JP2008203172A - Surface plasmon enhanced fluorescence detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To extremely sensitively detect fluorescence without filtering-processing a sample such as blood. <P>SOLUTION: The sample 1 is held by a metal film 20 formed on a surface of a dielectric block 13 comprising an excitation light transmissive material, and formed with an flexible film 31 comprising a hydrophobic material on its surface, an excitation light having 700-2000 nm of wavelength is made to get incident from a dielectric block 13 side, a substance contained in the sample 1 is excited by an evanescent wave 11 effused on a surface of the metal film 20 enhanced with a surface plasmon, and the fluorescence emitted from the substance by the excitation is detected by a photodetector 9. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a fluorescence sensor for detecting a specific substance in a sample by a fluorescence method, and more particularly to a fluorescence detection method by surface plasmon enhancement.

  Conventionally, the fluorescence method has been widely used as a highly sensitive and simple measurement method in biomeasurement and the like. This fluorescence method irradiates a sample that is considered to contain a detection target substance that emits fluorescence when excited by light of a specific wavelength, and detects the presence of the detection target substance by detecting the fluorescence at that time. It is a method to confirm. When the detection target substance is not a fluorescent substance, this binding, that is, detection is performed by contacting the sample with a substance that is labeled with the fluorescent substance and specifically binds to the detection target substance, and then detecting fluorescence in the same manner as described above. The existence of the target substance is also widely confirmed.

  FIG. 3 schematically shows an example of a sensor that performs the fluorescence method using the labeled substance. As an example, this fluorescent sensor is for detecting the antigen 2 contained in the sample 1, and the substrate 3 is coated with a primary antibody 4 that specifically binds to the antigen 2. Then, the sample 1 is flowed in the sample holding part 5 provided on the substrate 3, and then the secondary antibody 6 that is similarly labeled with the phosphor 10 and specifically binds to the antigen 2 is flowed. Thereafter, excitation light 8 is emitted from the light source 7 toward the surface portion of the substrate 3, and fluorescence is detected by the photodetector 9. At this time, if the predetermined fluorescence is detected by the photodetector 9, the binding between the secondary antibody 6 and the antigen 2, that is, the presence of the antigen 2 in the sample 1 can be confirmed.

  In the above example, it is the secondary antibody 6 that is actually confirmed by fluorescence detection. However, if the secondary antibody 6 does not bind to the antigen 2, it is washed away and is present on the substrate 3. Since it cannot be obtained, the presence of the antigen 2 as the detection target substance is indirectly confirmed by confirming the presence of the secondary antibody 6.

  However, in the conventional fluorescence sensor as shown in FIG. 3, reflected light / scattered light of excitation light at the interface between the substrate and the sample, or scattered light due to impurities other than the detection target substance / floating matter M or the like becomes noise. Therefore, the actual situation is that the S / N in fluorescence detection is not improved even if the performance of the photodetector is improved.

  As a solution to this, for example, an evanescent fluorescence method as shown in Non-Patent Document 1, that is, a fluorescence method using an evanescent wave has been proposed. An example of a fluorescence sensor that implements this fluorescence method is schematically shown in FIG. In FIG. 4, the same components as those in FIG. 3 are denoted by the same reference numerals, and description thereof will be omitted unless necessary (the same applies hereinafter).

  In this fluorescent sensor, a prism (dielectric block) 13 is used as an alternative to the substrate 3 described above, and the excitation light 8 from the light source 7 is totally reflected at the interface between the prism 13 and the sample 1. Irradiated through the prism 13. In this configuration, when the excitation light 8 is totally reflected at the interface, the secondary antibody 6 is excited by the evanescent wave 11 that oozes out in the vicinity of the interface. Fluorescence detection is performed by a photodetector 9 arranged on the opposite side of the sample 1 from the prism 13 (upward in the drawing).

  In this fluorescence sensor, since the excitation light 8 is totally reflected downward in the figure, the excitation light detection component does not become a background for the fluorescence detection signal in fluorescence detection from above. Further, since the evanescent wave 11 only reaches a region of several hundred nm from the interface, scattering from the impurities / floating matter M in the sample can be almost eliminated. Therefore, this evanescent fluorescence method is attracting attention as a method that can significantly reduce (light) noise as compared with the conventional fluorescence method and can measure the fluorescence of the detection target substance in units of one molecule.

