JP2011026433A - Fluorescence labeling reagent - Google Patents
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- JP2011026433A JP2011026433A JP2009173091A JP2009173091A JP2011026433A JP 2011026433 A JP2011026433 A JP 2011026433A JP 2009173091 A JP2009173091 A JP 2009173091A JP 2009173091 A JP2009173091 A JP 2009173091A JP 2011026433 A JP2011026433 A JP 2011026433A
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 42
- 238000001215 fluorescent labelling Methods 0.000 title claims abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 title abstract 3
- 239000002245 particle Substances 0.000 claims abstract description 21
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 6
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 6
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 229920000856 Amylose Polymers 0.000 claims description 4
- 238000002372 labelling Methods 0.000 claims description 4
- 229920000936 Agarose Polymers 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 2
- 229920001353 Dextrin Polymers 0.000 claims description 2
- 239000004375 Dextrin Substances 0.000 claims description 2
- 229920002873 Polyethylenimine Polymers 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 235000019425 dextrin Nutrition 0.000 claims description 2
- -1 polyvinylamine Substances 0.000 claims description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 2
- 239000013076 target substance Substances 0.000 abstract description 21
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 239000000975 dye Substances 0.000 description 27
- 230000005284 excitation Effects 0.000 description 22
- 239000000203 mixture Substances 0.000 description 15
- 239000000126 substance Substances 0.000 description 14
- 239000011230 binding agent Substances 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 8
- 239000008187 granular material Substances 0.000 description 8
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 6
- 239000006185 dispersion Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 239000000049 pigment Substances 0.000 description 5
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- 239000000470 constituent Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 150000004662 dithiols Chemical class 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000010954 inorganic particle Substances 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 239000001007 phthalocyanine dye Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 2
- 239000004925 Acrylic resin Substances 0.000 description 1
- 229920000178 Acrylic resin Polymers 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229920000297 Rayon Polymers 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000001000 anthraquinone dye Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000000987 azo dye Substances 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
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- 230000006866 deterioration Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 239000001013 indophenol dye Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
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- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
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- 239000000523 sample Substances 0.000 description 1
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- 238000003756 stirring Methods 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
Description
本発明は、蛍光を発光する蛍光ラベル化剤に関する。 The present invention relates to a fluorescent labeling agent that emits fluorescence.
従来から、バイオセンシング、バイオラベリング、バイオイメージングや環境検査などにおいて、サンプリング対象となる物質を標識（ラベル）化し、ラベル化された物質に関する様々な測定が行われている。 Conventionally, in biosensing, biolabeling, bioimaging, environmental inspection, and the like, a substance to be sampled is labeled (labeled), and various measurements relating to the labeled substance have been performed.
このような測定において、ラベル化された物質として蛍光物質が使用されることが多い。この場合、まず、ラベル化された蛍光物質を目的物質として反応させた後、非特異物質を洗浄、除去する。その後、洗浄等されたその目的物質に励起光を照射することによって励起状態とし、その状態で目的物質の蛍光波長や蛍光強度等の出力を測定している。 In such a measurement, a fluorescent substance is often used as a labeled substance. In this case, first, a labeled fluorescent substance is reacted as a target substance, and then a non-specific substance is washed and removed. Thereafter, the target substance that has been washed or the like is irradiated with excitation light to be in an excited state, and in this state, outputs such as the fluorescence wavelength and fluorescence intensity of the target substance are measured.
また、上述した蛍光物質としては、従来からＵＶ（UltraViolet ray）励起可視発光の蛍光体が用いられてきた。しかし、生体試料の分析においては、多量の夾雑物質が存在することにより、バックグラウンドノイズが高くなってしまう場合があるという問題があった。 In addition, as the above-described fluorescent material, a phosphor of UV (UltraViolet ray) excitation visible light emission has been conventionally used. However, in the analysis of biological samples, there is a problem that the background noise may increase due to the presence of a large amount of contaminants.
このような問題を回避する手法として、例えば、特許文献１に記載された技術のように、ＨＰＬＣ（High Performance Liquid Chromatography）を検出するための物質としてポリメチン色素を利用したり、あるいは特許文献２に記載された技術のように、ＤＮＡ（DeoxyriboNucleic Acid）を検出するための物質として有機近赤外蛍光色素を利用することが開示されている。 As a technique for avoiding such a problem, for example, a polymethine dye is used as a substance for detecting HPLC (High Performance Liquid Chromatography) as in the technique described in Patent Document 1, or in Patent Document 2 Like the described technique, it is disclosed that an organic near-infrared fluorescent dye is used as a substance for detecting DNA (DeoxyriboNucleic Acid).
