JP2012042290A - Device and method for measuring amount of micro objects - Google Patents
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本発明は、微小物体量測定装置および微小物体量測定方法に関するものである。 The present invention relates to a minute object amount measuring apparatus and a minute object amount measuring method.
水中に存在する微小物体、具体的には大きさ０．１μｍから１０μｍ程度の細菌やプラスチック粒子の量を測定する方法として、電気浸透流を用いた検出方法や誘電泳動力を用いた検出方法が提案されている。 Detection methods using electroosmotic flow and detection methods using dielectrophoretic force are methods for measuring the amount of minute objects present in water, specifically bacteria and plastic particles having a size of about 0.1 μm to 10 μm. Proposed.
誘電泳動力とは、不均一電場中において微小物体に誘起されたダイポールモーメントに働く力である。その強度は電場強度の勾配に比例おり、微小物体の誘電率と溶媒の誘電率の大小関係および電極に印加する電圧の周波数によって、電極に対して引力または斥力が働く。例えば、ポリスチレンの微粒子を純水中に懸濁させたポリスチレン懸濁液の場合、この力は１００ｋＨｚで引力となり、２ＭＨｚで斥力となる。引力となる場合は、電場強度が強い電極のエッジ部近傍に微粒子が捕集され、斥力が働く場合は、電場強度強い電極近傍から、微小物体は離れていき、電場強度が弱い位置に集まる。上記の引力の働く場合を正の誘電泳動いい、斥力が働く場合を負の誘電泳動という。 The dielectrophoretic force is a force acting on a dipole moment induced in a minute object in a non-uniform electric field. The strength is proportional to the gradient of the electric field strength, and an attractive force or a repulsive force acts on the electrode depending on the magnitude relationship between the dielectric constant of the micro object and the dielectric constant of the solvent and the frequency of the voltage applied to the electrode. For example, in the case of a polystyrene suspension in which polystyrene fine particles are suspended in pure water, this force is attractive at 100 kHz and repulsive at 2 MHz. When attracting, fine particles are collected in the vicinity of the edge portion of the electrode having a high electric field strength. When repulsive force is applied, the minute object moves away from the vicinity of the electrode having a strong electric field strength and collects at a position where the electric field strength is weak. The case where the above attractive force works is called positive dielectrophoresis, and the case where repulsive force works is called negative dielectrophoresis.
また、電気浸透流とは、電極近傍に形成される電気二重層に存在するイオンに電気力が働き、当該イオンが動き出すことによって生じる流体の流れのことである。電極に電圧が印加されると印加された電圧と反対の極性を持つイオンが電極近傍に集まり、印加された電圧と同極性をもつイオンは遠ざけられ、電極近傍に電気二重層が形成される。当該イオンには、電極印加された電圧から生じた電場により電気力が働き、電極面近傍で流体の流れが生じる。この流れが電気浸透流である。ここで電極近傍に微小物体があると、上記により発生した流体の流れにより、微小物体は流される。電気浸透流による力の大きさは電極のエッジ部近傍で特に大きく電極中心部に近づくにつれて小さくなる。よって、微小物体は電極上の中央近傍に誘導される。 The electroosmotic flow is a fluid flow generated by an electric force acting on ions existing in an electric double layer formed in the vicinity of an electrode and the ions starting to move. When a voltage is applied to the electrode, ions having a polarity opposite to the applied voltage gather near the electrode, ions having the same polarity as the applied voltage are moved away, and an electric double layer is formed near the electrode. An electric force acts on the ions due to an electric field generated from the voltage applied to the electrode, and a fluid flows near the electrode surface. This flow is an electroosmotic flow. Here, if there is a minute object in the vicinity of the electrode, the minute object is caused to flow by the fluid flow generated as described above. The magnitude of the force due to electroosmotic flow is particularly large in the vicinity of the edge portion of the electrode and decreases as the electrode central portion is approached. Therefore, the minute object is guided near the center on the electrode.
水中の微小物体に働く力は、上記誘電泳動力と電気浸透流による力の合力が働くことになる。これらの力はそれぞれ印加される交流電圧の周波数の関数となっているため、交流電圧の周波数によって支配的になる力が異なる。例えば、純水中において、電極に印加する電圧の周波数を１００Ｈｚ〜１０ｋＨｚとすると、微小物体に働く力は電気浸透流による力が他の力と比較して支配的となる。 The force acting on the minute object in water is the resultant force of the dielectrophoretic force and the force generated by electroosmotic flow. Since each of these forces is a function of the frequency of the applied AC voltage, the force that is dominant depends on the frequency of the AC voltage. For example, in pure water, when the frequency of the voltage applied to the electrode is 100 Hz to 10 kHz, the force acting on the minute object is dominant as compared with the other forces.
特許文献１には、表面プラズモン共鳴により微小物体の検出を行う方法が記載されている。表面プラズモンとは、金属膜上に励起される自由電子と光の結合モードである。プリズム上に金属膜が配置されており、金属膜上に被検出物が接している場合を考える。プリズムを介して金属膜に光を照射すると、全反射角以上の入射角で入射された光は、全反射され、エバネッセント波が生じる。エバネッセント波は金属膜を減衰しながら透過し、金属膜と被検出物の接する界面まで到達する。このうち特定の入射角（以下、表面プラズモン共鳴角と言う）で入射された場合、金属膜と被検出物の界面に表面プラズモンが励起される。この特定の入射角で波長程度の光の染み出しが生じる。このとき、光の波数と表面プラズモンの波数および周波数が整合しており、共鳴状態にある。表面プラズモンは、金属膜と被検出物の界面を減衰しながら伝搬するため、表面プラズモン共鳴角における反射光の強度は減少する。表面プラズモン共鳴角は、金属膜の表面近傍の誘電率変化に応じて敏感に変化する。従って、表面プラズモン共鳴角の変化量を測定することで、金属表面に付着した物質の濃度を高感度に定量することができる。 Patent Document 1 describes a method of detecting a minute object by surface plasmon resonance. Surface plasmon is a coupled mode of free electrons and light excited on a metal film. Consider a case where a metal film is disposed on a prism and an object to be detected is in contact with the metal film. When the metal film is irradiated with light through the prism, light incident at an incident angle greater than the total reflection angle is totally reflected to generate an evanescent wave. The evanescent wave passes through the metal film while being attenuated, and reaches the interface between the metal film and the object to be detected. Among these, when incident at a specific incident angle (hereinafter referred to as a surface plasmon resonance angle), the surface plasmon is excited at the interface between the metal film and the object to be detected. At this specific incident angle, the light leaks out at the wavelength. At this time, the wave number of the light and the wave number and frequency of the surface plasmon are matched and are in a resonance state. Since the surface plasmon propagates while attenuating the interface between the metal film and the object to be detected, the intensity of the reflected light at the surface plasmon resonance angle decreases. The surface plasmon resonance angle changes sensitively according to a change in dielectric constant near the surface of the metal film. Therefore, by measuring the amount of change in the surface plasmon resonance angle, the concentration of the substance attached to the metal surface can be quantified with high sensitivity.
