WO2022222237A1 - 卤代荧光素螺环内硫酯探针及其制备方法与在金属离子检测中的应用 - Google Patents

卤代荧光素螺环内硫酯探针及其制备方法与在金属离子检测中的应用 Download PDF

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WO2022222237A1
WO2022222237A1 PCT/CN2021/098271 CN2021098271W WO2022222237A1 WO 2022222237 A1 WO2022222237 A1 WO 2022222237A1 CN 2021098271 W CN2021098271 W CN 2021098271W WO 2022222237 A1 WO2022222237 A1 WO 2022222237A1
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probe
solution
fluorescence
detection
conversion
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PCT/CN2021/098271
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English (en)
French (fr)
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王筱梅
夏方媛
叶常青
陈硕然
梁作芹
李琳
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苏州科技大学
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Priority claimed from CN202110427141.1A external-priority patent/CN113698419B/zh
Priority claimed from CN202110427119.7A external-priority patent/CN113702342B/zh
Priority claimed from CN202110427136.0A external-priority patent/CN113354662B/zh
Application filed by 苏州科技大学 filed Critical 苏州科技大学
Publication of WO2022222237A1 publication Critical patent/WO2022222237A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

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  • the invention belongs to the technical field of up-conversion luminescence and heavy metal ion detection, and in particular relates to a halogenated fluorescein spiro thioester probe, a preparation method thereof, and an application in metal ion detection.
  • mercury ion is considered to be one of the most toxic metal ions.
  • Mercury ions can also cause a variety of human health problems at very low concentrations, including vision loss, severe cognitive and motor impairment, prenatal brain damage, and damage to the human heart, kidneys, stomach, etc., and even lead to mammalian death. Therefore, the development of efficient and sensitive compounds of copper and mercury ions has been extensively studied. At present, there are many methods for the determination of mercury ions, including: spectrophotometry, fluorescence analysis, electrochemical methods and atomic absorption spectrometry. Among them, fluorescence analysis has the advantages of high sensitivity (such as single-molecule detection) and selectivity, low cost, high cost performance, simple operation and wide application range.
  • the purpose of the present invention is to provide a halogenated fluorescein spiro thioester probe and its preparation method and application in metal ion detection, specifically a method for detecting mercury ions in a solution by using fluorescence conversion; the probe molecule used Mercury ions can be detected by rapidly enhancing the response of up/down-conversion fluorescence, with little damage to cells, and has potential application value in living cells.
  • the technical scheme adopted in the present invention is: a halogenated fluorescein spiro thioester probe, which has the following chemical structural formula: .
  • the invention discloses a preparation method of the above halogenated fluorescein spirolactone probe, which uses tetrahalogenated fluorescein, phosphorus oxychloride and sodium sulfide as raw materials to react to prepare the halogenated fluorescein spirolactone probe ;
  • the tetrahalofluorescein is tetrabromofluorescein or tetraiodofluorescein.
  • the invention discloses the application of the above halogenated fluorescein spirolactone probe in biological imaging; or the invention discloses the application of the above halogenated fluorescein spirolactone probe in the preparation of biological imaging reagents.
  • the biological imaging is cellular imaging.
  • the invention discloses a mercury ion detection test paper, which is composed of the test paper and the above halogenated fluorescein spirolactone probe; the preparation method is as follows: soaking the test paper with the halogenated fluorescein spirolactone probe The solution was dried to obtain mercury ion detection test paper.
  • the invention discloses a method for detecting mercury ions in a solution by utilizing fluorescence conversion, comprising the following steps: (1) using tetrahalogenated fluorescein, phosphorus oxychloride and sodium sulfide as raw materials, and reacting to prepare a probe.
  • step (2), step (3), and step (4) are parallel schemes, and the details are as follows.
  • a method for detecting mercury ions in a solution using fluorescence conversion comprising the following steps.
  • a method for detecting mercury ions in a solution using fluorescence conversion comprising the following steps.
  • a method for detecting mercury ions in a solution using fluorescence conversion comprising the following steps: (1) using tetrahalogenated fluorescein, phosphorus oxychloride and sodium sulfide as raw materials, and reacting to prepare a probe.
  • the tetrahalofluorescein is first reacted with phosphorus oxychloride to obtain the tetrahalofluorescein acid chloride; and then the tetrahalofluorescein acid chloride is reacted with sodium sulfide to obtain the probe.
  • the dosage ratio of tetrahalofluorescein, phosphorus oxychloride and sodium sulfide is 1 mmol: (0.8 ⁇ 1.2 mL): 10 mmol.