しかしながら、特許文献１および特許文献２に記載された技術では、上述したバックグラウンドノイズを抑制して、一時的に目的物質の蛍光波長や蛍光強度等の出力を高めることはできても、安定してその状態を維持することができないという問題があった。特に、目的物質が水溶性の場合には、有機近赤外蛍光色素の特性上、溶液中では容易に溶解するため、通常は分子状態となっていることが多く、そのような安定した状態を維持することが困難であるという問題があった。 However, the techniques described in Patent Document 1 and Patent Document 2 are stable even though the background noise described above can be suppressed and the output of the fluorescence wavelength and fluorescence intensity of the target substance can be temporarily increased. There is a problem that the state cannot be maintained. In particular, when the target substance is water-soluble, due to the characteristics of organic near-infrared fluorescent dyes, it is easily dissolved in solution and is usually in a molecular state. There was a problem that it was difficult to maintain.
本発明は、上記に鑑みてなされたものであって、バックグラウンドノイズを抑制した状態を維持し、目的物質を高感度で分析することができる蛍光ラベル化剤を提供することを目的とする。 This invention is made | formed in view of the above, Comprising: It aims at providing the fluorescence labeling agent which can maintain the state which suppressed the background noise and can analyze a target substance with high sensitivity.
上述した目的を達成するために、本発明に係る蛍光ラベル化剤は、水不溶性の光で励起して所定の波長の領域において蛍光発光する有機蛍光色素が液剤中に粒子状に分散した分散粒子を含むことを特徴とする。好ましくは、前記光は近赤外光であり、前記領域は近赤外領域である。また、好ましくは、７００ｎｍ〜１０００ｎｍの波長の前記近赤外光で励起し、７５０ｎｍ〜２０００ｎｍの前記近赤外領域で蛍光発光する有機蛍光色素が前記液剤中に粒子状に分散した前記分散粒子を含む蛍光ラベル化剤である。さらに、好ましくは、前記分散粒子のサイズが１０ｎｍ〜２００ｎｍの範囲である蛍光ラベル化剤である。また、好ましくは、前記液剤は、水または生理食塩水である蛍光ラベル化剤である。さらに、好ましくは、ポリビニルアルコール、デキストリン、アミロース、アガロース、ポリビニルピロリドン、ポリエチレンイミン、ポリビニルアミン、アルギニンから少なくとも１種以上選ばれる水溶性樹脂を含む蛍光ラベル化剤である。 In order to achieve the above-described object, the fluorescent labeling agent according to the present invention is a dispersed particle in which an organic fluorescent dye that is excited by water-insoluble light and emits fluorescence in a predetermined wavelength region is dispersed in a liquid form. It is characterized by including. Preferably, the light is near infrared light and the region is a near infrared region. Preferably, the dispersed particles in which an organic fluorescent dye that is excited by the near-infrared light having a wavelength of 700 nm to 1000 nm and fluoresces in the near-infrared region of 750 nm to 2000 nm is dispersed in the liquid form. A fluorescent labeling agent. Further preferably, the fluorescent labeling agent has a size of the dispersed particles in the range of 10 nm to 200 nm. Preferably, the liquid agent is a fluorescent labeling agent which is water or physiological saline. Furthermore, it is preferably a fluorescent labeling agent containing at least one water-soluble resin selected from polyvinyl alcohol, dextrin, amylose, agarose, polyvinylpyrrolidone, polyethyleneimine, polyvinylamine, and arginine.
本発明によれば、バックグラウンドノイズを抑制した状態を維持し、目的物質を高感度で分析することができる。 ADVANTAGE OF THE INVENTION According to this invention, the state which suppressed the background noise can be maintained and a target substance can be analyzed with high sensitivity.
以下に、この発明にかかる蛍光ラベル化剤の実施の形態を詳細に説明する。以下の説明では、一例として、蛍光ラベル化剤を有機蛍光色素に適用した場合について説明している。 Hereinafter, embodiments of the fluorescent labeling agent according to the present invention will be described in detail. In the following description, a case where a fluorescent labeling agent is applied to an organic fluorescent dye is described as an example.