図１０に、特許文献１に開示された定性定量分析装置に係る模式図を示す。図１０において、光源１０１から発せられた入射光１０５は、第一のレンズ１０２、第二のレンズ１０３、プリズム１０４、ガラス１０６を経由して第一の金属薄膜１０７に照射される。第一の金属薄膜１０７で反射された反射光１０８は、再度ガラス１０６、プリズム１０４を透過し光検出器１０９へと照射され、ここで入射角毎の光量検出が行われる。またガラス１０６は、被分析溶液１１０を保持する容器１１１の一部を成している。この光学測定系を用い、反射光１０８の光量が最も減少する角度、すなわち表面プラズモン共鳴が発生する条件を満たす入射光１０５の角度を測定する。 ガラス１０６が被分析溶液１１０と接触する面上には第一の金属薄膜１０７のほかに、第二の金属薄膜１１２と第三の金属薄膜１１３とが保持されており、これら二つの金属薄膜１１２、１１３はそれぞれ交流電源１１４と接続されていて電極対を成している。これら電極対間に交流電圧を印加し、被分析溶液１１０に含まれている分子や粒子に誘電泳動力を働かせて、表面プラズモン共鳴を発生する第一の金属薄膜１０７上に分子や粒子を集める。以上のような構成を用いることで、分子や粒子の濃度を局所的に高くさせ、より高感度な表面プラズモン共鳴を利用した屈折率測定を行うことができる。 FIG. 10 is a schematic diagram related to the qualitative quantitative analysis apparatus disclosed in Patent Document 1. In FIG. 10, incident light 105 emitted from a light source 101 is applied to a first metal thin film 107 via a first lens 102, a second lens 103, a prism 104, and a glass 106. The reflected light 108 reflected by the first metal thin film 107 passes through the glass 106 and the prism 104 again and is irradiated to the photodetector 109, where the light amount is detected for each incident angle. The glass 106 forms a part of the container 111 that holds the solution 110 to be analyzed. Using this optical measurement system, the angle at which the amount of the reflected light 108 decreases most, that is, the angle of the incident light 105 that satisfies the condition for generating surface plasmon resonance is measured. In addition to the first metal thin film 107, a second metal thin film 112 and a third metal thin film 113 are held on the surface where the glass 106 is in contact with the analysis solution 110, and these two metal thin films 112 are held. , 113 are each connected to an AC power source 114 to form an electrode pair. An alternating voltage is applied between these electrode pairs, and dielectrophoretic force is applied to the molecules and particles contained in the solution to be analyzed 110 to collect the molecules and particles on the first metal thin film 107 that generates surface plasmon resonance. . By using the configuration as described above, it is possible to locally increase the concentration of molecules and particles and perform refractive index measurement using surface plasmon resonance with higher sensitivity.
また、光検出器で得られた測定結果から屈折率の演算を行うための制御演算装置を用い、電極間に印加する交流電圧の周波数と、この交流電圧の電圧値と、この交流電圧の印加時間と、光検出器で得られた測定結果から求めた屈折率とから、被分析溶液中の分子や粒子の量を計測することを行うことができる。 Also, using a control arithmetic unit for calculating the refractive index from the measurement result obtained by the photodetector, the frequency of the AC voltage applied between the electrodes, the voltage value of this AC voltage, and the application of this AC voltage The amount of molecules and particles in the solution to be analyzed can be measured from the time and the refractive index obtained from the measurement result obtained by the photodetector.
さらに、室温の変動や通電による被分析溶液１１０の温度変動を補正する目的で、温度計測器１１６に接続された温度計測プローブ１１５が第一の金属薄膜１０７の極近傍に設けられている。 Further, a temperature measuring probe 115 connected to the temperature measuring instrument 116 is provided in the immediate vicinity of the first metal thin film 107 for the purpose of correcting the temperature fluctuation of the solution 110 to be analyzed due to the fluctuation of the room temperature or the energization.
特許文献１記載の技術は、屈折率に基づき、被分析溶液中の分子または粒子の量を計測する。前記計測においては、被分析溶液が、計測対象である微小物体（特許文献１における粒子または分子）と溶媒との混合物であり、かつ、微小物体の誘電率および溶媒の誘電率が所定の値であることを前提とする。 The technique described in Patent Document 1 measures the amount of molecules or particles in a solution to be analyzed based on the refractive index. In the measurement, the solution to be analyzed is a mixture of a minute object (particles or molecules in Patent Document 1) to be measured and a solvent, and the dielectric constant of the minute object and the dielectric constant of the solvent are predetermined values. It is assumed that there is.
微小物体の誘電率および溶媒の誘電率が所定の値であり、かつ微小物体の誘電率と溶媒の誘電率とが異なれば、微小物体と溶媒との混合比によって、被分析溶液全体の誘電率は一意に定まる。また、屈折率は誘電率の１／２乗に比例する。従って、微小物体の誘電率、溶媒の誘電率、被分析溶液の屈折率により、微小物体と溶媒との混合比、すなわち、被分析溶液における微小物体の量を求めることができる。 If the dielectric constant of the minute object and the dielectric constant of the solvent are the predetermined values, and the dielectric constant of the minute object and the solvent are different, the dielectric constant of the entire solution to be analyzed depends on the mixing ratio of the minute object and the solvent. Is uniquely determined. The refractive index is proportional to the 1/2 power of the dielectric constant. Therefore, the mixing ratio between the micro object and the solvent, that is, the amount of the micro object in the solution to be analyzed can be obtained from the dielectric constant of the micro object, the dielectric constant of the solvent, and the refractive index of the solution to be analyzed.