  A surface plasmon enhanced fluorescence sensor as shown in FIG. 5 is also known as a sensor capable of measuring fluorescence with higher sensitivity. This surface plasmon enhanced fluorescence sensor is described in, for example, Patent Document 1, and is basically different from the fluorescence sensor of FIG. 4 in that a metal film 20 is formed on the prism 13. That is, by forming such a metal film 20, surface plasmons are generated in the metal film 20 when the excitation light 8 is irradiated, and fluorescence is amplified by the electric field amplification action. According to a simulation, it has been found that the fluorescence intensity in that case is amplified to about 1000 times.

  However, in the surface plasmon enhanced fluorescence sensor as described above, as shown in Non-Patent Document 2, if the phosphor in the sample and the metal film are too close, the energy excited in the phosphor Transition to the metal film before generating fluorescence, and a phenomenon that fluorescence does not occur (so-called metal quenching) may occur.

  In order to cope with this metal quenching, Non-Patent Document 2 forms a SAM (self-assembled film) on the metal film, thereby separating the phosphor and the metal film in the sample by more than the thickness of the SAM. It has been proposed to let In FIG. 5 as well, this SAM is indicated by number 21. Further, in Non-Patent Document 3, in relation to the metal quenching, the dependence of the fluorescence intensity enhanced by the surface plasmon on the distance from the metal film is examined.

  In recent years, the performance of photodetectors has been improved, such as the development of cooled CCDs. In particular, in the visible region, dyes with high efficiency (quantum yield) such as FITC (fluorescence 525 nm, quantum yield 0) .6) and Cy5 (fluorescence of 680 nm, quantum yield of 0.3), dyes with a quantum efficiency exceeding 0.2, which are practical guidelines, were developed, and the fluorescence method described above is indispensable for bioresearch. It is a tool.

  By the way, when performing a precise diagnosis in a hospital or the like, fluorescence detection of an analyte in blood such as a virus is widely performed. However, since blood generally has a red or reddish brown color, excitation with visible light is difficult, and fluorescence signals are also absorbed by blood, making detection with high sensitivity difficult. In addition, when irradiated with visible light, a large amount of biological material that emits autofluorescence such as albumin exists in blood, and this becomes optical noise, which greatly reduces the detection limit.

For this reason, when highly sensitive detection of an analyte in blood is performed, a filtering process such as passing through a mesh-like film is widely performed. This is generally called blood cell separation and mainly removes red blood cells. As a result, the sample (blood component) becomes almost transparent, and fluorescence measurement using visible light becomes possible. However, the three-dimensional structure of the analyte may be destroyed or denatured by passing through the membrane, and the inherent biological function may be impaired. In some cases, the analyte itself also remains in the membrane, so the concentration in the blood drops, making detection difficult. Furthermore, there is a problem that it takes time and labor since one extra work is added.
Japanese Patent No. 3562912 “Understanding this with bioimaging” p.104-113 Akihiro Shiomi et al. Yodosha W. Knoll et al., Analytical Chemistry (Anal. Chem.) 75 (2003) p.2610 W. Knoll et al., Colloids and Surfaces. A. (Colloids Surf. A), 171 (2000) p.115

  As shown in FIG. 6, the hemoglobin of erythrocytes occupying most of the blood, whether it is oxidized hemoglobin (solid line) or reduced hemoglobin (dotted line), has a visible absorption region in the near infrared region longer than 700 nm. Therefore, it is considered that fluorescence detection is possible using near infrared light.

  However, near-infrared dyes that are currently in practical use are not suitable for high-sensitivity detection because the quantum yield is low and the fluorescence signal is weak. In fact, the quantum yield of the near-infrared fluorescent dye ICG (fluorescence 820 nm), which is widely used for blood volume measurement, cardiac function tests and liver function tests, as well as fluorescent fundus angiography, is about 0.1, and the signal is It is too weak to detect.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a surface plasmon enhanced fluorescence detection method capable of detecting fluorescence with extremely high sensitivity without filtering a sample such as blood. To do.

  In the surface plasmon enhanced fluorescence detection method of the present invention, a sample is held by a metal film formed on the surface of a dielectric block made of a material that transmits excitation light, and excitation light is incident from the dielectric block side, In the surface plasmon-enhanced fluorescence detection method, a substance contained in a sample is excited by an evanescent wave that exudes to the surface of the metal film enhanced by surface plasmon, and the fluorescence emitted by the substance is detected by the excitation. An inflexible film made of a hydrophobic material is formed on the surface, and the excitation light is near-infrared light having a wavelength of 700 to 2000 nm.

