷となっている。 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.

特許文献２には、微生物の構成成分と結合する抗体を固定化した捕捉体を用いて微生物を捕捉し、更に、捕捉し洗浄された微生物から抽出されたDNAをPCR法により増幅した検出方法が公開されているが、これらの方法も抽出や増幅などの高度な技術が必要となり、簡易かつ安価に検査結果を得ることは難しい。 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. Although publicly available, these methods also require advanced techniques such as extraction and amplification, and it is difficult to obtain test results easily and inexpensively.

このような課題を解決する手法として、単一の共振器内にプローブ光を入射し循環させる光学モードを利用したバイオセンサーが提供される。この光学共振モードおよび共振は、ウィスパリングギャラリーモード（WGM）、または、形態依存共振（MDR）と呼ばれ、プローブ光で入射した光が共振器の境界面で全反射し閉じ込められた場合に発生する。 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 (WGM) or form dependent resonance (MDR), and occur when the light incident on the probe light is totally reflected and confined at the interface of the resonator. To do.

WGMは表面の状態に非常に敏感であるために、微小回転楕円体の表面に標的分析物と結合する結合パートナーを固定化し、WGMのプロファイルピークを検出する方法が提案されている（例えば、特許文献３〜４参照）。また、検体溶液が検出器表面を流れ過ぎる前に、導波路と結合した光学微小共振器を含めることで、検出される信号光の割合を増加させるバイオセンサー（例えば、特許文献５参照）、および、光導波基板上に複数の微小光共振体を配置したセンシング装置（例えば、特許文献６参照）や、対物レンズを利用した全反射減衰配置による検出手法（例えば、特許文献７参照）が提案されている。 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). Also, a biosensor that increases the proportion of detected signal light by including an optical microresonator coupled to the waveguide before the analyte solution flows too much over the detector surface (see, for example, Patent Document 5), and A sensing device (for example, see Patent Document 6) in which a plurality of minute optical resonators are arranged on an optical waveguide substrate, and a detection method (for example, see Patent Document 7) using a total reflection attenuation arrangement using an objective lens have been proposed. ing.

しかしながら、これらのWGMの共振ピーク波長のシフトを検出するには、高分解能を要する検出器が必要となり、また測定時の迷光を拾い易いために測定誤差と波長シフトの判定ができないという問題もある。さらには、基板上に配置された微小球のWGMは様々な方向に周回し、微小球の表面状態に非常に敏感であるため、共振ピーク波長のシフト量から、微小球表面の付着物の厚みと屈折率を算出する手法（例えば、非特許文献１参照）では、検出される共振ピーク波長のS/N比の向上やスプリット現象（例えば、非特許文献２参照）の影響を少なくする必要がある。 However, in order to detect the shift of the resonance peak wavelength of these WGMs, 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. . Furthermore, since the WGM of the microspheres arranged on the substrate circulates in various directions and is very sensitive to the surface state of the microspheres, the thickness of the deposit on the microsphere surface is determined from the shift amount of the resonance peak wavelength. And the refractive index calculation method (for example, see Non-Patent Document 1), it is necessary to reduce the influence of the S / N ratio of the detected resonance peak wavelength and the split phenomenon (for example, Non-Patent Document 2). is there.

したがって、高価な測定装置を用いることなく、迅速で簡便な測定方法と測定装置を開発するため、共振ピーク波長のS/N比とシフト精度を向上した検出方法および装置が望まれている。 Therefore, in order to develop a quick and simple measurement method and apparatus without using an expensive measurement apparatus, a detection method and apparatus with improved resonance peak wavelength S / N ratio and shift accuracy are desired.

特開２００６−８１５０６号公報JP 2006-81506 A 特開２００７−９７５５１号公報JP 2007-97551 A 特開２０１２−１３７４９０号公報JP 2012-137490 A 特表２０１２−５０９０７０号公報Special table 2012-509070 gazette 特表２００８−５１３７７６号公報Special table 2008-513776 特開２０１３−９６７０７号公報JP 2013-96707 A 特開２０１４−１７８１５１号公報JP 2014-178151 A

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

抗原抗体反応した微小球のWGMは、基板との接触面積が増加するため、基板側へ光が放射され、微小球の散乱光スペクトルから検出される共振ピーク波長は、S/N比が減衰してしまう。また、微小球表面を周回する光の軌道が変化してしまうため、TE偏光とTM偏光に対応したスプリットピークが同時に発生する問題が生じていた。この共振ピーク波長のスプリット間隔とシフト量は同程度であるため、共振ピーク波長から検出するシフト量は精度が低くなる。また、検出で使用する照射光の温度変化によって、検出中の微小球が移動する問題も生じていた。 The antigen-antibody-reacted microsphere WGM increases the contact area with the substrate, so light is emitted to the substrate side, and the S / N ratio attenuates the resonance peak wavelength detected from the scattered light spectrum of the microsphere. End up. In addition, since the trajectory of the light traveling around the surface of the microsphere changes, there has been a problem that split peaks corresponding to TE polarized light and TM polarized light are generated simultaneously. 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 is less accurate. In addition, there is a problem that the microsphere being detected moves due to the temperature change of the irradiation light used for detection.