有機蛍光色素は、近赤外線（波長７８０ｎｍ以上）により励起されて赤外光領域に発光中心波長を有するものである。有機蛍光色素の代表的なものとしては、ポリメチン系色素、アントラキノン系色素、ジチオ―ル金属塩系色素、フタロシアニン系色素、インドフエノ―ル系色素、シアニン系色素、アミニウム系色素、ジイモニウム系色素、アゾ系色素などが好ましく用いられる。 The organic fluorescent dye is excited by near infrared rays (wavelength of 780 nm or more) and has an emission center wavelength in the infrared light region. Typical organic fluorescent dyes include polymethine dyes, anthraquinone dyes, dithiol metal salt dyes, phthalocyanine dyes, indophenol dyes, cyanine dyes, aminium dyes, diimonium dyes, azo dyes. A system dye or the like is preferably used.
具体的には、ポリメチン系色素として旧コダックラボラトリ―ケミカル社製の「ＩＲ−１４０」、日本化薬社製の「ＩＲ−８２０Ｂ」などが存在し、アントラキノン系色素として日本化薬社製の「ＩＲ−７５０」などが存在する。また、シアニン系色素の具体例としては、日本感光色素社製ＮＫ−１１４４、ＮＫ−１２３、ＮＫ−７８などが存在し、ジチオ―ル金属塩系色素として三井化学社製の「テトラブチルホソホニウム（１，２−ベンゼンチオラ―ト）ニコレ―トなど、フタロシアニン系色素としてＺｎ−フタロシアニンなどが挙げられる。 Specifically, there are “IR-140” manufactured by the former Kodak Laboratory Chemical Co., “IR-820B” manufactured by Nippon Kayaku Co., Ltd. as the polymethine dye, and “ IR-750 "exists. Specific examples of cyanine dyes include NK-1144, NK-123, and NK-78 manufactured by Nippon Photosensitive Dye Co., Ltd. “Tetrabutyl Phosofol” manufactured by Mitsui Chemicals, Inc. as a dithiol metal salt dye. Examples of phthalocyanine dyes such as nitro (1,2-benzenethiolate) nicolate include Zn-phthalocyanine.
上述したように、このような有機蛍光色素は耐候性に劣っている。特に、目的物質が液体状態である場合や、ラベル化された状態で外部から近赤外光等の光を照射した場合、その光の発光強度は、時間が経過する程容易に低下してしまう。この現象は、特に、目的物質がラベル化された状態において著しい。 As described above, such organic fluorescent dyes are inferior in weather resistance. In particular, when the target substance is in a liquid state or is irradiated with light such as near infrared light from the outside in a labeled state, the emission intensity of the light easily decreases as time passes. . This phenomenon is particularly remarkable in a state where the target substance is labeled.
すなわち、液体状態では、色素と外気との間に溶媒が介在し、色素と外気との直接の接触を防ぐことができる。しかし、目的物質がラベル化された状態では、溶媒などの介在物が存在せず、色素の分解性がラベル化の安定性に直接影響してしまう。この現象は、無機質や有機質の粒子表面に色素を吸着させたときでも同様であり、上記粒子表面では色素が溶媒中での溶解状態と同様に分子状態で存在している。したがって、このようなラベル化された目的物質では、色素が外気と直接接触することによって、有機蛍光色素の分解による劣化を抑制できない。また、目的物質の種類によっては表面に種々の官能基を持つものがあり、この官能基が有機蛍光色素の分解を促進して、室温で放置した場合でも発光出力の顕著な低下をもたらしやすい。 That is, in the liquid state, a solvent is interposed between the pigment and the outside air, and direct contact between the pigment and the outside air can be prevented. However, when the target substance is labeled, there is no inclusion such as a solvent, and the degradability of the dye directly affects the stability of labeling. This phenomenon is the same even when a dye is adsorbed on the surface of inorganic or organic particles, and the dye exists on the surface of the particles in a molecular state as well as a dissolved state in a solvent. Therefore, with such a labeled target substance, deterioration due to decomposition of the organic fluorescent dye cannot be suppressed by direct contact of the dye with the outside air. Some types of target substances have various functional groups on the surface, and these functional groups promote the decomposition of the organic fluorescent dye and tend to cause a significant decrease in light emission output even when left at room temperature.
