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JP-7856210-B2 - Method for measuring relative fluorescence intensity

JP7856210B2JP 7856210 B2JP7856210 B2JP 7856210B2JP-7856210-B2

Inventors

  • 竹澤 豊
  • 櫻井 直人
  • 豊村 恭一

Assignees

  • ＤＩＣ株式会社

Dates

Publication Date
20260511
Application Date
20240221
Priority Date
20230306

Claims (19)

  1. A method for measuring the relative fluorescence intensity of a test sample, The maximum fluorescence wavelength of the test sample is within the range of 650 nm to 1000 nm. A plate-shaped or film-shaped molded body of a resin composition obtained by melt-kneading a raw material mixture containing a near-infrared fluorescent dye and an amorphous resin is used as a standard plate. A plate-shaped or film-shaped molded body of a resin composition obtained by melt-kneading a raw material mixture having the same composition as the raw material mixture except that it does not contain the aforementioned near-infrared fluorescent dye is used as a blank plate. The test sample is a molded body of a resin composition containing the same or different near-infrared fluorescent dye as the near-infrared fluorescent dye in the standard plate. A molded body having the same composition as the test sample except that it does not contain the near-infrared fluorescent dye contained in the test sample is used as the test sample blank. The fluorescence intensity F1 of the test sample at wavelength λ1, the fluorescence intensity F1B of the test sample blank at wavelength λ1B , the fluorescence intensity FS of the standard plate at wavelength λS , and the fluorescence intensity FSB of the blank plate at wavelength λSB are measured, and the wavelength λ1 is within the range of 650 to 1000 nm, the difference between wavelength λ1 and wavelength λ1B is within 10 nm, the wavelength λS is within the range of 650 to 1000 nm, and the difference between wavelength λS and wavelength λSB is within 10 nm. A method for measuring relative fluorescence intensity, wherein ( F1 - F1B )/(Fs - FSB ) is defined as the relative fluorescence intensity of the test sample.
  2. The method for measuring relative fluorescence intensity according to claim 1, wherein the fluorescence intensity of the test sample, the test sample blank, the standard plate, and the blank plate is measured using a spectrofluorometer.
  3. The method for measuring relative fluorescence intensity according to claim 1, wherein the amorphous resin is a transparent resin.
  4. The method for measuring relative fluorescence intensity according to claim 1, wherein the amorphous resin is one or more selected from the group consisting of polycarbonate resins, polystyrene resins, acrylic resins, polyoxymethylene resins, polyester resins, and vinyl chloride resins.
  5. The method for measuring relative fluorescence intensity according to claim 1, wherein the standard plate is a plate with a thickness of 1 μm to 15 mm.
  6. The method for measuring relative fluorescence intensity according to claim 1, wherein the maximum fluorescence wavelength of the test sample is 700 nm or higher.
  7. The method for measuring relative fluorescence intensity according to claim 1, wherein the test sample is a molded article obtained by melt-molding a resin composition obtained by melt-kneading a mixture containing a near-infrared fluorescent dye as a raw material.
  8. The aforementioned test sample is a molded body used as a medical device, The method for measuring relative fluorescence intensity according to claim 7, wherein the quality control of the test sample is performed by measuring the relative fluorescence intensity of the test sample in vitro .
  9. The method for measuring relative fluorescence intensity according to claim 8, wherein at least a portion of the test sample is a medical device used in a patient's body.
  10. The aforementioned near-infrared fluorescent dye, The following general formula (I 1 ) [In formula (I 1 ), Ra and Rb , together with the nitrogen atom to which Ra is bonded and the carbon atom to which Rb is bonded, form an aromatic five-membered ring, an aromatic six-membered ring, or a condensed aromatic ring formed by the condensation of two to three five-membered or six-membered rings; Rc and Rd , together with the nitrogen atom to which Rc is bonded and the carbon atom to which Rd is bonded, form an aromatic five-membered ring, an aromatic six-membered ring, or a condensed aromatic ring formed by the condensation of two or three five-membered or six-membered rings; Re and Rf represent halogen atoms or oxygen atoms; R g represents a hydrogen atom or an electron-withdrawing group. However, if Re and Rf are oxygen atoms, Re , the boron atom bonded to Re , Ra , and the nitrogen atom bonded to Ra may all form a ring, and Rf, the boron atom bonded to Rf, Rc , and the nitrogen atom bonded to Rc may all form a ring. If Re is an oxygen atom and does not form a ring, then Re is a substituted oxygen atom, and if Rf is an oxygen atom and does not form a ring, then Rf is a substituted oxygen atom. Compounds represented