よって、本発明は、前述した従来の検出方法の場合に生じる課題を解決するために、共振ピーク波長の減衰やスプリット現象を無くし、さらには、微小球の移動を抑えることにより、共振ピーク波長の変化を高感度かつ高精度に検出する方法および装置を提供することを目的とする。 Therefore, the present invention eliminates the resonance peak wavelength attenuation and split phenomenon, and further suppresses the movement of the microspheres in order to solve the problems caused by the above-described conventional detection method. It is an object of the present invention to provide a method and apparatus for detecting a change with high sensitivity and high accuracy.

このような目的を達成するために、本発明は第一に、基板上に微小球を配置し、基板から染み出したエバネセント光により微小球表面に励起されるウィスパリングギャラリーモードを共振し、微小球の散乱光を検出する方法および装置であって、前記基板と微小球のウィスパリングギャラリーモードの軌道が接触しないように基板上に微小球を配置することを特徴とする散乱光の検出方法および装置を提供する。 In order to achieve such an object, the present invention firstly arranges a microsphere on a substrate, resonates a whispering gallery mode excited on the surface of the microsphere by evanescent light oozing out from the substrate, A method and apparatus for detecting scattered light from a sphere, wherein the microsphere is arranged on the substrate so that the whispering gallery mode trajectory of the substrate and the microsphere is not in contact, and Providing equipment.

散乱光の検出方法および装置は、溝が形成された基板上に微小球を配置することを特徴とし、溝が微小球径よりも幅狭に形成することで、該溝上に配置された微小球のズレを抑制することができる。微小球表面のウィスパリングギャラリーモードの軌道が基板と非接触状態であるため、微小球表面の付着物の増加による影響を受けないセンサーチップが作製できる。 The method and apparatus for detecting scattered light is characterized in that microspheres are arranged on a substrate on which grooves are formed, and the microspheres arranged on the grooves are formed by forming the grooves narrower than the diameter of the microspheres. Can be suppressed. Since the whispering gallery mode trajectory on the surface of the microsphere is not in contact with the substrate, a sensor chip that is not affected by an increase in the amount of deposits on the surface of the microsphere can be manufactured.

上記の散乱光の検出方法および装置は、光源からの照射光を微小球径以下に集光しエバネセント光を発生できる励起用対物レンズと、微小球からの散乱光スペクトルを検出できる検出用対物レンズを備えウィスパリングギャラリーモードの共振ピーク波長を検出できる分解能を有する分光器を備えた微小球共振センサーを用いることが望ましい。 The above scattered light detection method and apparatus include an excitation objective lens that can collect evanescent light by condensing the irradiation light from a light source to a diameter of a microsphere or less, and a detection objective lens that can detect a scattered light spectrum from the microsphere. It is desirable to use a microsphere resonance sensor equipped with a spectrometer having a resolution capable of detecting the resonance peak wavelength of whispering gallery mode.

また、本発明は第二に、上記の散乱光の検出方法を用いた対象溶液中の微生物汚染の検出方法および装置であって、抗体が固定化された微小球、および、抗体が固定化された微小球に溶液を接触させた微小球のウィスパリングギャラリーモードの共振ピーク波長を検出し、その共振ピーク波長の変化を検出することを特徴とする対象溶液中の微生物汚染の検出方法および装置を提供する。 The second aspect of the present invention is a method and apparatus for detecting microbial contamination in a target solution using the above-described method for detecting scattered light, wherein microspheres to which antibodies are immobilized, and antibodies are immobilized. A method and apparatus for detecting microbial contamination in a target solution, characterized by detecting a resonance peak wavelength of whispering gallery mode of a microsphere in which the solution is brought into contact with the microsphere and detecting a change in the resonance peak wavelength. provide.

共振ピーク波長の変化を高感度かつ高精度に検出する方法および装置を提供し、迅速簡便に目的の微生物汚染を検出する安価な微小球共振センサーを使用する微生物汚染の検出装置を作製することが可能となる。 To provide a method and apparatus for detecting a change in resonance peak wavelength with high sensitivity and high accuracy, and to produce a detection apparatus for microbial contamination using an inexpensive microsphere resonance sensor that detects a target microbial contamination quickly and easily. It becomes possible.