そこで、本発明者らは、鋭意検討した結果、有機蛍光色素の外気からの影響を低減するとともに、目的物質をラベル化した場合に官能基による作用を低減して有機蛍光色素の分解性を抑制するために、有機蛍光色素をバルク状態とすることによって、目的物質が液体状態の場合だけでなく、そのような目的物質をラベル化とした場合でも、高い発光出力を維持できることを見出した。 Therefore, as a result of intensive studies, the present inventors have reduced the influence of the organic fluorescent dye from the outside air and reduced the action of the functional group when the target substance is labeled to suppress the decomposability of the organic fluorescent dye. Therefore, it has been found that by setting the organic fluorescent dye in a bulk state, a high light emission output can be maintained not only when the target substance is in a liquid state but also when such a target substance is labeled.
なお、ラベル化とは、ある物質の場所を同定するための物質を目的物質に備えさせることを意味している。ラベル化には、上述した蛍光物質のほか、放射能や酵素等が利用されるが、ここでは蛍光物質の中の有機蛍光色素を利用している。このようにラベル化された物質を用いることにより、目的物質に含まれる物質（検査対象となる物質）の定性的、定量的な検出を行うことができる。 The labeling means that a target substance is provided with a substance for identifying the location of a certain substance. For labeling, in addition to the fluorescent material described above, radioactivity, enzyme, and the like are used. Here, an organic fluorescent dye in the fluorescent material is used. By using such a labeled substance, qualitative and quantitative detection of a substance (substance to be inspected) contained in the target substance can be performed.
すなわち、以下の説明では、上記の有機蛍光色素を使用するにあたり、この色素を水または生理食塩水中に粒径１０〜４５０ｎｍの粒状物として存在させることにより、液体状態のみならず、ラベル化とした場合でも、優れた保存安定性が得られることを見出したものである。なお、溶媒中の粒径は動的光散乱光度計により測定可能である。 That is, in the following description, when using the above organic fluorescent dye, the dye is allowed to exist as a granular material having a particle diameter of 10 to 450 nm in water or physiological saline, thereby forming a label as well as a liquid state. Even in this case, it has been found that excellent storage stability can be obtained. The particle size in the solvent can be measured with a dynamic light scattering photometer.
このような保存安定性が得られる理由は、以下のように考えられる。有機蛍光色素を粒径１０〜４５０ｎｍ（より効果的には１０ｎｍ〜２００ｎｍ）のバルク状態の粒状物とすると、これを用いて目的物質をラベル化した場合に、上記色素が外気またはサンプルの官能基と接触しても、粒状物の表面のみが劣化するに止まり、内部では活性な色素が存在し、これにより長期保存後でも発光出力の低下が抑制されるものと思われる。 The reason why such storage stability can be obtained is considered as follows. When the organic fluorescent dye is a granular material in a bulk state having a particle diameter of 10 to 450 nm (more effectively, 10 nm to 200 nm), when the target substance is labeled using the organic fluorescent dye, the dye may be outside air or a functional group of the sample. Even if it comes into contact, only the surface of the granular material is deteriorated, and an active dye is present inside, which is considered to suppress a decrease in light emission output even after long-term storage.
以下に示す実施例では、近赤外光の励起波長を８２０ｎｍとしているが、励起波長が７００ｎｍ〜１０００ｎｍ程度の間であって、有機蛍光色素が７５０ｎｍ〜２０００ｎｍ程度の間の近赤外領域で蛍光発光するものであればよい。 In the following examples, the excitation wavelength of near-infrared light is 820 nm, but the excitation wavelength is between 700 nm and 1000 nm, and the organic fluorescent dye is fluorescent in the near infrared region between about 750 nm and 2000 nm. Any device that emits light may be used.