ここで問題となるのは、溶媒の誘電率および屈折率である。従来技術においては、溶媒の誘電率が所定の値であることを前提としているため、溶媒の誘電率が特定できない場合には、被分析溶液における微小物体の量を求めることはできない。 The problem here is the dielectric constant and refractive index of the solvent. In the prior art, since it is assumed that the dielectric constant of the solvent is a predetermined value, if the dielectric constant of the solvent cannot be specified, the amount of minute objects in the solution to be analyzed cannot be obtained.
溶媒の誘電率および屈折率が変化することは珍しいことでは無い。例えば、表面プラズモンが励起される金属部分は，自由電子によるジュール損があるため温度が上昇し、溶媒の温度も上昇する。溶媒が水の場合、温度が１℃上昇すると屈折率は０．００００１低下する。また，被分析溶液が血液である場合は、人によって溶媒である血漿の誘電率および屈折率が異なる。また、溶媒が水道水である場合は、その取水場所等によってイオン濃度等が異なるため、溶媒の誘電率および屈折率が異なる。従って、特許文献１記載の技術においては、溶媒の誘電率が変化したことにより、測定結果に誤差が生じることが珍しくなかった。 It is not uncommon for the dielectric constant and refractive index of the solvent to change. For example, the metal portion where surface plasmons are excited has a Joule loss due to free electrons, so the temperature rises and the temperature of the solvent also rises. When the solvent is water, the refractive index decreases by 0.00001 when the temperature rises by 1 ° C. Further, when the solution to be analyzed is blood, the dielectric constant and refractive index of plasma, which is a solvent, vary depending on the person. Further, when the solvent is tap water, since the ion concentration and the like vary depending on the place of water intake and the like, the dielectric constant and refractive index of the solvent differ. Therefore, in the technique described in Patent Document 1, it is not uncommon for errors in measurement results to occur due to changes in the dielectric constant of the solvent.
以上の問題に鑑み、本発明は、溶媒の温度が変化した場合やイオン濃度が異なる溶媒を用いた場合にあっても、被分析溶液中の微小物体の量を正確に測定することができる微小物体量測定装置ならびに微小物体量測定方法を提供することを目的とする。 In view of the above problems, the present invention is capable of accurately measuring the amount of minute objects in a solution to be analyzed even when the temperature of the solvent changes or when solvents having different ion concentrations are used. An object of the present invention is to provide an object amount measuring apparatus and a minute object amount measuring method.
上記課題を解決するために、本発明の微小物体量測定装置は、前記液体に接するように配置された第１電極と、前記液体に接するように配置された第２電極と、前記第１電極または前記第２電極に交流電圧を印加する交流電源と、前記交流電源の周波数を制御する周波数制御手段と、前記第１電極の測定部に接する前記液体の反射光の強度を検出する検出手段と、を備え、前記検出手段により、前記第１電極または第２電極に、前記測定部から微小物体を排除するような第１の周波数の交流電圧を印加して、第１信号を検出し、前記第１電極または第２電極に、前記測定部に微小物体を凝集するような第２の周波数の交流電圧を印加して、前記検出手段により第２信号を検出し、前記第１信号と前記第２信号により微小物体の量を算出することを特徴とする。 In order to solve the above problems, a minute object amount measuring apparatus according to the present invention includes a first electrode disposed so as to be in contact with the liquid, a second electrode disposed so as to be in contact with the liquid, and the first electrode. Or an AC power supply for applying an AC voltage to the second electrode, a frequency control means for controlling the frequency of the AC power supply, and a detection means for detecting the intensity of the reflected light of the liquid in contact with the measurement part of the first electrode. The first signal or the second electrode is applied to the first electrode or the second electrode with an AC voltage having a first frequency that excludes a minute object from the measurement unit, and the first signal is detected. An AC voltage having a second frequency that agglomerates a minute object on the measurement unit is applied to the first electrode or the second electrode, the second signal is detected by the detection means, and the first signal and the second electrode are detected. Calculate the amount of minute objects using two signals The features.
また、前記検出手段は、表面プラズモン共鳴を用いたものであることを特徴とする。 The detecting means uses surface plasmon resonance.
また、前記第２の周波数は電気浸透流が発生する周波数であることを特徴とする。 The second frequency is a frequency at which electroosmotic flow is generated.
また、前記第１の周波数を印加した時間が長い場合に、第１信号と前記第２信号との差に対して微小物体の量を少なく算出することを特徴とする。 In addition, when the time during which the first frequency is applied is long, the amount of the minute object is calculated to be small with respect to the difference between the first signal and the second signal.
また、本発明の微小物体量測定方法は、前記液体に接するように配置された第１電極と、
前記液体に接するように配置された第２電極と、前記第１電極または前記第２電極の少なくとも一方に交流電圧を印加する交流電源と、前記交流電源の周波数を選択する周波数制御手段と、前記第１電極の測定部Ｓに接する前記液体の誘電率に関係する信号を検出する検出手段と、を備える微小物体量測定装置を用いて、微小物体が懸濁した液体における微小物体の量を測定する微小物体量測定方法であって、前記第１電極または第２電極の少なくとも一方に第１の周波数の交流電圧を印加して、前記測定部Ｓから微小物体を排除するステップと、前記検出手段により第１信号を検出するステップと、前記第１電極または第２電極の少なくとも一方に第２の周波数の交流電圧を印加して、前記測定部Ｓに微小物体を凝集するステップと、前記検出手段により第２信号を検出するステップと、前記第１信号と前記第２信号の差により微小物体の量を算出するステップとを備えること特徴とする。
Further, the method for measuring a minute object amount according to the present invention includes a first electrode disposed so as to be in contact with the liquid,
A second electrode disposed in contact with the liquid, an AC power supply for applying an AC voltage to at least one of the first electrode or the second electrode, a frequency control means for selecting a frequency of the AC power supply, And a detecting unit that detects a signal related to the dielectric constant of the liquid in contact with the measuring unit S of the first electrode, and measures the amount of the minute object in the liquid in which the minute object is suspended. A method for measuring a minute object amount, the step of applying an alternating voltage of a first frequency to at least one of the first electrode or the second electrode to exclude the minute object from the measuring unit S, and the detecting means Detecting a first signal by applying an alternating voltage of a second frequency to at least one of the first electrode or the second electrode to agglomerate micro objects on the measurement unit S; Detecting a second signal by means output, and wherein further comprising the step of calculating the amount of the minute object by the difference between the first signal and the second signal.