本発明にかかる微小球共振センサーを使用する微生物汚染の検出方法および装置は、単一の微小球により感度の増幅を行うため、検査工程において培養法を利用する必要がない。また、切削した凹形状の溝から切削方向にエバネセント光を微小球表面に作用させるだけで、共振ピーク波長の減衰やスプリット現象を抑えることができるため、抗原抗体反応による共振ピーク波長のS/N比とシフト精度を向上し、微量な標的物質の評価が可能となる。すなわち、本発明手法を利用することで、標的とする微生物汚染を高感度で高精度に判定することが可能となり、使い捨て用の安価なセンサーチップや安価な小型装置の開発も可能となる。 Since the detection method and apparatus for microbial contamination using the microsphere resonance sensor according to the present invention amplifies the sensitivity with a single microsphere, it is not necessary to use a culture method in the inspection process. In addition, by simply applying evanescent light to the surface of the microsphere from the cut concave groove in the cutting direction, attenuation and splitting of the resonance peak wavelength can be suppressed. The ratio and shift accuracy are improved, and a trace amount of target substance can be evaluated. That is, by using the method of the present invention, it becomes possible to determine target microbial contamination with high sensitivity and high accuracy, and it becomes possible to develop a disposable inexpensive sensor chip and an inexpensive small device.

また、この発明は、食品分野をはじめ、環境や、医療など微生物検査が必要な分野において、バイオセンサー、化学センサー、マイクロ流路などの簡便、迅速、安価な検査判定として利用することが可能となる。 In addition, the present invention can be used as simple, quick, and inexpensive test judgments for biosensors, chemical sensors, microchannels, etc. in the field of food, the environment, and fields that require microbial testing such as medicine. Become.

（ａ）は、平面状基板上の微小球をエバネセント光で励起し表面を周回するWGMの模式図であり、（ｂ）は、抗原抗体反応後のWGMと再放射光を示す模式図である。(A) is a schematic diagram of WGM that excites microspheres on a planar substrate with evanescent light and circulates around the surface, and (b) is a schematic diagram showing WGM after antigen-antibody reaction and re-radiated light. . Mie散乱理論より算出した微小球の表面状態におけるWGMの共振ピーク波長を示すグラフである。It is a graph which shows the resonance peak wavelength of WGM in the surface state of the microsphere computed from the Mie scattering theory. （ａ）は、平面状基板の凹状溝上に固定された微小球のWGMを示す模式図であり、（ｂ）は、平面状基板の凹状溝に固定された微小球表面の抗原抗体反応を示す断面図である。(A) is a schematic diagram showing the WGM of a microsphere fixed on the concave groove of the planar substrate, and (b) shows an antigen-antibody reaction on the surface of the microsphere fixed in the concave groove of the planar substrate. It is sectional drawing. 本発明の微小球の励起と観測、および、散乱光の検出を示す概略図である。It is the schematic which shows excitation and observation of the microsphere of this invention, and detection of a scattered light. 平面状基板への凹状溝が有無の場合における抗原抗体反応後のWGMの共振ピーク波長を示すグラフである。It is a graph which shows the resonance peak wavelength of WGM after an antigen antibody reaction in the case of the presence or absence of the concave groove | channel on a planar substrate. 微生物汚染の検出方法および装置の実施例を示す図である。It is a figure which shows the Example of the detection method and apparatus of microbial contamination.

本発明において、標的の微生物汚染は大腸菌群とし、大腸菌群が産生する酵素のβガラクトシダーゼを検出する。平面状基板にもうけた凹形状の上に微小球を配置し、高感度・高精度に散乱光を検出する方法を説明する。以下、本発明について実施例を用いて詳細に説明するが本発明はこれらの実施例に限定されるものではない。 In the present invention, the target microbial contamination is coliform bacteria, and β-galactosidase, an enzyme produced by the coliform bacteria, is detected. A method for detecting scattered light with high sensitivity and high accuracy by arranging microspheres on a concave shape provided on a planar substrate will be described. EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these Examples.