（実施例１） 水９０部に、バインダ（結合剤）としてポリビニルアルコ―ル（クラレ社製のＰＶＡ）５部を溶解し、これに、近赤外線によって励起されて発光する色素（日本化薬社製の商品名「ＩＲ−８２０Ｂ」、励起波長：８２０ｎｍ、発光波長：９００ｎｍ）０．５部を加え、遊星ボ―ルミルにより４時間分散処理して、上記色素が粒径２８０〜３２０ｎｍの粒状物として存在する近赤外線励起型の組成物を調製した。 (Example 1) In 90 parts of water, 5 parts of polyvinyl alcohol (PVA manufactured by Kuraray Co., Ltd.) as a binder (binder) is dissolved, and a dye (Nihon Kayaku Co., Ltd.) that is excited by near infrared rays to emit light. 0.5 parts of the product name “IR-820B” manufactured by the manufacturer, excitation wavelength: 820 nm, emission wavelength: 900 nm), and dispersion treatment for 4 hours with a planetary ball mill, the above pigment is a granular material having a particle size of 280 to 320 nm A near-infrared excitation type composition was prepared.
（実施例２） 遊星ボ―ルミルによる分散処理時間を８時間とした以外は、実施例１と同様にして、色素が粒径１１０〜２５０ｎｍの粒状物として存在する近赤外線励起型の組成物を調製した。 (Example 2) A near-infrared excitation type composition in which a pigment is present as a granular material having a particle size of 110 to 250 nm is prepared in the same manner as in Example 1 except that the dispersion treatment time by the planetary ball mill is set to 8 hours. Prepared.
（実施例３） 水９０部に、バインダ（結合剤）としてアミロース５部を溶解し、これに、近赤外線によって励起されて発光する色素（日本化薬社製の商品名「ＩＲ−８２０Ｂ」、励起波長：８２０ｎｍ、発光波長：９００ｎｍ）０．５部を加え、遊星ボ―ルミルにより４時間分散処理して、上記色素が粒径２８０〜３２０ｎｍの粒状物として存在する近赤外線励起型の組成物を調製した。 (Example 3) In 90 parts of water, 5 parts of amylose is dissolved as a binder (binder), and a dye that emits light when excited by near infrared rays (trade name “IR-820B” manufactured by Nippon Kayaku Co., Ltd.) Excitation wavelength: 820 nm, emission wavelength: 900 nm) 0.5 part) and dispersion treatment with a planetary ball mill for 4 hours, near-infrared excitation type composition in which the dye is present as a particle having a particle size of 280 to 320 nm Was prepared.
（実施例４） 水９０部に、バインダ（結合剤）としてアガロース５部を溶解し、これに、近赤外線によって励起されて発光する色素（日本化薬社製の商品名「ＩＲ−８２０Ｂ」、励起波長：８２０ｎｍ、発光波長：９００ｎｍ）０．５部を加え、遊星ボ―ルミルにより４時間分散処理して、上記色素が粒径２８０〜３２０ｎｍの粒状物として存在する近赤外線励起型の組成物を調製した。 (Example 4) In 90 parts of water, 5 parts of agarose as a binder (binder) is dissolved, and a pigment that emits light when excited by near infrared rays (trade name “IR-820B” manufactured by Nippon Kayaku Co., Ltd.) Excitation wavelength: 820 nm, emission wavelength: 900 nm) 0.5 part) and dispersion treatment with a planetary ball mill for 4 hours, near-infrared excitation type composition in which the dye is present as a particle having a particle size of 280 to 320 nm Was prepared.
（実施例５） 生理食塩水９０部に、バインダ（結合剤）としてアミロース５部を溶解し、これに、近赤外線によって励起されて発光する色素（日本化薬社製の商品名「ＩＲ−８２０Ｂ」、励起波長：８２０ｎｍ、発光波長：９００ｎｍ）０．５部を加え、遊星ボ―ルミルにより４時間分散処理して、上記色素が粒径２８０〜３２０ｎｍの粒状物として存在する近赤外線励起型の組成物を調製した。 (Example 5) In 90 parts of physiological saline, 5 parts of amylose was dissolved as a binder (binder), and a dye (trade name “IR-820B, manufactured by Nippon Kayaku Co., Ltd.”, which was excited by near infrared rays to emit light. ”, Excitation wavelength: 820 nm, emission wavelength: 900 nm), 0.5 part) and dispersion treatment with a planetary ball mill for 4 hours to obtain a near-infrared excitation type in which the dye is present as a granular material having a particle size of 280 to 320 nm. A composition was prepared.
