Fluorescent probe, fluorescence detection method, and method for using fluorescent probe
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A fluorescent probe and fluorescence detection technology, applied in the field of fluorescent probes, can solve problems such as difficult to confirm fluorescently labeled cells, unable to detect fluorescence, etc.
Inactive Publication Date: 2018-12-21
NAGOYA UNIVERSITY +1
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In the case of near-infrared fluorescent probes for living body observation, it is currently impossible to detect fluorescence with in vitro fluorescence imaging devices such as fluorescence microscopes and flow cytometers, and it is difficult to confirm fluorescent labels that bind to near-infrared fluorescent probes in vitro cell
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Embodiment 1
[0116] (production of fluorescent probes)
[0117]
[0118] (1) APTES (462 μmol) was added to a DMF solution (3.8 mM, 32 mL) of TCPP obtained by dissolving TCPP in DMF, followed by N,N′-dipropylcarbodiimide (480 μmol) and N- Hydroxysuccinimide (480 μmol), stirred at 50° C. for 24 hours.
[0119] (2) 200 µL of the above-obtained porphyrin-silane DMF solution (0.75 µmol of porphyrin-silane) was mixed with 5 µL (0.027 mmol) of MPTMS. 500 μL of DMF and 300 μL of ammonia water (concentration: 28% by mass, pH 11) were added to the obtained mixed solution, and the reaction was carried out at 80° C. for 24 hours.
[0120] (3) After 24 hours, the product was recovered as a precipitate by centrifugation (15000 rpm, 20 minutes), washed with water and ethanol several times, and finally dispersed in 1 mL of water.
[0121] (4) The obtained substance is a compound of polysiloxane and porphyrin with polysiloxane as the carrier molecule, porphyrin as the fluorescent pigment a, covalently ...
Embodiment 2
[0127] (production of fluorescent probes)
[0128]
[0129] PPS HNPs were obtained in the same manner as in Example 1.
[0130]
[0131] (1) Add 251 μL of FA-PEG-Mal aqueous solution (concentration: 2 mg / mL, FA-PEG-Mal: 1.48×10 -4 mmol) and 63 μL of an aqueous solution of ICG-Mal (concentration: 2 mg / mL, ICG-Mal: 1.48×10 -4 mmol), stirred at 30°C for 3 hours.
[0132] (2) After the reaction, the product was recovered as a precipitate by centrifugation (15000 rpm, 20 minutes), washed with water several times, and finally dispersed in 1 mL of water.
[0133] (3) The obtained material is polysiloxane as the carrier molecule, porphyrin as the fluorescent pigment a, indocyanine green as the fluorescent pigment b, and folic acid as the cell surface binding substance. A complex (fluorescence probe) in which porphyrin, indocyanine green, and folic acid were bonded was measured by a dynamic light scattering method ("DelsaMax PRO" manufactured by Beckman Coulter), and it was a fl...
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Abstract
The present invention relates to a fluorescent probe which includes: a carrier molecule; and fluorescent dye a and fluorescent dye b which are bonded to the carrier molecule. Fluorescent dye a and fluorescent dye b have different excitation wavelengths. Fluorescence resonance energy transfer (FRET) does not occur between fluorescent dye a and fluorescent dye b. The present invention also relates to a fluorescence detection method which includes: a step in which target cells are labelled with the fluorescent probe; and a step in which the target cells labelled with the fluorescent probe are irradiated with excitation light, and fluorescence from the fluorescent probe is observed. Furthermore, the present invention relates to a method for using the fluorescent probe, said method including: astep in which cells are fluorescence labelled using the fluorescent probe; a step in which the fluorescence-labelled cells are screened using a flow cytometer or a fluorescence microscope; a step inwhich the screened fluorescence-labelled cells are transplanted into a living organism; and a step in which the fluorescence-labelled cells in the living organism are observed using an in vivo fluorescence imaging device.
Description
technical field [0001] The invention relates to a fluorescent probe containing two fluorescent pigments, a fluorescent detection method and a use method of the fluorescent probe. Background technique [0002] In recent years, in vivo fluorescence imaging analysis using animals such as mice has been widely performed in the field of cancer and genesis research. Fluorescence imaging analysis is the main method for analyzing information on where cells such as stem cells such as iPS cells and ES cells transplanted into mice, cancer cells, and disease-related cells such as liver cirrhosis cells undergo differentiation, growth, and metastasis after transplantation. method. In particular, in order to observe the progress of transplanted cells while keeping animals such as mice alive, there is currently only a method using fluorescence imaging analysis, which is positioned as a particularly important analysis method. [0003] Fluorescent imaging analysis of transplanted cells in vi...
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