  Here, the above “inflexibility” means that the film has rigidity enough to prevent deformation so that the film thickness changes during normal surface plasmon enhanced fluorescence detection.

  The film thickness of the inflexible film is preferably in the range of 10 to 100 nm. As the inflexible film, a polymer film is preferably used. When an inflexible film made of this polymer is applied, a hydrophilic linker that binds to a specific substance is preferably formed on the inflexible film. In particular, the surface plasmon enhanced fluorescence detection method of the present invention is suitable when the sample is blood.

  In the surface plasmon enhanced fluorescence detection method of the present invention, it is preferable to use a dielectric block in which capture molecules that bind to a specific substance are immobilized on the inflexible film. In that case, it is desirable that the capture molecule binds to a second messenger in the living body.

  When such a dielectric block is used, a labeled specific substance obtained by integrating a substance that directly binds to the capture molecule and a fluorescent component that can be excited by an evanescent wave is included in the sample. Thus, it is desirable to detect the fluorescence.

  In the surface plasmon enhanced fluorescence detection method of the present invention, a sample is held by a metal film formed on the surface of a dielectric block made of a material that transmits excitation light, and excitation light is incident from the dielectric block side, In a surface plasmon enhanced fluorescence detection method in which a substance contained in a sample is excited by an evanescent wave that exudes to the surface of a metal film enhanced by surface plasmon, and the fluorescence emitted by the substance is detected by this excitation, the surface of the metal film is hydrophobic. Since the inflexible film made of a conductive material is formed and the excitation light is a near-infrared light having a wavelength of 700 to 2000 nm, it is a dye in the near-infrared region where the fluorescence signal becomes weak because the quantum yield is low. Even if it exists, it is possible to implement | achieve highly sensitive detection.

  That is, fluorescence detection is possible for samples such as blood using near-infrared light, but fluorescence detected by excitation of near-infrared light is weak. In the case of detecting sensitivity, visible light must be used as excitation light, and for this reason, it has been forced to perform a filtering process that may reduce the blood concentration of an analyte or the like. According to the surface plasmon enhanced fluorescence detection method, excitation is performed by an evanescent wave that oozes on the surface of the metal film enhanced by the surface plasmon, and the fluorescence emitted from the substance is detected by this excitation. Sensitivity can be detected, and pre-processing such as filtering is not necessary, so that measurement can be greatly simplified.

  Note that by using near-infrared light having a long wavelength as excitation light, scattered light due to Rayleigh scattering in the substrate can be largely suppressed, so that optical noise can be reduced and the S / N ratio can be improved. Although a glass substrate with little scattering is usually used as the substrate, there are some directions where it is desired to use a resin substrate (such as PMMA) in consideration of cost, and according to the surface plasmon enhanced fluorescence detection method of the present invention, there is much scattering. Even a resin substrate can be detected with high sensitivity, and the cost can be greatly reduced.

  In addition, according to the research of the present inventor, in the conventional surface plasmon enhanced fluorescence sensor provided with the above-mentioned SAM, the sensitivity of fluorescence detection is not so improved because the SAM is so-called “fluffy”. It was found that the thickness fluctuates easily, and the phosphor in the sample solution may be close to the metal film to the extent that metal quenching occurs.

  Furthermore, in the conventional surface plasmon enhanced fluorescence sensor provided with this SAM, the reason why the sensitivity of fluorescence detection is not improved so much is that this SAM is so-called “slight”. It has also been found that molecules that cause quenching, such as dissolved oxygen, can penetrate into the SAM and deprive the excitation energy of the excitation light.

  In the surface plasmon enhanced fluorescence detection method according to the present invention, an inflexible film is provided on the metal film in view of the above-mentioned new knowledge, so that the phosphor in the sample solution is not quenched by metal with respect to the metal film. Since proximity to the extent to which it occurs is prevented, the above-described metal quenching is not caused, and the electric field amplification effect by the surface plasmon is reliably obtained, and the fluorescence can be detected with extremely high sensitivity.

  In addition, in the surface plasmon enhanced fluorescence detection method according to the present invention, in view of the above-mentioned new knowledge, the inflexible film is formed of a hydrophobic material, so that quenching such as metal ions and dissolved oxygen present in the sample liquid is possible. The causative molecules do not penetrate into the inflexible film, so that the molecules are prevented from depriving the excitation energy of the excitation light. Therefore, according to the surface plasmon enhanced fluorescence sensor according to the present invention, extremely high excitation energy is ensured, and fluorescence can be detected with extremely high sensitivity.