図１（ａ）に示すように、マイクロキャビティー共振器として真球性のあるポリスチレンの微小球１、または、蛍光ポリスチレンの微小球１を用いる。入射光３が平面状基板７の裏面から入射し表面で全反射した時に発生するエバネセント光２により微小球表面が励起され、微小球表面内に光を閉じ込め循環するWGMが発生する。このとき、微小球表面から発する散乱光からは、WGMに起因した周期的な共振ピーク波長を有するスペクトルが検出される。この共振ピーク波長は、微小球の直径や屈折率、微小球周辺の状態などにより非常に敏感であるため、微小球１の表面を利用した高感度なセンサーを作製することができる。表面修飾が無い微小球１は、平面状基板７との接触部４が少ないため、-X方向に運動量を有するエバネセント光２が作用すると、主にXZ平面を時計回りに周回する励起方向WGM５が励起される。同時に斜め方向WGM６も励起されるが、励起方向WGM５の成分が大きいため、散乱光は励起方向WGM５に起因した共振ピーク波長が取得される。 As shown in FIG. 1A, a spherical microsphere 1 of polystyrene or a microsphere 1 of fluorescent polystyrene is used as a microcavity resonator. The surface of the microsphere 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 surface, and WGM that confines and circulates the light within the surface of the microsphere is generated. At this time, a spectrum having a periodic resonance peak wavelength caused by WGM is detected from the scattered light emitted from the surface of the microsphere. Since this resonance peak wavelength is very sensitive to the diameter and refractive index of the microsphere, the state around the microsphere, etc., a highly sensitive sensor using the surface of the microsphere 1 can be produced. Since the microsphere 1 without surface modification has few contact portions 4 with the planar substrate 7, when the evanescent light 2 having momentum in the -X direction acts, the excitation direction WGM5 that mainly circulates clockwise around the XZ plane Excited. At the same time, the oblique direction WGM 6 is also excited, but since the component of the excitation direction WGM 5 is large, the resonance peak wavelength resulting from the excitation direction WGM 5 is acquired for the scattered light.

図１（ｂ）は抗原抗体反応後の微小球表面を周回するWGMの状態を示す。微小球１に標的酵素９と反応する抗体８を固定化することで微生物汚染の判定が可能となる。エバネセント光などにより励起され、微小球１の表面内に光を閉じ込め循環するWGMにより、微小球１の表面から散乱光が発せられ、周期的な波長のスペクトルが形成される。抗原抗体反応により共振条件の変化が発生するため、WGMの共振波長のシフト変化から、目的とする検出物質の評価が可能となる。この評価手法は、単一の微小球をバイオプローブとして利用するためセンサーチップが安価に作製でき、また使い捨ても可能となる。また、汚染されたセンサーを再利用しないために食品検査において安全安心な検査方法として提供できる。 FIG. 1 (b) shows the state of WGM orbiting the microsphere surface after the antigen-antibody reaction. By immobilizing the antibody 8 that reacts with the target enzyme 9 on the microsphere 1, it is possible to determine microbial contamination. Scattered light is emitted from the surface of the microsphere 1 by the WGM excited by evanescent light and confined and circulated within the surface of the microsphere 1 to form a periodic spectrum of wavelengths. Since the resonance condition changes due to the antigen-antibody reaction, the target detection substance can be evaluated from the shift change of 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 a low cost and can be disposable. Moreover, since the contaminated sensor is not reused, it can be provided as a safe and secure inspection method in food inspection.

しかし、図１（ｂ）は抗原抗体反応により、微小球表面と平面状基板７との接触部４が増加するため、XZ平面を周回する励起方向WGM５は、基板との相互作用が大きくなる。そのため、ＷＧＭが微小球の上方へ散乱光されるよりも、平面状基板７と接触部４からの再放射光１０が大きくなる。一方で、斜め方向WGM６は平面状基板７と接触部４の影響を受けないため、微小球１からの散乱光は、微小球表面を斜め方向WGM６に起因した共振ピーク波長が主に取得され、TE偏光とTM偏光に対応した共振ピーク波長が同時に発生しやすい。 However, in FIG. 1B, the contact portion 4 between the surface of the microsphere and the planar substrate 7 increases due to the antigen-antibody reaction, so that the excitation direction WGM 5 that goes around the XZ plane has a larger interaction with the substrate. Therefore, the re-radiated light 10 from the planar substrate 7 and the contact portion 4 becomes larger than the WGM scattered light above the microsphere. On the other hand, since the oblique direction WGM 6 is not affected by the planar substrate 7 and the contact portion 4, the scattered light from the microsphere 1 mainly obtains the resonance peak wavelength due to the oblique direction WGM 6 on the surface of the microsphere, Resonant peak wavelengths corresponding to TE polarized light and TM polarized light are likely to occur simultaneously.