また、前記微小物体の量を算出するステップにおいて、前記第１の周波数を印加した時間が長い場合に微小物体の量を少なく算出することを特徴とする。 In the step of calculating the amount of the minute object, the amount of the minute object is calculated to be small when the time during which the first frequency is applied is long.
溶媒の温度が変化した場合やイオン濃度が異なる溶媒を用いた場合にあっても、被分析溶液中の微小物体の量を正確に測定することができる。 Even when the temperature of the solvent changes or when solvents having different ion concentrations are used, the amount of minute objects in the solution to be analyzed can be accurately measured.
以下、本発明の実施形態である微小物体量測定装置および微小物体量測定方法について、以下に詳細に説明する。 Hereinafter, a minute object amount measuring apparatus and a minute object amount measuring method according to embodiments of the present invention will be described in detail.
（装置の構成）
図１は本実施例に係る微小物体量測定装置の側方断面図を概略的に示したものである。本実施例に係る微小物体量測定装置は光源１とコリメートレンズ２と集光レンズ３とプリズム４とプリズム４上に配置された第１電極５と第２電極６とコリメートレンズ７と光検出部８と周波数制御手段９と交流電源１０とを備える。
(Device configuration)
FIG. 1 schematically shows a side sectional view of a minute object amount measuring apparatus according to the present embodiment. The minute object amount measuring apparatus according to the present embodiment includes a light source 1, a collimating lens 2, a condenser lens 3, a prism 4, a first electrode 5, a second electrode 6, a collimating lens 7, and a light detecting unit disposed on the prism 4. 8, frequency control means 9, and AC power supply 10.
第１電極５および第２電極６が配置された面には、ミクロセル（図示せず）または、フローセル（図示せず）が配置され、ミクロセルまたはフローセルに被測定物である液体が収納された場合に、第１電極５および第２電極６が前記液体に接するよう配置される。 When a micro cell (not shown) or a flow cell (not shown) is arranged on the surface on which the first electrode 5 and the second electrode 6 are arranged, and a liquid to be measured is stored in the micro cell or the flow cell. In addition, the first electrode 5 and the second electrode 6 are arranged in contact with the liquid.
光源１は、半導体レーザ、外部共振半導体レーザ、固体レーザ、ガスレーザのいずれを用いてもよい。また、単色性の良いものであれば、ＬＥＤであってもよい。 As the light source 1, any of a semiconductor laser, an external resonance semiconductor laser, a solid-state laser, and a gas laser may be used. Further, an LED may be used as long as it has good monochromaticity.
プリズム４は、ガラス、プラスチック、いずれの材料でもよい。光源１が出射する光に対する吸収率が低い材料を用いれば検出感度が良くなり、また屈折率の高い材料を用いれば検出精度が良くなるので、光源１が発する光量と波長、被測定物である液体およびその濃度、要求する検出精度、検出感度等に応じて適切な材料を選択すれば良い。 The prism 4 may be made of any material such as glass or plastic. The detection sensitivity is improved by using a material having a low absorptance with respect to the light emitted from the light source 1, and the detection accuracy is improved by using a material having a high refractive index. An appropriate material may be selected according to the liquid and its concentration, the required detection accuracy, detection sensitivity, and the like.
コリメートレンズ２、集光レンズ３およびプリズム４は、光源１から発する光線ＬＢの経路Ｃ１上に配置されている。コリメートレンズ２により光線ＬＢは平行光となり集光レンズ３に入射し集光されつつプリズム４に入射し、プリズム４上に配置された第１電極５のセンサ部の中央に位置する測定部Ｓに集光される。センサ部に入射する光線はＰ偏光とする。 The collimating lens 2, the condenser lens 3, and the prism 4 are disposed on the path C <b> 1 of the light beam LB emitted from the light source 1. The collimating lens 2 converts the light beam LB into parallel light that enters the condensing lens 3, enters the prism 4 while being collected, and enters the measuring unit S located at the center of the sensor unit of the first electrode 5 disposed on the prism 4. Focused. The light beam incident on the sensor unit is P-polarized light.
測定部Ｓに集光された光線は、測定部Ｓに反射され、光検出部８に受光される。光検出部８はＣＣＤやＣＭＯＳの１次元センサが適している。ＣＣＤやＣＭＯＳの２次元のセンサでもよい。また、検出面が複数に分割されたフォトダイオードであってもよい。 The light beam focused on the measurement unit S is reflected by the measurement unit S and received by the light detection unit 8. A CCD or CMOS one-dimensional sensor is suitable for the light detection unit 8. A two-dimensional sensor such as a CCD or CMOS may be used. Moreover, the photodiode by which the detection surface was divided | segmented into plurality may be sufficient.
光源１、コリメートレンズ２、集光レンズ３、プリズム４、第１電極５の測定部Ｓ、光検出部８は、全体として表面プラズモン共鳴センサを構成する。表面プラズモン共鳴センサは、測定部Ｓに接する液体の誘電率に関係する信号を検出する検出手段である。 The light source 1, the collimating lens 2, the condensing lens 3, the prism 4, the measuring unit S of the first electrode 5, and the light detecting unit 8 constitute a surface plasmon resonance sensor as a whole. The surface plasmon resonance sensor is detection means for detecting a signal related to the dielectric constant of the liquid in contact with the measurement unit S.
図２、図３、図４は第１電極５および第２電極６の構造を概略的に示した上面図の例である。第１電極５はセンサ部５ａと配線部５ｂとからなる。センサ部５ａの形状は、図２、図３においてはひし形、図４においては円形であるが、そのいずれの形状でも良い。また、多角形等の他の形状であってもよい。 2, 3, and 4 are examples of top views schematically showing the structures of the first electrode 5 and the second electrode 6. The first electrode 5 includes a sensor part 5a and a wiring part 5b. The shape of the sensor unit 5a is a rhombus in FIGS. 2 and 3 and a circle in FIG. 4, but any shape may be used. Moreover, other shapes, such as a polygon, may be sufficient.