by ] The following general formula (I 2 ) Compounds represented by the following general formula ( I3 ): [In formula ( I2 ), Ra a to R f are the same as in formula ( I1 ).] [In formula (I 3 ), Rh and Ri , together with the nitrogen atom to which Rh is bonded and the carbon atom to which Ri is bonded, form an aromatic five-membered ring, an aromatic six-membered ring, or a condensed aromatic ring formed by the condensation of two or three five-membered or six-membered rings; R j and R k , together with the nitrogen atom to which R j is bonded and the carbon atom to which R k is bonded, form an aromatic five-membered ring, an aromatic six-membered ring, or a condensed aromatic ring formed by the condensation of two or three five-membered or six-membered rings; R l , R m , R n , and R o independently represent a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group; R p and R q independently represent a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group. R r and R s independently represent a hydrogen atom or an electron-withdrawing group. [The compound represented by ] and the following general formula (I 4 ) [In formula ( I4 ), Rh to Rq are the same as in formula ( I3 ).] One or more compounds selected from the group consisting of compounds represented by: The method for measuring relative fluorescence intensity according to claim 1.
  11. The aforementioned near-infrared fluorescent dye is given by the following general formula (I 1 - 0) [In formula (I 1 -0), R1 , R2 , and R3 are, (p1) Independently representing a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group, (p2) R1 and R2 both form an aromatic five-membered ring or an aromatic six-membered ring, and R3 represents a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group, or (p3) R2 and R3 both form an aromatic five-membered ring or an aromatic six-membered ring, and R1 represents a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group. R4 , R5 , and R6 are, (q1) Each independently represents a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group. (q2) R4 and R5 both form an aromatic five-membered ring or an aromatic six-membered ring, and R6 represents a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group, or (q3) R5 and R6 both form an aromatic five-membered ring or an aromatic six-membered ring, and R4 represents a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group. R7 and R8 represent halogen atoms or oxygen atoms; R9 represents a hydrogen atom or an electron-withdrawing group. However, if R7 and R8 are oxygen atoms, R7 , the boron atom bonded to R7 , the nitrogen atom bonded to the boron atom, R1 , and the carbon atom bonded to R1 may all form a ring, and R8 , the boron atom bonded to R8 , the nitrogen atom bonded to the boron atom, R4 , and the carbon atom bonded to R4 may all form a ring. If R7 is an oxygen atom and does not form a ring, then R7 is a substituted oxygen atom, and if R8 is an oxygen atom and does not form a ring, then R8 is a substituted oxygen atom. Compounds represented by ] and the following general formula ( I2-0 ) A method for measuring relative fluorescence intensity according to claim 10, wherein the compound is one or more compounds selected from the group consisting of compounds represented by formula ( I2-0 ), where R1 to R8 are the same as in formula ( I1-0 ).
  12. In the above general formula ( I1-0 ) or the above general formula ( I2-0 ), R1 and R2 form a ring, and R4 and R5 form a ring, or R2 and R3 form a ring, and R5 and R6 form a ring, The aforementioned ring is given by the following general formulas (C-1) to (C-9) A method for measuring relative fluorescence intensity according to claim 11, represented by any of the following formulas: [In formulas (C-1) to (C-9), Y1 to Y8 independently represent a sulfur atom, an oxygen atom, a nitrogen atom, or a phosphorus atom, and R11 to R22 independently represent a hydrogen atom or any group that does not inhibit the fluorescence of the compound.]
  13. The aforementioned near-infrared fluorescent dye is expressed by the following general formulas ( I3-1 ) to ( I3-6 ) [In formula (I 3 -1), R23 , R24 , R25 , and R26 independently represent a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group; R 27 and R 28 independently represent a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group; R 29 and R 30 independently represent a hydrogen atom or an electron-withdrawing group; Y9 and Y10 independently represent a sulfur atom, an oxygen atom, a nitrogen atom, or a phosphorus atom; R31 and R32 are, (p4) Independently, each represents a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group; or (p5) R31 and R32 together form an optionally substituted aromatic five-membered ring or an optionally substituted aromatic six-membered ring; R33 and R34 are, (q4) Independently, each represents a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group, or (q5) R33 and R34 together form an optionally substituted aromatic five-membered ring or an optionally substituted aromatic six-membered ring. [In equations ( I3-2 ) to ( I3-6 ), R23 to R30 are the same as in equation ( I3-1 ); X1 and X2 independently represent a nitrogen atom or a phosphorus atom; R35 , R36 , R37 , and R38 are, (p6) Each independently represents a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group. (p7) R 35 and R 36 both form optionally substituted aromatic five-membered rings or optionally substituted aromatic six-membered rings, and R 37 and R 38 independently represent a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group. (p8) R 36 and R 37 together form optionally substituted aromatic five-membered rings or optionally substituted aromatic six-membered rings, and R 35 and R 38 independently represent a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group, or (p9) R 37 and R 38 together form optionally substituted aromatic five-membered rings or optionally substituted aromatic six-membered rings, and R 35 and R 36 independently represent a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group; R39 , R40 , R41 , and R42 are, (q6) Each independently represents a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group. (q7) R 39 and R 40 both form optionally substituted aromatic five-membered rings or optionally substituted aromatic six-membered rings, and R 41 and R 42 independently represent a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group. (q8) R 40 and R 41 both form optionally substituted aromatic five-membered rings or optionally substituted aromatic six-membered rings, and R 39 and R 42 independently represent a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group, or (q9) R 41 and R 42 both form optionally substituted aromatic five-membered rings or optionally substituted aromatic six-membered rings, and R 39 and R 40 independently represent a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group. Compounds represented by either of the following general formulas (I 4-1 ) to (I 4-6 ) A method for measuring relative fluorescence intensity according to claim 10 , wherein the compound is one or more compounds selected from the group consisting of compounds represented by any of the following formulas: [In formulas ( I4-1 ) to ( I4-6 ), R23 to R28 are the same as in formula ( I3-1 ). In formula ( I4-1 ), R31 to R34 , Y9 , and Y10 are the same as in formula ( I3-1 ). In formulas ( I4-2 ) to ( I4-6 ), R35 to R42 are the same as in formula (I3-2). In formulas ( I4-3 ) to (I4-6), X1 and X2 are the same as in formula (I3-3).]
  14. The aforementioned near-infrared fluorescent dyes are those of the following general formulas: ( I1-1-1 ) to ( I1-1-6 ), ( I1-2-1 ) to (I1-2-12), ( I2-1-1 ) to ( I2-1-6 ), and ( I2-2-1 ) to ( I2-2-12 ) [In the formula, Y 11 and Y 12 independently represent either an oxygen atom or a sulfur atom; Y21 and Y22 independently represent a carbon atom or a nitrogen atom; Q11 represents a trifluoromethyl group, a cyano group, a nitro group, or a phenyl group; X independently represents a halogen atom, a C1-20 alkoxy group, an aryloxy group, or an acyloxy group; P11 to P14 and P17 independently represent a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an amino group, a monoalkylamino group, or a dialkylamino group; A11 to A14 independently represent a phenyl group which may have 1 to 3 substituents selected from the group consisting of a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an amino group, a monoalkylamino group, and a dialkylamino group, or a heteroaryl group which may have 1 to 3 substituents selected from the group consisting of a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an amino group, a monoalkylamino group, and a dialkylamino group; n11 to n14 and n17 represent integers from 0 to 3, independently of each other; m1 represents either 0 or 1. The method for measuring relative fluorescence intensity according to claim 10, wherein the compound is one or more compounds selected from the group consisting of compounds represented by any of the following.
  15. The aforementioned near-infrared fluorescent dyes are those of the following general formulas ( I3-7 ) to ( I3-9 ) and ( I4-7 ) to ( I4-9 ) [In the formula, Y23 and Y24 independently represent a carbon atom or a nitrogen atom; Y13 and Y14 independently represent either an oxygen atom or a sulfur atom; Y25 and Y26 independently represent a carbon atom or a nitrogen atom; R 47 and R 48 independently represent a hydrogen atom or an electron-withdrawing group; R 43 , R 44 , R 45 , and R 46 represent halogen atoms or optionally substituted aryl groups; P15 and P16 independently represent a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an amino group, a monoalkylamino group, or a dialkylamino group; n15 and n16 represent integers between 0 and 3, independently of each other; A15 and A16 independently represent a phenyl group which may have 1 to 3 substituents selected from the group consisting of a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an amino group, a monoalkylamino group, and a dialkylamino group. The method for measuring relative fluorescence intensity according to claim 10, wherein the compound is one or more compounds selected from the group consisting of compounds represented by any of the following.