図２は、ポリスチレン微小球（以下PS微小球と略称）の表面状態における散乱断面積スペクトルを示す。無修飾のPS微小球の直径と屈折率を１０．０４μmと１．５９、純水の屈折率を１．３３とし、PS微小球表面に抗体(anti-β-Galactosidase)や抗原が吸着していくと、微小球表面を周回するWGMの共振ピーク波長は長波長側へシフトすることがわかる。ここでは、PS微小球表面に抗体「Y」が単一層になって固定化されているとし、抗原抗体層の屈折率は１．５０、抗体厚みは１４nm、抗原厚みは１６nmとして計算している。PS微小球表面に抗体や抗原が吸着すると、共振ピーク波長が約１〜4nm長波長側へシフトするため、表面に付着するタンパク質の屈折率や厚みを推定するには、TE偏光とTM偏光に対応した共振ピーク波長を高感度かつ高精度に評価することが望ましい。 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 set to 10.04 μm and 1.59, the refractive index of pure water is 1.33, and antibodies (anti-β-Galactosidase) and antigens are adsorbed on the surface of PS microspheres. It can be seen that the resonance peak wavelength of WGM orbiting the surface of the microsphere shifts to the longer wavelength side. Here, it is assumed that the antibody “Y” is immobilized as a single layer on the surface of the PS microsphere, and 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 antibodies and antigens are adsorbed on the surface of PS microspheres, the resonance peak wavelength shifts to the long wavelength side by about 1 to 4 nm. Therefore, to estimate the refractive index and thickness of proteins attached to the surface, use TE polarized light and TM polarized light. It is desirable to evaluate the corresponding resonance peak wavelength with high sensitivity and high accuracy.

そこで本発明では、図３（ａ）に示すように、平面状基板７に先端直径５μmのプローブで凹状形状溝１１を切削し、微小球１を配置できるセンサーチップを考案した。全反射減衰配置により凹形状面から染み出したエバネセント光２が、XZ平面を周回する励起方向WGM５を励起しても、図３（ｂ）に示すように、微小球表面に吸着した抗体８と標的酵素９は凹状形状溝１１にあるため、平面状基板７との接触部４の影響を受けることがない。このため、接触部４からの再放射光１０が減少し、S/N比が向上すると同時に共振ピーク波長のスプリットも抑えられるため、高感度かつ高精度に評価することが可能となる。さらには、凹状形状溝１１に微小球１を固定化できるため、入射光３の温度上昇によって生じる微小球１の移動を抑えることも可能となる。 In view of this, in the present invention, as shown in FIG. 3A, a sensor chip was devised in which the microsphere 1 can be arranged by cutting the concave groove 11 on the planar substrate 7 with a probe having a tip diameter of 5 μm. Even if the evanescent light 2 oozing out from the concave surface due to the total reflection attenuation arrangement excites the excitation direction WGM5 that goes around the XZ plane, as shown in FIG. 3B, 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 planar substrate 7. For this reason, the re-radiated 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 it is possible to evaluate with high sensitivity and high accuracy. Furthermore, since the microsphere 1 can be fixed to the concave groove 11, the movement of the microsphere 1 caused by the temperature rise of the incident light 3 can be suppressed.

図４は本発明の微小球１の励起と観測、および、散乱光の検出を示す概略図を示す。励起用光源１２には白色光源を用い、偏光子１３によってTE偏光とTM偏光の励起方向の切替え、反射ミラー１４によって倒立型の励起用対物レンズ１５へ入射光３を照射させる。微小球１は、ディッシュ底面に貼りつけた平面状基板７に滴下し配置させる。入射光３は大きな開口数（NA）を持つ励起用対物レンズ１５により入射するため、平面状基板７への入射光３の角度は全反射角度以上となり、平面状基板７の表面上に局在波であるエバネセント光２が発生する。励起用対物レンズ１５と平面状基板７の間をイマージョンオイル１６で満たして、焦点が微小球１に合うようにするために、平面状基板７の厚みは１７０μm以下であることが望ましい。大きな開口数（NA）を持つ励起用対物レンズ１５を用いることで、入射光３のスポット径は微小球１の直径以下となり、微小球１以外からの迷光を検出せず、高いS/N比で検出が可能となった。また、標的酵素９を滴下した検出部を封止ガラス１７で被うことで、溶液の蒸発と厚みムラを抑えることが可能となり、高精度なスペクトルの検出が可能となった。微小球１からの散乱光は上部に取り付けられた検出用レンズ１８により集光し、X軸移動ミラー１９によりCCDカメラ２０の観測と分光器２１のスペクトル検出に切替えながら評価する。 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 excitation light is switched between TE polarized light and TM polarized light by the polarizer 13, and the incident light 3 is irradiated to the inverted excitation objective lens 15 by the reflection mirror 14. The microsphere 1 is dropped and disposed on the planar substrate 7 attached to the bottom of the dish. Since the incident light 3 is incident by the excitation objective lens 15 having a large numerical aperture (NA), the angle of the incident light 3 on the planar substrate 7 is equal to or greater than the total reflection angle, and is localized on the surface of the planar substrate 7. Evanescent light 2 that is a wave is generated. In order to fill the space between the excitation objective lens 15 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 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. Detection became possible. Further, by covering the detection portion where 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 to detect the spectrum with high accuracy. Scattered light from the microsphere 1 is collected by a detection lens 18 mounted on the upper part, and evaluated while switching between observation by a CCD camera 20 and spectrum detection by a spectroscope 21 by an X-axis moving mirror 19.