  In addition, although a polymer is mentioned as a preferable material of an inflexible film | membrane, the protein etc. which are contained in a sample liquid in many cases are easy to carry out nonspecific adsorption | suction easily. If so, for example, when detecting an antigen specifically adsorbed to the antibody applied to the surface of the inflexible film by a fluorescence method, this non-specific adsorption is in a state similar to that causing specific adsorption, This leads to false detection.

  Therefore, in the surface plasmon enhanced fluorescence detection method of the present invention, in particular, when an inflexible film made of a polymer is applied, if a hydrophilic linker is formed on the inflexible film, the linker blocks the protein or the like. Therefore, proteins and the like are not adsorbed nonspecifically on the inflexible film, and the above-described erroneous detection is prevented. On the other hand, an antibody or the like that should originally be arranged on the surface portion of the inflexible film can be specifically bound to the linker and captured on the surface portion of the inflexible film.

  Preferred examples of the inflexible film in the present invention include a film containing a hydrophobic polymer and an inorganic oxide.

  The hydrophobic polymer that can be used in the present invention preferably contains 50% by weight or more of a monomer having a solubility in water of 20% by weight or less.

  The solubility of the monomer forming the hydrophobic polymer in water at 25 ° C. can be measured by the method described in New Experimental Chemistry Course Basic Operation 1 (Maruzen Chemical, 1975). When measured by this method, the solubility of the monomer in the present invention in water at 20 ° C. is, for example, 0.002% by weight for 2-ethylhexyl methacrylate, 0.03% by weight for styrene, 1.35% by weight for methyl methacrylate, and butyl acrylate. 0.32% by weight and 0.03% by weight for butyl methacrylate. The solubility index for water as the hydrophobic polymer membrane in the present invention is more preferably 10% by weight or less, and further preferably 1% by weight or less.

  Specific examples of the monomer having a water solubility of 20% by weight or less used in the present invention include vinyl esters, acrylic esters, methacrylic esters, olefins, styrenes, crotonic esters, itaconic acid diesters. , Maleic acid diesters, fumaric acid diesters, allyl compounds, vinyl ethers, vinyl ketones, etc., styrene, methyl methacrylate, hexafluoropropane methacrylate, vinyl acetate, acrylonitrile, etc. are preferably used It is done. The hydrophobic polymer compound may be a homopolymer composed of one type of monomer or a copolymer composed of two or more types of monomers.

  In the present invention, a polymer compound obtained by copolymerizing a monomer having a solubility in water of 20% by weight or more may be used in combination with the monomer having a solubility in water of 20% by weight or less. Specific examples of the monomer having a solubility in water of 20% by weight or more include 2-hydroxyethyl methacrylate, methacrylic acid, acrylic acid, and allyl alcohol.

  As the inorganic oxide usable in the present invention, silica, alumina, titania, zirconia, ferrite, and composite materials and derivatives thereof can be selected. As a film forming method, a conventional method can be used. For example, a sol-gel method, a sputtering method, a vapor deposition method, a plating method, or the like can be employed.

  As a compound which forms a water non-swellable film | membrane in this invention, polyacrylic acid ester, polymethacrylic acid ester, polyester, and polystyrene are preferable. By doing so, film formation becomes easy and it becomes easy to expose a functional group for immobilizing a physiologically active substance on the surface. For example, a film made of polyacrylic acid ester, polymethacrylic acid ester, or polyester can easily expose the carboxyl group and hydroxyl group on the surface by hydrolyzing the surface with acid or base, and is made of polystyrene. The film can be easily exposed to carboxylic acid by subjecting it to an oxidation treatment such as UV / ozone treatment.

  On the other hand, in the surface plasmon enhanced fluorescence detection method according to the present invention, when using a dielectric block in which a capture molecule that binds to a specific substance such as a second messenger in a living body is immobilized on an inflexible film, a so-called “ The “competitive format” fluorescence detection can be suitably employed. That is, more specifically, after the sample includes a labeled specific substance in which a substance such as the second messenger that directly binds to the capture molecule and a fluorescent component that can be excited by an evanescent wave are integrated. By performing fluorescence detection, it is possible to implement a competitive type detection method in which a target substance and a labeled specific substance are competitively bound to a capture molecule to detect the labeled specific substance.