第１電極５は厚さ１００ｎｍ以下の金属の薄膜である。材質としては、金または銀が適しているが、他の金属であっても良い。第１電極５の最適な厚さはその材質によって異なるが、金の場合は、５０ｎｍが最適である。また、第１電極５とプリズム４との密着性を高めるため、間にＴｉやＮｉＣｒ等の下地膜を形成しても良い。 The first electrode 5 is a metal thin film having a thickness of 100 nm or less. As the material, gold or silver is suitable, but other metals may be used. The optimum thickness of the first electrode 5 differs depending on the material, but in the case of gold, 50 nm is optimum. Further, in order to improve the adhesion between the first electrode 5 and the prism 4, a base film such as Ti or NiCr may be formed therebetween.
第２電極６は第１電極５のセンサ部５ａの外周と対向するように、第１電極５と同一面内に配置される。第１電極５の材質・厚さと、第２電極６の材質・厚さとは異なっても良いが、同一の材質・厚さであれば、同一の電極作成プロセスで製造することができる。 The second electrode 6 is disposed in the same plane as the first electrode 5 so as to face the outer periphery of the sensor portion 5 a of the first electrode 5. The material / thickness of the first electrode 5 and the material / thickness of the second electrode 6 may be different, but if they are the same material / thickness, they can be manufactured by the same electrode production process.
第１電極５と第２電極６とのうち、少なくとも一方は交流電源１０に接続されており、交流電圧を印加することができる。他方の電極は接地してもよいが、第１電極５を交流電源１０に接続し、第２電極６を交流電源１０に接続し、第１電極５と第２電極６とに位相の反転した交流電圧を印加可能に構成することが好ましい。交流電圧の波形は、Ｓｉｎ波がもっとも適しているが、矩形波、三角波でも使用可能である。交流電源１０は周波数制御手段９からの命令を受けて周波数を変化させることができる。 At least one of the first electrode 5 and the second electrode 6 is connected to an AC power source 10 and can apply an AC voltage. The other electrode may be grounded, but the first electrode 5 is connected to the AC power source 10, the second electrode 6 is connected to the AC power source 10, and the phase is inverted between the first electrode 5 and the second electrode 6. It is preferable that an AC voltage can be applied. As the AC voltage waveform, a Sin wave is most suitable, but a rectangular wave and a triangular wave can also be used. The AC power supply 10 can change the frequency in response to a command from the frequency control means 9.
なお、第１電極５および第２電極６を配置した基板を設けて、該基板をプリズム４に密着させた構成としてもよい。この場合、プリズム４の屈折率と近い屈折率を有する材料を、基板の材料として用いる。センサ部５ａ以外での反射光を抑制し、迷光や干渉性雑音の影響を除去するため、プリズム４の屈折率または基板の屈折率と屈折率が近い接着材またはマッチングオイルをプリズム４と基板との間に塗布してもよい。 In addition, it is good also as a structure which provided the board | substrate which has arrange | positioned the 1st electrode 5 and the 2nd electrode 6, and made this board | substrate adhere to the prism 4. FIG. In this case, a material having a refractive index close to that of the prism 4 is used as the substrate material. In order to suppress the reflected light other than the sensor unit 5a and remove the influence of stray light and coherent noise, an adhesive or matching oil having a refractive index close to that of the prism 4 or the substrate is used for the prism 4 and the substrate. You may apply between.
（本実施例の処理フロー）
図５に基づき、本実施例に係る微小物体の量を検出する処理フローについて説明する。本処理フローの開始時において、第１電極５および第２電極６が配置された面に配置されたミクロセルまたはフローセルには、微小物体が懸濁した液体が被測定物として収容されており、該液体は、第１電極５および第２電極６に接触している。
(Processing flow of this embodiment)
A processing flow for detecting the amount of a minute object according to the present embodiment will be described with reference to FIG. At the start of the processing flow, the microcell or flow cell disposed on the surface on which the first electrode 5 and the second electrode 6 are disposed contains a liquid in which a minute object is suspended as a measurement object. The liquid is in contact with the first electrode 5 and the second electrode 6.
まず初めに、周波数制御手段９が交流電源１０に第１の周波数を印加するよう信号を発し、これを受けて交流電源１０は第１の周波数とする交流電圧を第１電極５および／または第２電極６に印加する。第１の周波数は微小物体に対し正の誘電泳動を働かせる。正の誘電泳動は、電界強度が弱い所から電界強度の強い所に向かって働く力なので、電界強度の強い所、すなわち第１電極と第２電極との間で、第１電極５と第２電極６との間隔が狭くなっている所に微小物体は集まる（Ｓ１）。 First, the frequency control means 9 issues a signal so as to apply a first frequency to the AC power source 10, and in response to this, the AC power source 10 applies an AC voltage having the first frequency to the first electrode 5 and / or the first frequency. Applied to two electrodes 6. The first frequency exerts positive dielectrophoresis on the minute object. Since the positive dielectrophoresis is a force that works from a place where the electric field strength is weak to a place where the electric field strength is strong, the first electrode 5 and the second electrode 5 are placed between the place where the electric field strength is strong, that is, between the first electrode and the second electrode. The minute objects gather at a place where the distance from the electrode 6 is narrow (S1).
図６は正の誘電泳動により微小物体の捕集された様子を示した模式図である。センサ部５ａの中央に位置する測定部Ｓは電界強度が弱いため、本実施例の処理フロー開始前に測定部Ｓにあった微小物体も電界強度の強いセンサ部５ａの第１電極５と第２電極６との間隔が狭くなっている所に補集される。そのため、測定部Ｓから微小物体が排除される。 FIG. 6 is a schematic diagram showing a state in which minute objects are collected by positive dielectrophoresis. Since the measurement unit S located at the center of the sensor unit 5a has a low electric field strength, the minute object existing in the measurement unit S before the start of the processing flow of the present embodiment is also the first electrode 5 and the first electrode 5 of the sensor unit 5a having a high electric field strength. They are collected where the distance between the two electrodes 6 is narrow. Therefore, the minute object is excluded from the measurement unit S.