  • the reaction between tetrahalofluorescein and phosphorus oxychloride is a reflux reaction for 6-10 hours; the reaction between tetrahalofluorescein acid chloride and sodium sulfide is a reflux reaction under nitrogen for 20-30 hours.
  • the solvent used for the reaction of tetrahalofluorescein and phosphorus oxychloride is dichloroethane, the reaction is completed by rotary evaporation, and the obtained intermediate product does not need to be purified, and is directly reacted with sodium sulfide in a nitrogen atmosphere for reflux reaction. The solvent was removed by evaporation, and the probe was obtained by column chromatography and vacuum drying.
  • the luminescent agent is anthracene or an anthracene derivative, such as DPA.
  • the present invention uses anti-Stokes emission, that is, an up-conversion detection method of "long wavelength excitation, short wavelength emission". Because the long-wavelength light is used as the excitation light source, the penetration of the excitation light source in the medium can be deepened, and the detection depth is wide; at the same time, due to the low excitation energy required, it can effectively eliminate the background fluorescence of the organism and improve the detection resolution. In addition, because of the low excitation energy required and the low detection lethality to biological living cells, it has potential application value in biological imaging and cell environment detection. Therefore, compared with Stokes emission fluorescence (ie down-conversion fluorescence) detection technology, up-conversion detection technology has more attractive application value.
  • Stokes emission fluorescence ie down-conversion fluorescence
  • the mercury ions in the solution to be tested can be detected by fluorescence spectroscopy; the mercury ions in the solution to be tested can also be detected by visual observation with detection test paper.
  • the fluorescence spectrum is a down-conversion fluorescence spectrum or an up-conversion fluorescence spectrum.
  • the concentration of the probe in the solution of the probe is 1-50 ⁇ M.
  • the fluorescence spectrum is an up-conversion fluorescence spectrum
  • the up-conversion is OPA-UC up-conversion or TTA-UC up-conversion.
  • the solution of the probe is mixed with the solution to be tested, and OPA-UC is used for detection.
  • the concentration of the probe in the solution of the probe is 50-150 ⁇ M; the solution of the probe is mixed with the luminescence In the solution of the probe, the concentration of the probe is 1-20 ⁇ M, and the concentration of the luminescent agent is 0.1-10 mM, preferably 0.5-5 mM. .
  • the excitation light wavelength is 500 nm; when the fluorescence spectrum is up-conversion fluorescence spectrum, the excitation light wavelength is 655 nm.
  • the concentration of the probe in the solution of the probe is 0.1 ⁇ 10 -4 to 10 ⁇ 10 -4 M.
  • the color change is that when the color of the test paper changes from white to rose red or red under the naked eye, the solution to be tested contains mercury ions, and vice versa, it does not contain mercury ions.
  • the detection system of the probe is a neutral medium of water/DMSO (1/2, v/v), which has strong practicability; it is the first time to use the same compound to achieve three Channel fluorescence detection (Stokes fluorescence, OPA-UC fluorescence and TTA-UC fluorescence), the detection limit and range of traditional Stokes fluorescence for mercury ions are 8.617 ⁇ 10 -9 M and 0 ⁇ 10 ⁇ M, respectively, while TTA-UC
  • the fluorescence detection limit is at the nM level (1.48 ⁇ 10 -9 M), while the fluorescence detection range of OPA-UC is at the mM level (0 ⁇ 0.5 mM).
  • the detection of ions can achieve the requirements of high sensitivity (nM level) and wide detection range (mM level) for a single compound molecule at the same time, solving the problem of high sensitivity and wide detection range using conventional fluorescence detection methods.
  • the probe test paper can be used for naked-eye detection of high concentrations of mercury ions, and the probe test paper can quickly change color when encountering mercury, which is convenient and fast;
  • the instruments used in OPA-UC detection are small semiconductor lasers and optical fibers
  • the spectrometer does not require conventional large-scale fluorescence spectrometers, therefore, OPA-UC detection appears to be more economical and portable. Therefore, the up-conversion detection technology has more practical application value.
  • Figure 1 shows the hydrogen NMR spectrum of tetrahalofluorescein spirolactone (deuterated DMSO).
  • Figure 2 is a mass spectrum of tetrahalofluorescein spirolactone.
  • Figure 3 shows the absorption spectra (a), down-conversion fluorescence spectra (b) and up-conversion fluorescence spectra (c) of probes 1 and 2 (probe concentration: (a, b) 10 ⁇ M, (c) 100 ⁇ M, solvent: DMSO).