  By adopting this competitive fluorescence detection method, it is possible to detect fluorescence with high sensitivity by enhancing the surface plasmon. Therefore, B / F, which has been considered difficult by “sandwich-type” fluorescence detection using two types of capture molecules. Separation of separation (bound / free separation) and high sensitivity can be realized. If B / F separation can be made unnecessary, it is possible to prevent a decrease in sensitivity due to uneven cleaning that often occurs in competitive-type fluorescence detection methods.

  Also in this case, since the surface plasmon enhancing effect is obtained, detection and / or quantification of a low molecular weight compound, which is usually very difficult to perform by the sandwich type detection method, can be easily performed.

  The labeled specific substance as described above may be present in the sample in advance, or the sample may be mixed with the sample after contacting the sample with a capture molecule after a predetermined time has passed. The sample may be mixed with the sample after a predetermined period of time has elapsed from contact with the capture molecules.

The capture molecule is not particularly limited as long as it is a molecule that binds to a specific substance. For example, antibodies, fragments thereof, adapters composed of nucleic acids, inclusion compounds and template molecules constructed by molecular imprinting can be used. Although there is a dissociation equilibrium constant (Kd) as an index indicating the binding property of a capture molecule to a specific substance, in the present invention, Kd is preferably 10 ⁻⁵ or less, more preferably 10 ⁻⁶ or less, and even more preferably 10 ^−. Capture molecules of ⁷ or less, more preferably 10 ⁻⁸ or less are selected. In addition, it is desirable to selectively use a capture molecule having specificity that exhibits binding properties to specific molecules but does not exhibit binding properties to other substances.

  In particular, when the capture molecule binds to a second messenger in the living body, the activation or inhibition of the receptor by the ligand can be detected and / or quantified by measuring the amount of the second messenger. In this case as well, the second messenger can be detected and / or quantified with high sensitivity by enhancing the surface plasmon.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a schematic side view showing a surface plasmon enhanced fluorescence sensor (hereinafter simply referred to as a fluorescence sensor) used for carrying out the surface plasmon enhanced fluorescence detection method of the present invention. As shown in the drawing, this fluorescent sensor is composed of a light source 7 such as a semiconductor laser that emits excitation light 8 of near-infrared light, and a material that transmits the excitation light 8, and is arranged at a position where the excitation light 8 is incident from one end face. The prism (dielectric block) 13 formed, the metal film 20 formed on one surface 13 a of the prism 13, the inflexible film 31 made of polymer formed on the metal film 20, and the opposite of the prism 13 A sample holder 5 that holds the sample 1 so that the liquid sample 1 contacts the inflexible film 31 from the side, and a photodetector (fluorescence detection means) 9 disposed above the sample holder 5. It will be.

  In FIG. 1, the light source 7 is arranged so that the excitation light 8 is incident through the prism 13 so as to satisfy the total reflection condition toward the interface between the prism 13 and the metal film 20. That is, the light source 7 itself constitutes an incident optical system that makes the excitation light 8 incident on the prism 13 as described above. However, the present invention is not limited to this configuration, and there is no problem even if an incident optical system including a lens, a mirror, or the like that makes the excitation light 8 incident as described above is provided separately from the light source 7.

  For example, the prism 13 is made of ZEONEX (registered trademark) 330R (refractive index: 1.50) manufactured by Nippon Zeon Co., Ltd. On the other hand, the metal film 20 is formed by sputtering gold on the one surface 13a of the prism 13 and has a film thickness of 50 nm.

  The inflexible film 31 is formed by spin-coating a polystyrene polymer having a refractive index of 1.59 on the metal film 20, and here the film thickness is 20 nm. The inflexible film 31 is not limited to an organic material such as a polymer, and may be an inorganic material. The material of the prism is not limited to ZEONEX (registered trademark) 330R, and a resin such as polymethyl methacrylate (PMMA), polycarbonate (PC), and amorphous polyolefin (APO) containing cycloolefin may be used as a known resin or optical glass. May be used as appropriate, or an inorganic material such as optical glass may be used.

  Note that phosphor molecules existing in the vicinity of the metal are quenched by energy transfer to the metal. The degree of energy transfer is inversely proportional to the third power of the distance if the metal has a semi-infinite thickness, inversely proportional to the fourth power of the distance if the metal is infinitely thin, and It becomes smaller in inverse proportion to the sixth power. As described in Non-Patent Document 3 described above, in the case of a metal film, it is desirable to secure a distance between the metal and the fluorescent molecule of at least several nm or more, more preferably 10 nm or more. Therefore, the lower limit of the film thickness of the inflexible film 31 is preferably 10 nm.