図５に示すように、凹状形状溝１１がない平面状基板７の上で抗原抗体反応をした微小球１の散乱光を検出すると、TE偏光励起にも関わらず、TM偏光にも対応した共振ピーク波長が検出され易い。しかし、凹状形状溝１１を加工した上で微小球１を励起すると、偏光方向に対応した共振ピーク波長しか検出されず、シフト量を精度良く見積もることができる。また、平面状基板７の接触部４からの再放射光１０が少なくなるため、共振ピーク波長の強度が約２倍となり、S/N比を向上することができる。なお、分光器２１で検出した散乱光は、共振ピーク波長が約１〜４nm長波長側へシフトするため、分光器の分解能は１nm以下であることが望ましい。 As shown in FIG. 5, when the scattered light of the microsphere 1 that has undergone an antigen-antibody reaction is detected on a planar substrate 7 that does not have the concave groove 11, resonance corresponding to TM polarization is performed in spite of TE polarization excitation. The peak wavelength is easy to detect. However, when the microsphere 1 is excited after the concave groove 11 is processed, only the resonance peak wavelength corresponding to the polarization direction is detected, and the shift amount can be accurately estimated. Further, since the re-radiated 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. The scattered light detected by the spectroscope 21 has a resonance peak wavelength shifted to the longer wavelength side by about 1 to 4 nm. Therefore, the resolution of the spectroscope is desirably 1 nm or less.

図６は、微生物汚染の検出方法および装置の実施例を示している。WGM光検出部２２から取得した散乱光は光ファイバー２３で導光され、分光器２１で検出される。分光器２１により光信号から変換された電気信号は、波長変化検出部２４により共振ピーク波長の変化量が算出され、微生物汚染判定部２５により微小球１の表面に標的の汚染物質が結合したことを判定できる。 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. The electrical signal converted from the optical signal by the spectroscope 21 is calculated by the change in the resonance peak wavelength 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.

以下実証実験の状況を説明する。 The status of the demonstration experiment is described below.

（１）目的とする酵素
大腸菌群は食品の衛生分野において、汚染指標菌として広く用いられている。大腸菌群が糖を分解する時に産生する酵素の一種のβ-D-ガラクトシダーゼ（和光純薬工業社製）を使用した。 (1) The target enzyme coliform 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）抗体
目的とするβガラクトシダーゼ（β-galactosidase）と反応するためにRabbit IgGのポリクロナール抗体（医学生物学研究所社製）を使用した。 (2) Rabbit IgG polyclonal antibody (manufactured by Institute of Medical Biology) was used to react with the target β-galactosidase.

（３）使用する照射光
使用した励起用光源１２は、Technology社の白色光源(１７０〜２１００nm)を使用した。シグマ光機社製の偏光子１３(グラントムソンプリズム)を９０°回転することにより、ランダム偏光からTEまたはTMの偏光方向を選択した。また、タンパク質がダメージを受ける紫外光や、照射部の温度が上昇する赤外光は、シグマ光機社製のシャープカットフィルターとコールドフィルターを使用し、主に５００〜６００nmの波長域で評価を実施した。 (3) Excitation light source 12 used was a white light source (170-2100 nm) from Technology. The polarization direction of TE or TM was selected from random polarization by rotating a polarizer 13 (Gran Thompson prism) manufactured by Sigma Kogyo Co., Ltd. by 90 °. In addition, ultraviolet light that damages proteins and infrared light that raises the temperature of the irradiated area are evaluated using a sharp cut filter and cold filter manufactured by Sigma Koki Co., Ltd., mainly in the wavelength range of 500 to 600 nm. Carried out.

（４）平面状基板の切削方法
平面状基板７への凹状形状溝１１の切削は、マイクロサポート社のハードメタル製のプローブ(先端直径５μm)を使用した。ナリシゲ製の三次元液圧マイクロマニピュレータ(最小駆動距離１μm)に組み合わせ操作することで、線幅が5μm以下の凹状形状溝１１を作製した。 (4) Planar substrate cutting method The concave groove 11 was cut into the planar substrate 7 by using a hard metal probe (tip diameter: 5 μm) manufactured by Micro Support. 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) manufactured by Narishige.

（５）微小球プローブの作製と評価方法
直径１０μmのカルボキシル基が修飾されたPS微小球（micromod社製）を２回洗浄した後、水溶性のカルボジイミド（Polysciences社製）によりカルボキシル基を活性化させ、抗体を微小球の表面に固定化した。６０分間程度反応した後、遠心分離機で上澄み部を取り除くことで抗体が固定化した微小球を含む溶液を作製した。 (5) Preparation of microsphere probe and evaluation method After washing PS microsphere (manufactured by micromod) with 10 μm diameter carboxyl group modified twice, the carboxyl group is activated by water-soluble carbodiimide (manufactured by Polysciences). And 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 microspheres with immobilized antibodies.