（比較例１） メチルエチルケトン９０部に、実施例１と同じ色素０．５部を溶解し、これにさらにバインダ（結合剤）としてアクリル系樹脂（三菱レ―ヨン製の商品名「ダイヤナ―ルＢＲ−１０６」）９．５部を加えて、よく撹拌混合して溶解し、近赤外線励起型の組成物を調製した。 (Comparative Example 1) In 90 parts of methyl ethyl ketone, 0.5 part of the same dye as in Example 1 was dissolved, and an acrylic resin (trade name “Dianal BR made by Mitsubishi Rayon” was used as a binder. -106 ") 9.5 parts was added, and the mixture was thoroughly stirred and dissolved to prepare a near-infrared excitation type composition.
（比較例２） 遊星ボ―ルミルによる分散処理時間を１時間とした以外は、実施例１と同様にして、近赤外線励起型の組成物を調製した。動的光錯乱法によって測定した色素の粒径は４８０〜５５０ｎｍであつた。 (Comparative Example 2) A near-infrared excitation type composition was prepared in the same manner as in Example 1 except that the dispersion treatment time with the planetary ball mill was set to 1 hour. The particle size of the dye measured by the dynamic light scattering method was 480 to 550 nm.
（比較例３） メチルエチルケトン３部に実施例１と同じ色素０．５部を溶解した。つぎに、この色素溶解液を、水７７部に無機質粒子として平均粒径１００ｎｍのシリカ粒子１０部、バインダ（結合剤）としてポリビニルアルコ―ル（クラレ社製のＰＶＡ）９．５部を混合してなるスラリ―中に、配合し、これをよく撹拌混合して、近赤外線励起型の組成物を調製した。 Comparative Example 3 0.5 part of the same dye as in Example 1 was dissolved in 3 parts of methyl ethyl ketone. Next, in this dye solution, 77 parts of water was mixed with 10 parts of silica particles having an average particle diameter of 100 nm as inorganic particles and 9.5 parts of polyvinyl alcohol (PVA made by Kuraray Co., Ltd.) as a binder. The mixture was blended in the slurry and mixed well with stirring to prepare a near infrared excitation type composition.
このように、各実施例で説明した方法により組成物を調製した場合の蛍光発光強度、およびその維持率を各比較例と対比した結果は以下のとおりである。なお、以下に示す維持率は、励起レーザを目的物質に照射した後、さらにその１時間後に測定した発光強度の割合として示している。また、蛍光発光強度は、比較例１の場合を基準（１００）とした場合の数値となっている。 Thus, the result of having compared the fluorescence emission intensity at the time of preparing a composition with the method demonstrated in each Example, and its maintenance rate with each comparative example is as follows. In addition, the maintenance factor shown below is shown as a ratio of the emission intensity measured after one hour after irradiating the target material with the excitation laser. Further, the fluorescence emission intensity is a numerical value when the case of Comparative Example 1 is used as a reference (100).
蛍光発光強度 励起レーザ照射後の出力維持率（％）
実施例１ １８０ ８５
実施例２ １７５ ９０
実施例３ １７０ ８５
実施例４ １６５ ８５
実施例５ １７０ ８５
比較例１有機蛍光体 １００ ４
比較例２無機蛍光体 ５ １００
比較例３有機蛍光体 １２０ １６
以上のように、赤外線の励起により赤外光を発光する有機蛍光色素を本質的に溶解しない溶媒に、この溶媒に溶解する樹脂をあらかじめ溶解して、溶液組成物中にバルク状態で存在させる（例えば、水または生理食塩水中に微細に分散させて水溶液中にバルク状態で存在させる）ことにより、蛍光色素として安定に存在することが可能となる。
Fluorescence emission intensity Output retention rate after excitation laser irradiation (%)
Example 1 180 85
Example 2 175 90
Example 3 170 85
Example 4 165 85
Example 5 170 85
Comparative Example 1 Organic phosphor 100 4
Comparative Example 2 Inorganic phosphor 5 100
Comparative Example 3 Organic phosphor 120 16
As described above, the resin that dissolves in the solvent is dissolved in advance in a solvent that does not essentially dissolve the organic fluorescent dye that emits infrared light by infrared excitation, and is present in a bulk state in the solution composition ( For example, it can be stably present as a fluorescent dye by being finely dispersed in water or physiological saline and existing in an aqueous solution in a bulk state.