  On the other hand, the phosphor molecules are excited by evanescent waves that have been enhanced by surface plasmons and ooze out on the surface of the metal film. It is known that the reach of the evanescent wave (distance from the surface of the metal film) is at most about a wavelength, and the electric field strength attenuates exponentially and exponentially according to the distance from the surface of the metal film. Actually, in the near-infrared light having a wavelength of 808 nm, the evanescent wave oozes out at about the wavelength (808 nm), and the electric field strength rapidly attenuates when the wavelength exceeds 100 nm. Since it is desirable that the electric field intensity for exciting the phosphor molecules is larger, it is desirable to make the distance between the metal film surface and the phosphor molecules smaller than 100 nm for effective excitation. Accordingly, the upper limit value of the film thickness of the inflexible film 31 is preferably 100 nm.

  The prism 13 can be appropriately formed using a known resin or optical glass in addition to the above materials. From the point of cost, it can be said that resin is more preferable than optical glass. When formed from a resin, a resin such as polymethyl methacrylate (PMMA), polycarbonate (PC), or amorphous polyolefin (APO) containing cycloolefin can be suitably used.

  As the photodetector 9, for example, LAS-1000 (trade name) manufactured by FUJIFILM Corporation is used.

As an example, this fluorescent sensor detects CRP antigen 2 (molecular weight 110,000 Da), and a primary antibody (monoclonal antibody) 4 that specifically binds to CRP antigen 2 is immobilized on the inflexible film 31. Has been. The primary antibody 4 is fixed to the inflexible film 31 made of the polymer by, for example, an amine coupling method via PEG having a terminal carboxyl group. On the other hand, as the secondary antibody 6, a monoclonal antibody labeled with a phosphor (ICG) 10 represented by the following chemical formula (epitope; antigenic determinant> is different from the primary antibody 4) is used.

  The amine coupling method includes the following steps (1) to (3) as an example. This is an example in which a 30 μl (microliter) cuvette / cell is used.

(1) Activate the -COOH group at the linker tip (terminal): 0.1M (mol) NHS (N-hydrooxysuccinimide) and 0.4M EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) 30 μl of an equal volume mixed solution was added and allowed to stand at room temperature for 30 minutes (2) After washing 5 times with immobilized PBS buffer (pH 7.4) of primary antibody 4, 30 μl of primary antibody solution (500 μg / ml) was added, and 30 to Leave at room temperature for 60 minutes (3) Wash unreacted —COOH group 5 times with blocking PBS buffer (pH 7.4), add 30 μl of 1M ethanolamine (pH 8.5), and leave at room temperature for 20 minutes. Further, it was washed 5 times with PBS buffer (pH 7.4).

On the other hand, the light source 7 is not limited to the semiconductor laser, and other known light sources can be appropriately selected and used. The photodetector 9 is not limited to the above-described one, and a known one such as a CCD, a PD (photodiode), a photomultiplier tube, or a c-MOS can be appropriately selected and used. In addition to the above ICG, IRDye ^™ 700DX (excitation wavelength / fluorescence wavelength 689/700), IRDye ^™ 800DX (778/806), HiLyte Fluor750 (750/780), Cy7 (750/780) , Alexa Fluor (registered trademark) 700 (700/720), Alexa Fluor (registered trademark) 750 (750/780), DiR (DilC18 (7)) (750/780), TO-PRO-5 (750/770) Etc. can also be used.

  Hereinafter, the surface plasmon enhanced fluorescence detection method of the present invention using this fluorescence sensor will be described taking as an example the case of detecting CRP antigen 2 contained in blood (hereinafter referred to as blood 1) as sample 1. First, blood 1 is flowed in the sample holder 5, and then a secondary antibody 6 that is similarly labeled with the phosphor 10 and specifically binds to the CRP antigen 2 is flowed. The ICG is excited by excitation light 8 having a wavelength of 768 nm and emits fluorescence of 807 nm.