  16. A standard plate set for measuring relative fluorescence intensity, for measuring the relative fluorescence intensity of a test sample whose maximum fluorescence wavelength is in the range of 650 nm to 1000 nm, A standard plate comprising a plate-shaped or film-shaped molded body of a resin composition obtained by melt-kneading a raw material mixture containing a near-infrared fluorescent dye and an amorphous resin, The system comprises a blank plate made of a plate-shaped or film-shaped molded body of a resin composition obtained by melt-kneading a raw material mixture having the same composition as the raw material mixture except that it does not contain the aforementioned near-infrared fluorescent dye, A standard plate set for measuring relative fluorescence intensity, wherein the fluorescence intensity F1 at wavelength λ1 of a test sample made from a molded body of a resin composition containing the same or different near-infrared fluorescent dyes as the near-infrared fluorescent dye in the standard plate, the fluorescence intensity F1B at wavelength λ1B of a test sample blank made from a molded body having the same composition as the test sample except that it does not contain the near-infrared fluorescent dye contained in the test sample , the fluorescence intensity FS at wavelength λS of the standard plate , and the fluorescence intensity FSB at wavelength λSB of the blank plate, wherein when the wavelength λ1 is in the range of 650 to 1000 nm , the difference between wavelength λ1 and wavelength λ1B is within 10 nm, the wavelength λS is in the range of 650 to 1000 nm , and the difference between wavelength λS and wavelength λSB is within 10 nm, the relative fluorescence intensity of the test sample is measured as ( F1 - F1B ) / ( FS - FSB ).
  17. The aforementioned near-infrared fluorescent dye, The following general formula (I 1 ) [In formula (I 1 ), Ra and Rb , together with the nitrogen atom to which Ra is bonded and the carbon atom to which Rb is bonded, form an aromatic five-membered ring, an aromatic six-membered ring, or a condensed aromatic ring formed by the condensation of two or three five-membered or six-membered rings; Rc and Rd , together with the nitrogen atom to which Rc is bonded and the carbon atom to which Rd is bonded, form an aromatic five-membered ring, an aromatic six-membered ring, or a condensed aromatic ring formed by the condensation of two or three five-membered or six-membered rings; Re and Rf represent halogen atoms or oxygen atoms; R g represents a hydrogen atom or an electron-withdrawing group. However, if Re and Rf are oxygen atoms, Re , the boron atom bonded to Re , Ra , and the nitrogen atom bonded to Ra may all form a ring, and Rf, the boron atom bonded to Rf, Rc , and the nitrogen atom bonded to Rc may all form a ring. If Re is an oxygen atom and does not form a ring, then Re is a substituted oxygen atom, and if Rf is an oxygen atom and does not form a ring, then Rf is a substituted oxygen atom. Compounds represented by ] The following general formula (I 2 ) Compounds represented by the following general formula ( I3 ): [In formula ( I2 ), Ra a to R f are the same as in formula ( I1 ).] [In formula (I 3 ), Rh and Ri , together with the nitrogen atom to which Rh is bonded and the carbon atom to which Ri is bonded, form an aromatic five-membered ring, an aromatic six-membered ring, or a condensed aromatic ring formed by the condensation of two or three five-membered or six-membered rings; R j and R k , together with the nitrogen atom to which R j is bonded and the carbon atom to which R k is bonded, form an aromatic five-membered ring, an aromatic six-membered ring, or a condensed aromatic ring formed by the condensation of two or three five-membered or six-membered rings; R l , R m , R n , and R o independently represent a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group; R p and R q independently represent a hydrogen atom, a halogen atom, a C1-20 alkyl group, a C1-20 alkoxy group, an aryl group, or a heteroaryl group. R r and R s independently represent a hydrogen atom or an electron-withdrawing group. [The compound represented by ] and the following general formula (I 4 ) A standard plate set for measuring relative fluorescence intensity according to claim 16, comprising one or more compounds selected from the group consisting of compounds represented by [Formula ( I4 ), where Rh to Rq are the same as in Formula ( I3 )].
  18. The standard plate set for relative fluorescence intensity measurement according to claim 16, wherein the standard plate is a plate with a thickness of 1 μm to 15 mm.
  19. The standard plate set for relative fluorescence intensity measurement according to claim 16, wherein the amorphous resin is one or more selected from the group consisting of polycarbonate resins, polystyrene resins, acrylic resins, polyoxymethylene resins, polyester resins, and vinyl chloride resins.