作製した微小球は、図４のガラスベースディッシュ（Iwaki社製）の底面に貼りつけた平面状基板７の上に配置し、１００μl程度同量のβ-D-ガラクトシダーゼを滴下する。封止ガラス１７で検出部を被い白色光源で微小球を励起し、５７０〜６００nmの波長領域で散乱光スペクトルを検出した。 The produced microspheres are placed on the planar substrate 7 attached to the bottom of the glass base dish (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, the microsphere was excited with a white light source, and the scattered light spectrum was detected in a wavelength region of 570 to 600 nm.

β-D-ガラクトシダーゼを滴下し１５分間反応させた後、散乱光スペクトルの中に微小球の表面状態を反映した光共振反応を示すWGMの共振ピーク波長を検出できた。共振ピーク波長の位置は、TE偏光の共振ピーク波長がTM偏光の共振ピーク波長より２〜３nm長波長側に取得できる。 After β-D-galactosidase was added dropwise and allowed to react for 15 minutes, the resonance peak wavelength of WGM showing an optical resonance reaction reflecting the surface state of the microsphere in the scattered light spectrum could be detected. The position of the resonance peak wavelength can be acquired from the resonance peak wavelength of TE polarized light by 2 to 3 nm longer than the resonance peak wavelength of TM polarized light.

検出した共振ピーク波長は、図２のグラフで示したMie理論による散乱断面積とフィッティングすることで付着物の屈折率や厚みが見積れ、抗体や抗原の仕様とも一致した。図５のグラフに示すように、凹状形状溝１１の上で抗原抗体反応をした微小球１を励起し散乱光を検出すると、スプリット現象を抑えた共振ピーク波長が検出できる。さらには、平面状基板７の接触部４からの再放射光１０を抑えることができるため、散乱光から検出される共振ピーク波長の強度は約２倍となり、S/N比を向上することができた。 The detected resonance peak wavelength was fitted with the scattering cross section by the Mie theory shown in the graph of FIG. 2 to estimate the refractive index and thickness of the deposit, and matched the specifications of the antibody and antigen. As shown in the graph of FIG. 5, when the microsphere 1 that has undergone an antigen-antibody reaction on the concave groove 11 is excited and scattered light is detected, the resonance peak wavelength with the split phenomenon suppressed can be detected. Furthermore, since the re-radiated 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.

図６の波長変化検出部２４により、抗原抗体反応の前後で共振ピーク波長は約１〜４nm長波長側へシフトする。分光器２１の波長分解能を1nmに設定し、微生物検出判定部１８により微生物汚染を判定すると、判定時間は8分、検出下限濃度は5μg/mLとなる。 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 detection unit 24 of FIG. When the wavelength resolution of the spectroscope 21 is set to 1 nm and the microorganism detection determination unit 18 determines microorganism contamination, the determination time is 8 minutes and the detection lower limit concentration is 5 μg / mL.

上記に述べたように、本発明によって、高精度で高感度な微小球センサーの検出手法を用いることで迅速に微生物を検出できる装置を提供することが可能となった。 As described above, according to the present invention, it is possible to provide an apparatus capable of rapidly detecting microorganisms by using a highly accurate and sensitive microsphere sensor detection technique.

本発明は、微生物の検査が必要な食品分野における検査方法に関するものであるが、環境衛生分野、医薬品分野等での利用も可能であり、分野において、バイオセンサー、化学センサー、マイクロ流路などの簡便、迅速、安価な検査判定として利用することが可能となる。さらには、高価な検出装置等を用いることなく、高感度で高精度、しかも低コスト検査チップを実現できる微小球共振センサーを使用する微生物検査を提供することができる。 The present invention relates to an inspection method in the field of food that requires microbiological inspection, but can also be used in the environmental health field, pharmaceutical field, etc., and in the field, biosensors, chemical sensors, microchannels, etc. It can be used as simple, quick, and inexpensive inspection determination. Furthermore, it is possible to provide a microorganism test using a microsphere resonance sensor that can realize a high-sensitivity, high-precision, and low-cost test chip without using an expensive detection device or the like.