この状態で、センサ部５ａの中央に位置する測定部Ｓにおける反射光の強度の測定を行なう。具体的には、光源１が光を出射し、プリズム４とセンサ部５ａの測定部Ｓとの界面で反射した光を光検出部８で受光し、受光した光の強度に対応する信号を取得する。前記信号は、プリズム４と第１電極５との界面への入射角毎に測定する。これを第１信号とする（Ｓ２）。 In this state, the intensity of the reflected light is measured at the measurement unit S located at the center of the sensor unit 5a. Specifically, the light source 1 emits light, the light reflected at the interface between the prism 4 and the measurement unit S of the sensor unit 5a is received by the light detection unit 8, and a signal corresponding to the intensity of the received light is acquired. To do. The signal is measured at every incident angle to the interface between the prism 4 and the first electrode 5. This is the first signal (S2).
以下に、Ｓ２における光の強度の測定について、詳細に説明する。図１に示すように、光源１から発せられた光線ＬＢは経路Ｃ１を辿って、コリメートレンズ２を透過する。コリメートレンズ２により光線ＬＢは平行光となり集光レンズ３に入射し集光されつつプリズム４に入射し、プリズム４上に配置された第１電極５のセンサ部５ａの中央に位置する測定部Ｓに集光される。 Hereinafter, the measurement of the light intensity in S2 will be described in detail. As shown in FIG. 1, the light beam LB emitted from the light source 1 follows the path C <b> 1 and passes through the collimating lens 2. The light beam LB is converted into parallel light by the collimator lens 2, enters the condenser lens 3, enters the prism 4 while being condensed, and is measured at the center of the sensor unit 5 a of the first electrode 5 disposed on the prism 4. It is focused on.
センサ部５ａに入射された光は、集光された光であるため、さまざまな入射角の光を含んでいる。このうち、全反射する入射角で入射した光のうち表面プラズモン共鳴角を入射角として入射された光は、センサ部５ａと被測定物との界面に表面プラズモンを励起し、前記界面を減衰しながら伝搬するため、表面プラズモン共鳴角を入射角として入射された光の反射率は他の入射角で入射された光と比較して低下し、反射光の強度は低下する。 Since the light incident on the sensor unit 5a is condensed light, it includes light of various incident angles. Of these, the light incident with the surface plasmon resonance angle as the incident angle among the light incident at the total reflection incident angle excites the surface plasmon at the interface between the sensor unit 5a and the object to be measured, and attenuates the interface. Therefore, the reflectance of light incident with the surface plasmon resonance angle as an incident angle is lower than that of light incident at another incident angle, and the intensity of the reflected light is decreased.
センサ部５ａで反射された光は、コリメートレンズ７に入射し、平行光とされたのち経路Ｃ２を辿って光検出部８へ到達する。光検出部８は、プリズム４と第１電極５との界面への入射角毎に反射された光の強度を計測する。なお、光検出部８として、ＣＣＤやＣＭＯＳ等の２次元のセンサを用いる場合は、同一の入射角に係る信号を合算または平均化することにより、入射角毎の光の強度とする。 The light reflected by the sensor unit 5a is incident on the collimator lens 7 and is converted into parallel light, and then follows the path C2 to reach the light detection unit 8. The light detection unit 8 measures the intensity of light reflected at each incident angle to the interface between the prism 4 and the first electrode 5. When a two-dimensional sensor such as a CCD or CMOS is used as the light detection unit 8, the light intensity at each incident angle is obtained by adding or averaging signals related to the same incident angle.
次に、第１電極５および／または第２電極６に第２の周波数の電圧を印加する。これにより、第２電極６のエッジ部において電気浸透流が起こり、電位の勾配方向に沿って第１電極５のセンサ部５ａの外周近傍に捕集された微小物体はセンサ部５ａ上へ誘導される（Ｓ３）。 Next, a voltage having a second frequency is applied to the first electrode 5 and / or the second electrode 6. As a result, an electroosmotic flow occurs at the edge portion of the second electrode 6, and the minute object collected near the outer periphery of the sensor portion 5a of the first electrode 5 along the potential gradient direction is guided onto the sensor portion 5a. (S3).
図７は電気浸透流によりセンサ部５ａに微小物体が誘導された様子を示した模式図である。電気浸透流では、電位の勾配方向に沿って力が働くので、第１電極の外周に捕集された微小物体は、センサ部５ａの中央に位置する測定部Ｓに誘導される。 FIG. 7 is a schematic diagram showing a state in which a minute object is guided to the sensor unit 5a by electroosmotic flow. In the electroosmotic flow, force acts along the gradient direction of the electric potential, so that the minute object collected on the outer periphery of the first electrode is guided to the measurement unit S located at the center of the sensor unit 5a.
Ｓ２と同様に測定を行ない、第２信号とする（Ｓ４）。 Measurement is performed in the same manner as in S2 to obtain a second signal (S4).
次に第２信号と第１信号の差を算出する（Ｓ５）。図８（ａ）に入射角を横軸とし、光の強度を縦軸とする第１信号および第２信号のグラフを示す。第１信号、第２信号は共に、各々の表面プラズモン共鳴角を底とするＶ字型の谷型波形である。第１信号におけるグラフの谷は、微小物体はほとんど存在しない溶媒の表面プラズモン共鳴角であり、第２信号におけるグラフの谷は、微小物体が凝集された場合の表面プラズモン共鳴角である。測定部Ｓの上面近傍の微小物体の量の変化に伴い、測定部Ｓの上面近傍の誘電率が変化するため、表面プラズモン共鳴角は、第１信号、第２信号でそれぞれ異なる。その差を図８（ａ）のＡに示す。 Next, the difference between the second signal and the first signal is calculated (S5). FIG. 8A shows a graph of the first signal and the second signal with the incident angle as the horizontal axis and the light intensity as the vertical axis. Both the first signal and the second signal are V-shaped valley waveforms with the respective surface plasmon resonance angles as the bottom. The valley of the graph in the first signal is the surface plasmon resonance angle of the solvent in which there are almost no micro objects, and the valley of the graph in the second signal is the surface plasmon resonance angle when the micro objects are aggregated. The surface plasmon resonance angle differs between the first signal and the second signal because the dielectric constant in the vicinity of the upper surface of the measurement unit S changes with the change in the amount of minute objects in the vicinity of the upper surface of the measurement unit S. The difference is shown in A of FIG.