Description

This invention relates to a method for determining the relative fluorescence intensity of a test sample that emits near-infrared fluorescence. This application claims priority based on Japanese Patent Application No. 2023-033836, filed in Japan on March 6, 2023, and the contents of that application are incorporated herein by reference. Near-infrared fluorescent dyes are used in industrial products, primarily for product identification and anti-counterfeiting. In recent years, they have also been used in medical applications such as bioimaging probes and diagnostic reagents. The near-infrared wavelength range is known to be invisible to the naked eye, has minimal impact on living organisms, and exhibits high penetration into skin and other biological tissues. These characteristics can be utilized by incorporating near-infrared fluorescent dyes into medical devices themselves. For example, by incorporating near-infrared fluorescent dyes into medical devices such as shunt tubes, the location of implanted medical devices can be confirmed by irradiating them with near-infrared light from outside the body. If near-infrared fluorescent dyes can be mixed and dispersed in a resin, various molded articles that emit near-infrared fluorescence can be manufactured using that resin as a raw material. However, if the dispersibility of the near-infrared fluorescent dye in the resin is low, the near-infrared fluorescent dye may be unevenly distributed in the resin composition, and aggregates of the near-infrared fluorescent dye may form. Molded articles made from such resin compositions are prone to spot-like or streak-like surface defects. Therefore, it is desirable that the near-infrared fluorescent dye be uniformly dispersed in the resin. As a method for uniformly dispersing additive components in resin, melt kneading is widely used because it is suitable for actual production. However, even when melt kneading is performed at a temperature below the decomposition point of the dye, depending on the type of resin and dye and the kneading conditions, dispersion failure may occur or the dye may decompose, resulting in no fluorescence emission. For this reason, unless the near-infrared fluorescent dye has sufficient heat resistance and durability, it is difficult to uniformly disperse it in the resin by melt kneading. For example, Patent Document 1 discloses that by mixing and dispersing a BODIPY dye or DPP-based boron complex, which has excellent heat resistance and emission quantum yield and emits near-infrared fluorescence, in a resin, a near-infrared fluorescent resin composition with strong emission intensity and a molded article made by processing the composition can be obtained. On the other hand, products made from molded resin compositions containing near-infrared fluorescent dyes, like molded products containing other fluorescent dyes, must meet a predetermined fluorescence intensity value set in the product design for quality control purposes. Here, fluorescence intensity is easily affected by the measuring device and measurement conditions; therefore, a standard sample is generally established, and the fluorescence intensity of the sample is evaluated as a relative value to the fluorescence intensity of the standard sample. For fluorescence intensity measurements including the visible light region, rhodamine-based fluorescent dyes such as rhodamine B (CAS No.: 81-88-9) are often used as standard samples. Rhodamine-based fluorescent dyes have low heat resistance and cannot be melt-mixed into resins; in addition, their fluorescence intensity may decrease over time (Patent Document 2). Therefore, a solution diluted to an appropriate concentration at the time of measurement is usually used as the standard sample. International Publication No. 2015/056779Japanese Patent Publication No. 2010-223782 The method for measuring relative fluorescence intensity according to the present invention is a method for measuring the relative fluorescence intensity of a test sample that emits near-infrared fluorescence, characterized in that a plate-shaped or film-shaped molded body of a resin composition obtained by melt-kneading a raw material mixture containing a near-infrared fluorescent dye and an amorphous resin is used as a standard plate during fluorescence measurement. In this standard plate, since the near-infrared fluorescent dye is present in the molded body of the resin composition, it is more stable than in a solution state, and the fluorescence intensity of the standard plate is stable over time. For this reason, the same standard plate can be used in common for measurements with long intervals between measurements, eliminating the need for preparation each time and suppressing variability between measurements. In the method for measuring relative fluorescence intensity according to the present invention, the relative value of the fluorescence intensity of the test sample at a wavelength substantially the same as λ1 in t