さらには、赤外線の励起による赤外光領域での発光出力が向上し、かつ耐候性が改善され、目的物質が液体状態である場合やラベル化された場合であっても、バックグラウンドノイズを抑制した状態を維持し、継続して目的物質を高感度で分析することができる。例えば、高い検出感度を必要とし、多量の夾雑物質が存在する血漿においても高感度で分析することができる。 In addition, the light output in the infrared region by infrared excitation is improved, the weather resistance is improved, and background noise is suppressed even when the target substance is in a liquid state or labeled. The target substance can be continuously analyzed with high sensitivity. For example, it is possible to analyze with high sensitivity even in plasma that requires high detection sensitivity and contains a large amount of contaminants.
なお、上述した実施の形態においては、近赤外光を照射して有機蛍光色素が粒径１１０〜３２０ｎｍの粒状物として存在する近赤外線励起型の組成物を調製した。しかし、例えば、一般に毛細血管の太さが１０μｍ程度であり、毛細血管の細胞壁の大きさが５０ｎｍ程度であることから、このような粒状物のサイズを、５０ｎｍ〜１０μｍの大きさの組成物として調整することによって、調整した組成物をヒトの生体（例えば、静脈）に適用して、例えば、一般的な８０ｎｍ以上の大きさの癌細胞の有無等を検出することができる。 In the embodiment described above, a near-infrared excitation type composition in which the organic fluorescent dye exists as a granular material having a particle size of 110 to 320 nm by irradiating near-infrared light was prepared. However, for example, since the capillaries generally have a thickness of about 10 μm and the size of the cell wall of the capillaries is about 50 nm, the size of such a granular material is defined as a composition having a size of 50 nm to 10 μm. By adjusting, the adjusted composition can be applied to a human living body (for example, vein) to detect the presence or absence of cancer cells having a general size of 80 nm or more, for example.
また、上述した実施の形態においては、励起光として近赤外光を用いたが、このような特定の波長に限らず、例えば、紫外光や短波長の可視光を用いることによって、これらの光の波長領域で有機蛍光色素を蛍光発光させることとしてもよい。 In the embodiment described above, near-infrared light is used as excitation light. However, the light is not limited to such a specific wavelength. For example, ultraviolet light or short-wavelength visible light can be used. The organic fluorescent dye may be made to emit fluorescent light in the wavelength region.
なお、本発明は、上記実施の形態そのままに限定されるものではなく、実施段階ではその要旨を逸脱しない範囲で構成要素を変形して具体化することができる。また、上記実施の形態に開示されている複数の構成要素の適宜な組み合わせにより、種々の発明を形成することができる。例えば、実施の形態に示される全構成要素からいくつかの構成要素を削除してもよい。さらに、異なる実施の形態にわたる構成要素を適宜組み合わせても良い。 It should be noted that the present invention is not limited to the above-described embodiment as it is, and can be embodied by modifying the constituent elements without departing from the scope of the invention in the implementation stage. In addition, various inventions can be formed by appropriately combining a plurality of constituent elements disclosed in the above embodiments. For example, some components may be deleted from all the components shown in the embodiment. Furthermore, constituent elements over different embodiments may be appropriately combined.
Claims (6)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012215540A (en) * | 2011-03-28 | 2012-11-08 | Nitto Denko Corp | Spr sensor cell and spr sensor |
| JP2012215541A (en) * | 2011-03-28 | 2012-11-08 | Nitto Denko Corp | Spr sensor cell and spr sensor |
| JP2013117545A (en) * | 2011-03-28 | 2013-06-13 | Nitto Denko Corp | Spr sensor cell and spr sensor |
| JP2014016357A (en) * | 2011-03-28 | 2014-01-30 | Nitto Denko Corp | Spr sensor cell and spr sensor |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012215540A (en) * | 2011-03-28 | 2012-11-08 | Nitto Denko Corp | Spr sensor cell and spr sensor |
| JP2012215541A (en) * | 2011-03-28 | 2012-11-08 | Nitto Denko Corp | Spr sensor cell and spr sensor |
| JP2013117545A (en) * | 2011-03-28 | 2013-06-13 | Nitto Denko Corp | Spr sensor cell and spr sensor |
| JP2014016357A (en) * | 2011-03-28 | 2014-01-30 | Nitto Denko Corp | Spr sensor cell and spr sensor |
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