図８（ｂ）は、第２信号から第１信号を減算した差分を表す差分信号のグラフである。差分信号は、第１信号の表面プラズモン共鳴角付近で最大値となり、第２信号の表面プラズモン共鳴角付近で最小値となるグラフとなる。 FIG. 8B is a graph of a difference signal representing a difference obtained by subtracting the first signal from the second signal. The difference signal is a graph having a maximum value near the surface plasmon resonance angle of the first signal and a minimum value near the surface plasmon resonance angle of the second signal.
次に、最大値と最小値の差の絶対値Ｂを算出し、差分検出信号強度とする（Ｓ６）。また、最大値における入射角と最小値における入射角の差Ｃや、０を横切る点の傾きＤや、０を横切る点の入射角Ｅを差分検出信号強度としても良い。 Next, the absolute value B of the difference between the maximum value and the minimum value is calculated and set as the difference detection signal intensity (S6). Further, the difference C between the incident angle at the maximum value and the incident angle at the minimum value, the inclination D of the point crossing 0, or the incident angle E of the point crossing 0 may be used as the difference detection signal intensity.
また、第１信号における表面プラズモン共鳴角と第２信号における表面プラズモン共鳴角との差Ａを差分検出信号強度としても良く、その場合、図８（ｂ）に示す差分信号を求めなくても良い。 Further, the difference A between the surface plasmon resonance angle in the first signal and the surface plasmon resonance angle in the second signal may be used as the difference detection signal intensity. In this case, the difference signal shown in FIG. .
最後に、微小物体の量を算出する（Ｓ７）。ステップ（Ｓ７）において算出する量は、被測定物である液体の体積あたりの量であり、すなわち濃度である。 Finally, the amount of minute objects is calculated (S7). The amount calculated in step (S7) is the amount per volume of the liquid that is the object to be measured, that is, the concentration.
図９に差分検出信号強度と微小物体の量との関係をグラフで示す。図９に示すように差分検出信号強度と微小物体の量との間には有意な対応関係があるので、差分検出信号強度と微小物体の量との対応関係をあらかじめ数式またはテーブルとして記憶しておくことで、差分検出信号強度から微小物体の量を定量できる。なお、差分検出信号強度はステップＳ１において微小物体が濃縮された液体に係る信号強度であり、ステップ（Ｓ９）において算出すべき微小物体の量は、被測定物である液体における微小物体の濃度であるので、図９に示す対応関係には、ステップ（Ｓ１）における微小物体の濃縮率が含まれている。 FIG. 9 is a graph showing the relationship between the difference detection signal intensity and the amount of minute objects. As shown in FIG. 9, since there is a significant correspondence between the difference detection signal intensity and the amount of minute objects, the correspondence between the difference detection signal intensity and the amount of minute objects is stored in advance as a mathematical expression or a table. Thus, the amount of the minute object can be quantified from the difference detection signal intensity. The difference detection signal intensity is the signal intensity related to the liquid in which the minute object is concentrated in step S1, and the amount of the minute object to be calculated in step (S9) is the concentration of the minute object in the liquid to be measured. Therefore, the correspondence shown in FIG. 9 includes the minute object concentration rate in step (S1).
本実施例によれば、微小物体を排除した状態における誘電率を示す第１信号と、微小物体を凝集した状態における誘電率を示す第２信号とに基づいて、微小物体の量を算出する。第１信号は微小物体を含まない溶媒の誘電率を示すので、第２信号から第１信号を減じることによって、溶媒の誘電率の相違による第２信号の相違を補正することができる。従って、溶媒の温度が変化した場合やイオン濃度が異なる溶媒を用いた場合にあっても、被分析溶液中の微小物体の量を正確に測定することができる。 According to the present embodiment, the amount of the minute object is calculated based on the first signal indicating the dielectric constant in a state where the minute object is excluded and the second signal indicating the dielectric constant in the state where the minute object is aggregated. Since the first signal indicates the dielectric constant of the solvent that does not include the minute object, the difference in the second signal due to the difference in the dielectric constant of the solvent can be corrected by subtracting the first signal from the second signal. Therefore, even when the temperature of the solvent changes or when solvents having different ion concentrations are used, the amount of minute objects in the solution to be analyzed can be accurately measured.
また、本実施例によれば、第２の周波数は電気浸透流が発生する周波数である。電気浸透流は，流体の流れによるストークス抵抗力で電位の勾配方向に沿って微小物体を動かすため，微小物体を測定部Ｓに凝集させる時間を短縮することができる。 Further, according to this embodiment, the second frequency is a frequency at which electroosmotic flow is generated. Since the electroosmotic flow moves the minute object along the potential gradient direction by the Stokes resistance force caused by the fluid flow, the time for the minute object to aggregate in the measurement unit S can be shortened.
また、第１の周波数を印加する時間とステップ（Ｓ１）における濃縮率とは正の相関があるので、第１の周波数を印加する時間と濃縮率との関係を示す情報をあらかじめ記憶しておき、第１の周波数を印加する時間に基づいて濃縮率を特定し、差分検出信号強度から算出した濃縮後の微小物体の量を濃縮率で除して被測定物である液体における微小物体の量としてもよい。これにより、電圧印加時間が短くても、また長くても微小物体の量を正確に測定することができる。電圧印加時間を短くすれば短時間で測定することができる。また、電圧印加時間を長くすれば、電圧印加時間中により多くの微小物体を補集することができるので、より高感度で測定することができる。 In addition, since the time for applying the first frequency and the concentration rate in step (S1) have a positive correlation, information indicating the relationship between the time for applying the first frequency and the concentration rate is stored in advance. Then, the concentration rate is specified based on the application time of the first frequency, and the amount of the minute object after concentration calculated from the difference detection signal intensity is divided by the concentration rate, and the amount of the minute object in the liquid as the measurement object It is good. Thereby, the amount of minute objects can be accurately measured even when the voltage application time is short or long. If the voltage application time is shortened, it can be measured in a short time. Further, if the voltage application time is lengthened, more minute objects can be collected during the voltage application time, so that measurement can be performed with higher sensitivity.