１ 微小球
２ エバネセント光
３ 入射光
４ 接触部
５ 励起方向WGM
６ 斜め方向WGM
７ 平面状基板
８ 抗体
９ 標的酵素
１０ 再放射光
１１ 凹状形状溝
１２ 励起用光源
１３ 偏光子
１４ 反射ミラー
１５ 励起用対物レンズ
１６ イマージョンオイル
１７ 封止ガラス
１８ 検出用レンズ
１９ X軸移動ミラー
２０ CCDカメラ
２１ 分光器
２２ WGM光検出部
２３ 光ファイバー
２４ 波長変化検出部
２５ 微生物汚染判定部

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-shaped 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 Spectrometer 22 WGM light detection unit 23 Optical fiber 24 Wavelength change detection unit 25 Microorganism contamination determination unit

Claims (5)

基板上に微小球を配置し、基板から染み出したエバネセント光により微小球表面に励起されるウィスパリングギャラリーモードを共振し、微小球の散乱光を検出する方法および装置であって、
前記基板と微小球のウィスパリングギャラリーモードの軌道が接触しないように基板上に微小球を配置することを特徴とする散乱光の検出方法および装置。 A method and apparatus for detecting scattered light of a microsphere by arranging a microsphere on a substrate, resonating a whispering gallery mode excited on the surface of the microsphere by evanescent light oozing out from the substrate,
A method and apparatus for detecting scattered light, wherein microspheres are arranged on a substrate so that a whispering gallery mode orbit of the substrate and microspheres does not contact each other. 溝が形成された基板上に微小球を配置することを特徴とする請求項１記載の散乱光の検出方法および装置。 2. The method and apparatus for detecting scattered light according to claim 1, wherein microspheres are arranged on a substrate on which grooves are formed. 溝が微小球径よりも幅狭に形成され、該溝上に配置された微小球のズレを抑制することを特徴とする請求項２記載の散乱光の検出方法および装置。 3. The scattered light detection method and apparatus according to claim 2, wherein the groove is formed to be narrower than the diameter of the microsphere, and the displacement of the microsphere arranged on the groove is suppressed. 光源からの照射光を微小球径以下に集光しエバネセント光を発生できる励起用対物レンズと、微小球からの散乱光スペクトルを検出できる検出用対物レンズを備えウィスパリングギャラリーモードの共振ピーク波長を検出できる分解能を有する分光器を備えた微小球共振センサーを用いることを特徴とする請求項１〜３のいずれか記載の散乱光の検出方法および装置。 Equipped with an excitation objective lens that can generate evanescent light by condensing the light emitted from the light source below the microsphere diameter, and a detection objective lens that can detect the scattered light spectrum from the microsphere. The method and apparatus for detecting scattered light according to any one of claims 1 to 3, wherein a microsphere resonance sensor including a spectroscope having a resolution capable of detection is used. 請求項１〜４のいずれか記載の散乱光の検出方法を用いた対象溶液中の微生物汚染の検出方法および装置であって、
抗体が固定化された微小球、および、抗体が固定化された微小球に溶液を接触させた微小球のウィスパリングギャラリーモードの共振ピーク波長を検出し、その共振ピーク波長の変化を検出することを特徴とする対象溶液中の微生物汚染の検出方法および装置。 A method and apparatus for detecting microbial contamination in a target solution using the method for detecting scattered light according to any one of claims 1 to 4,
Detecting the resonance peak wavelength of whispering gallery mode of microspheres with immobilized antibodies and microspheres with solutions in contact with microspheres with immobilized antibodies, and detecting changes in the resonance peak wavelength A method and apparatus for detecting microbial contamination in a target solution.
JP2016049128A 2016-03-14 2016-03-14 Method and apparatus for highly sensitive detection of microsphere resonance sensor Expired - Fee Related JP6682307B2 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109990975A (en) * 2019-04-10 2019-07-09 暨南大学 Detection system, debugging system and sensor based on optical microcavity mechanical mode
CN110596814A (en) * 2018-06-12 2019-12-20 中国计量大学 Optical fiber corrosion groove type echo wall resonator based on microspheres
CN112683793A (en) * 2020-12-09 2021-04-20 哈尔滨工程大学 Sensor for detecting concentration of liquid drops based on double-microsphere coupling mode splitting

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110596814A (en) * 2018-06-12 2019-12-20 中国计量大学 Optical fiber corrosion groove type echo wall resonator based on microspheres
CN110596814B (en) * 2018-06-12 2021-06-15 中国计量大学 Optical fiber corrosion groove type echo wall resonator based on microspheres
CN109990975A (en) * 2019-04-10 2019-07-09 暨南大学 Detection system, debugging system and sensor based on optical microcavity mechanical mode
CN109990975B (en) * 2019-04-10 2021-04-23 暨南大学 Detection system, debugging system and sensor based on optical microcavity mechanical mode
CN112683793A (en) * 2020-12-09 2021-04-20 哈尔滨工程大学 Sensor for detecting concentration of liquid drops based on double-microsphere coupling mode splitting

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