  Thereafter, the excitation light 8 is emitted from the light source 7 toward the prism 13, and the fluorescence is detected by the photodetector 9. At this time, the evanescent wave 11 oozes out from the interface between the prism 13 and the metal film 20. Therefore, if CRP antigen 2 is bound to primary antibody 4, secondary antibody 6 is further bound to CRP antigen 2, and phosphor 10, which is a label for secondary antibody 6, is excited by evanescent wave 11. The Rukoto. The excited phosphor 10 emits fluorescence having a predetermined wavelength, and the fluorescence is detected by the photodetector 9. Thus, when the photodetector 9 detects fluorescence of a predetermined wavelength, it confirms that the secondary antibody 6 is bound to the CRP antigen 2, that is, the blood 1 contains the CRP antigen 2. It becomes possible.

  As described above, in the surface plasmon enhanced fluorescence detection method of the present invention, near-infrared light can be used, so that a substance in blood is directly detected with high sensitivity without performing filtering processing such as blood cell separation. Is possible.

  The evanescent wave 11 reaches only a region of about several hundred nm from the interface between the prism 13 and the metal film 20. For this reason, almost no scattering from impurities / floating matters in the blood 1 can be achieved. In addition, in this fluorescent sensor, light scattered by the impurity N in the prism 13 (this is normal propagation light) is blocked by the metal film 20 and does not reach the photodetector 9. For this reason, in the surface plasmon enhanced fluorescence detection method of the present invention, optical noise can be reduced to almost none, and extremely high S / N fluorescence detection is possible.

  Further, in the surface plasmon enhanced fluorescence detection method of the present invention, surface plasmon is excited on the one surface 13a of the prism 13 by the metal film 20, and the fluorescence is amplified by the electric field amplification action of this surface plasmon. Method, for example, a detection limit of 5 ng / ml (about 180 pM) in “Serum (plasma)” shown in TABLE 1 (p.55) of LA Tempelman et al., Analytical Biochemistry, 233 (1996) p.50-57. 2), it is possible to improve the 2-3 digit detection sensitivity.

  In addition, in the surface plasmon enhanced fluorescence detection method of the present invention, since the inflexible film 31 is provided on the metal film 20, the phosphor 10 in the blood 1 performs metal quenching on the metal film 20. Proximity to the extent that it occurs is prevented. Therefore, according to this fluorescent sensor, the above-described metal quenching is not caused, and the electric field amplification action by the surface plasmon is reliably obtained, and the fluorescence can be detected with extremely high sensitivity.

  Since the inflexible film 31 is formed of a polystyrene-based polymer that is a hydrophobic material, molecules that cause quenching such as metal ions and dissolved oxygen present in the blood 1 are contained in the inflexible film 31. Therefore, these molecules are prevented from depriving the excitation energy of the excitation light 8. Therefore, according to this fluorescence sensor, extremely high excitation energy is ensured, and fluorescence can be detected with extremely high sensitivity.

  In this fluorescent sensor, the secondary antibody 6 that does not bind to the CRP antigen 2 and is separated from the surface of the inflexible film 31 does not emit fluorescence because the evanescent wave 11 does not reach there. Therefore, even if such secondary antibody 6 is floating in blood 1, there is no problem in measurement. Therefore, it is not necessary to perform washing, that is, B / F separation (bound / free separation) for each measurement.

  The secondary antibody 6 and the antigen 2 are easily non-specifically adsorbed on the inflexible film 31 made of a polystyrene-based polymer acting as described above. If this is the case, a specific adsorption between the antibodies 4 and 6 and the antigen 2 occurs, and this leads to erroneous detection of the antigen 2. For this reason, you may use the fluorescence sensor by which the hydrophilic linker 32 is couple | bonded on the inflexible film | membrane 31 as shown in FIG.

  In the fluorescent sensor shown in FIG. 2, a hydrophilic linker 32 that binds to the primary antibody 4 is formed on the inflexible film 31, and the linker 32 blocks the secondary antibody 6 and the antigen 2. Non-specific adsorption to the inflexible film 31 is eliminated, and erroneous detection as described above is prevented. On the other hand, the primary antibody 4 that should originally be disposed on the surface portion of the inflexible film 31 can be specifically adsorbed with the linker 32 and captured on the surface portion of the inflexible film 31.

  As described above, according to the surface plasmon enhanced fluorescence detection method of the present invention, excitation is performed by an evanescent wave that oozes on the surface of the metal film enhanced by the surface plasmon, and the fluorescence emitted from the substance is detected by this excitation. It is possible to perform high-sensitivity detection using infrared light, and it is not necessary to perform preprocessing such as filtering, so that measurement can be greatly simplified.