また、差分検出信号強度と電圧印加時間と微小物体の量との関係を、濃縮率を含めた情報としてあらかじめ記憶しておき，第１の周波数を印加した時間が長い場合に差分検出信号強度に対し微小物体の量を少なく、第１の周波数を印加した時間が短い場合に差分検出信号強度に対し微小物体の量を多く算出して良い。 In addition, the relationship between the difference detection signal intensity, the voltage application time, and the amount of minute objects is stored in advance as information including the concentration rate, and the difference detection signal intensity is obtained when the time period during which the first frequency is applied is long. On the other hand, when the amount of the minute object is small and the time during which the first frequency is applied is short, the amount of the minute object may be calculated larger than the difference detection signal intensity.
本発明は微小物体量測定装置及び微小物体量測定方法に係るものであり、水中の微生物の量等の、微小物体の量を検出する水質センサや上記水質センサを搭載した分析装置、浄水器、洗浄機、浄水プラントなどに利用することができる。 The present invention relates to a minute object amount measuring apparatus and a minute object amount measuring method, and includes a water quality sensor that detects the amount of minute objects, such as the amount of microorganisms in water, an analysis device equipped with the water quality sensor, a water purifier, It can be used for washing machines and water purification plants.
１ 光源
２ コリメートレンズ
３ 集光レンズ
４ プリズム
５ 第１電極
６ 第２電極
７ コリメートレンズ
８ 光検出部
９ 周波数制御手段
１０ 交流電源
DESCRIPTION OF SYMBOLS 1 Light source 2 Collimating lens 3 Condensing lens 4 Prism 5 1st electrode 6 2nd electrode 7 Collimating lens 8 Light detection part 9 Frequency control means 10 AC power supply
Claims (6)
前記微小物体量測定装置は、
前記液体に接するように配置された第１電極と、
前記液体に接するように配置された第２電極と、
前記第１電極または前記第２電極に交流電圧を印加する交流電源と、
前記交流電源の周波数を制御する周波数制御手段と、
前記第１電極の測定部に接する前記液体の反射光の強度を検出する検出手段と、
を備え、
前記検出手段により、
前記第１電極または第２電極に、前記測定部から微小物体を排除するような第１の周波数の交流電圧を印加して、第１信号を検出し、
前記第１電極または第２電極に、前記測定部に微小物体を凝集するような第２の周波数の交流電圧を印加して、前記検出手段により第２信号を検出し、
前記第１信号と前記第２信号により微小物体の量を算出すること
を特徴とする微小物体量測定装置。 A minute object amount measuring apparatus for measuring the amount of minute objects in a liquid in which minute objects are suspended,
The minute object amount measuring device is:
A first electrode disposed in contact with the liquid;
A second electrode disposed in contact with the liquid;
An AC power supply for applying an AC voltage to the first electrode or the second electrode;
Frequency control means for controlling the frequency of the AC power supply;
Detection means for detecting the intensity of reflected light of the liquid in contact with the measurement part of the first electrode;
With
By the detection means,
An AC voltage having a first frequency that excludes a minute object from the measurement unit is applied to the first electrode or the second electrode, and a first signal is detected.
An AC voltage having a second frequency that agglomerates a minute object on the measurement unit is applied to the first electrode or the second electrode, and a second signal is detected by the detection unit,
A minute object amount measuring apparatus that calculates the amount of a minute object from the first signal and the second signal.
前記液体に接するように配置された第２電極と、
前記第１電極または前記第２電極の少なくとも一方に交流電圧を印加する交流電源と、
前記交流電源の周波数を選択する周波数制御手段と、
前記第１電極の測定部に接する前記液体の誘電率に関係する信号を検出する検出手段と、
を備える微小物体量測定装置を用いて、
微小物体が懸濁した液体における微小物体の量を測定する微小物体量測定方法であって、
前記第１電極または第２電極の少なくとも一方に第１の周波数の交流電圧を印加して、前記測定部から微小物体を排除するステップと、
前記検出手段により第１信号を検出するステップと、
前記第１電極または第２電極の少なくとも一方に第２の周波数の交流電圧を印加して、前記測定部に微小物体を凝集するステップと、
前記検出手段により第２信号を検出するステップと、
前記第１信号と前記第２信号の差により微小物体の量を算出するステップと、
を備えること特徴とする微小物体量測定方法。 A first electrode disposed in contact with the liquid;
A second electrode disposed in contact with the liquid;
An AC power supply for applying an AC voltage to at least one of the first electrode or the second electrode;
Frequency control means for selecting the frequency of the AC power source;
Detection means for detecting a signal related to a dielectric constant of the liquid in contact with the measurement unit of the first electrode;
Using a minute object amount measuring apparatus comprising
A method for measuring the amount of a minute object that measures the amount of a minute object in a liquid in which the minute object is suspended,
Applying an alternating voltage of a first frequency to at least one of the first electrode or the second electrode to exclude a minute object from the measurement unit;
Detecting the first signal by the detecting means;
Applying an alternating voltage of a second frequency to at least one of the first electrode or the second electrode to agglomerate micro objects on the measurement unit;
Detecting the second signal by the detecting means;
Calculating an amount of a minute object based on a difference between the first signal and the second signal;
A method for measuring the amount of a minute object.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015194513A1 (en) * | 2014-06-18 | 2015-12-23 | コニカミノルタ株式会社 | Measurement chip, measurement method, and measurement device |
| WO2021131043A1 (en) * | 2019-12-27 | 2021-07-01 | 株式会社ジェイエイアイコーポレーション | Prism device providing method and prism device |
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2010
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015194513A1 (en) * | 2014-06-18 | 2015-12-23 | コニカミノルタ株式会社 | Measurement chip, measurement method, and measurement device |
| JPWO2015194513A1 (en) * | 2014-06-18 | 2017-04-20 | コニカミノルタ株式会社 | Measuring chip, measuring method and measuring device |
| WO2021131043A1 (en) * | 2019-12-27 | 2021-07-01 | 株式会社ジェイエイアイコーポレーション | Prism device providing method and prism device |
| EP4109144A4 (en) * | 2019-12-27 | 2023-10-18 | JAI Ltd. | Prism device providing method and prism device |
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