  Next, another embodiment of the surface plasmon enhanced fluorescence detection method according to the present invention will be described with reference to FIG. The fluorescent sensor used here is basically different from that shown in FIG. 1 in that the capture molecules 40 are immobilized on the inflexible film 31.

  The method of this embodiment performs so-called competitive-type fluorescence detection when detecting and / or quantifying a substance contained in the sample 1. Hereinafter, the fluorescence detection will be described. In the present embodiment, as an example, the second messenger 41 existing in the living body is set as a detection target, and the capture molecule 40 that directly binds to the second messenger 41 is applied.

  Prior to fluorescence detection, the sample 1 contains a labeled specific substance formed by integrating the phosphor 10 and the second messenger 41 as described above. Such a labeled specific substance may be present in the sample 1 in advance, or may be mixed in the sample 1 after the sample 1 has been brought into contact with the capture molecule 40 and a predetermined time has passed, Alternatively, the labeled specific substance may be brought into contact with the capture molecule 40 and the sample 1 may be mixed after a predetermined time has elapsed. Such a labeled specific substance is preferably sold together with the fluorescent sensor or as a kit dedicated to the fluorescent sensor, because it is easily available to the sensor user and can easily carry out competitive-type fluorescence detection.

  In this competitive form of fluorescence detection, the more specific second messengers 41 to be detected are present in the sample 1, the fewer labeled specific substances (that is, the phosphors 10) are bound to the capture molecules 40. That is, as the amount of the second messenger 41 in the sample 1 increases, the intensity of the fluorescence detected by surface plasmon enhancement decreases, and therefore the second messenger 41 can be detected and / or quantified based on this principle.

  Also in this case, since the surface plasmon enhancement effect described above can be obtained in the same manner, the second messenger 41 can be detected with high sensitivity. Therefore, detection of low molecular weight compounds that are usually very difficult to perform with the sandwich type detection method. And / or quantification can be easily performed.

Schematic side view showing a surface plasmon enhanced fluorescence sensor capable of performing the fluorescence detection method of the present invention The schematic side view which shows the surface plasmon enhancement fluorescence sensor of another aspect which can implement the fluorescence detection method of this invention Schematic side view showing an example of a conventional fluorescent sensor Schematic side view showing another example of a conventional fluorescent sensor Schematic side view showing still another example of a conventional fluorescent sensor Graph showing hemoglobin absorption wavelength spectrum Schematic side view showing another example of a surface plasmon enhanced fluorescence sensor capable of performing the fluorescence detection method of the present invention

Explanation of symbols

1 Sample 2 Antigen 4 Primary Antibody 6 Secondary Antibody 7 Light Source 8 Excitation Light 9 Photodetector 10 Phosphor 13 Prism (Dielectric Block)
20 Metal film 31 Inflexible film 32 Hydrophilic linker 40 Capture molecule 41 Second messenger

Claims (8)

The sample is held by a metal film formed on the surface of the dielectric block made of a material that transmits the excitation light, and the excitation light is incident from the dielectric block side so that the substance contained in the sample is made by surface plasmon. In the surface plasmon enhanced fluorescence detection method, which is excited by an evanescent wave that oozes out on the surface of the enhanced metal film, and detects fluorescence emitted by the substance by the excitation,
A surface plasmon enhanced fluorescence detection method, wherein an inflexible film made of a hydrophobic material is formed on a surface of the metal film, and the excitation light is near infrared light having a wavelength of 700 to 2000 nm.   2. The surface plasmon enhanced fluorescence detection method according to claim 1, wherein the inflexible film has a thickness in a range of 10 to 100 nm.   3. The surface plasmon enhanced fluorescence detection method according to claim 1, wherein the inflexible film is made of a polymer.   4. The surface plasmon enhanced fluorescence detection method according to claim 3, wherein a hydrophilic linker that binds to a specific substance is formed on the inflexible film made of the polymer.   The surface plasmon enhanced fluorescence detection method according to any one of claims 1 to 4, wherein the sample is blood.   6. The surface plasmon enhanced fluorescence detection method according to claim 1, wherein a dielectric block in which capture molecules that bind to a specific substance are immobilized on the inflexible film is used.   The surface plasmon enhanced fluorescence detection method according to claim 6, wherein the capture molecule binds to a second messenger in a living body.   The fluorescence is detected after a labeled specific substance formed by integrating a substance that directly binds to the capture molecule and a fluorescent component that can be excited by the evanescent wave is included in the sample. The method for detecting surface plasmon enhanced fluorescence according to claim 6 or 7.

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