  • Figure 4 shows the changes of the fluorescence spectrum (a) and the maximum fluorescence peak intensity (b) of probe 1 at different pH values (wherein, the probe concentration is 10 ⁇ M, water/DMSO: 1/2, v/v, excitation Wavelength: 500 nm).
  • Figure 5 shows the changes of the fluorescence spectrum (a) and the maximum fluorescence peak intensity (b) of probe 2 at different pH values (wherein, the probe concentration is 10 ⁇ M, water/DMSO: 1/2, v/v, excitation Wavelength: 500 nm).
  • Figure 6 shows the down-conversion fluorescence enhancement spectrum (a) and the corresponding fluorescence response change histogram of probe 1 after adding 16 kinds of metal cations (b, ordinate F/F 0 is the probe at 560 nm before and after adding Hg 2+ fluorescence intensity) (wherein, the cation concentration is 100 ⁇ M and the probe concentration is 10 ⁇ M).
  • Figure 7 shows the down-conversion fluorescence enhancement spectrum (a) and the corresponding fluorescence response change histogram of probe 2 after adding 16 kinds of metal cations (b, ordinate F/F 0 is the probe at 577 nm before and after adding Hg 2+ fluorescence intensity) (wherein, the cation concentration is 100 ⁇ M and the probe concentration is 10 ⁇ M).
  • Figure 8 shows the upconversion fluorescence enhancement spectrum (a) and the corresponding upconversion enhancement response histogram of probe 1 after adding 16 kinds of metal cations (b, the ordinate UCPL/UCPL 0 is the probe before and after adding Hg 2+ at 576 nm up-converted fluorescence intensity) (wherein, the cation concentration is 300 ⁇ M and the probe concentration is 300 ⁇ M).
  • Figure 9 shows the upconversion fluorescence enhancement spectrum (a) and the corresponding upconversion enhancement response histogram of probe 2 after adding 16 kinds of metal cations (b, ordinate UCPL/UCPL 0 is the probe before and after adding Hg 2+ at 592 nm up-converted fluorescence intensity) (wherein the cation concentration is 100 ⁇ M and the probe concentration is 100 ⁇ M).
  • Figure 10 shows the down-conversion fluorescence spectrum (a) and the corresponding working curve (b) of probe 1 with the addition of different concentrations of mercury ions ( 0 ⁇ 10 ⁇ M) nm fluorescence intensity) (wherein, the probe concentration is 10 ⁇ M, and the excitation wavelength is 500 nm).
  • Figure 11 shows the down-conversion fluorescence spectrum (a) and the corresponding working curve (b) of probe 2 with the addition of different concentrations of mercury ions ( 0-10 ⁇ M). nm fluorescence intensity) (wherein, the probe concentration is 10 ⁇ M, and the excitation wavelength is 500 nm).
  • Figure 12 shows the up-conversion fluorescence spectrum (a) and the corresponding working curve (b) of probe 1 with the addition of different concentrations of mercury ions ( 0-100 ⁇ M). nm fluorescence intensity) (wherein, the probe concentration is 300 ⁇ M and the excitation wavelength is 655 nm).
  • Figure 13 shows the up-conversion fluorescence spectrum (a) and the corresponding working curve (b) of probe 2 with the addition of different concentrations of mercury ions (0-60 ⁇ M), the ordinate UCPL/ UCPL 0 is the probe at 588 nm fluorescence intensity) (wherein, the probe concentration is 100 ⁇ M, and the excitation wavelength is 655 nm).
  • Figure 19 shows the color change of Hg 2+ detected by probe 2 test strip (Hg 2+ concentration from left to right: 0, 0.1, 0.5, 1, 10, unit: mM).
  • FIG. 20 is a graph showing the down-conversion fluorescence enhancement response of Luc-1 to metal ions.
  • FIG. 21 is a graph showing the down-conversion fluorescence enhancement response of Luc-4 to metal ions.
  • FIG. 22 is a graph showing the down-conversion fluorescence enhancement response of Luc-5 to metal ions.
  • Figure 23 shows the TTA-UC fluorescence selectivity of Luc-7 for cations (Luc-7/DPA: 10 ⁇ M/1 mM, ion: 10 ⁇ M).
  • Figure 24 shows the TTA-UC fluorescence spectrum of Luc-7 after adding different concentrations of mercury ions and its working curve (Luc-7/DPA: 10 ⁇ M/1 mM, DMSO).
  • the tetraiodofluorescein spirolactone type fluorescent probe molecule disclosed in the invention has the characteristic of "up/down conversion fluorescence" fast enhanced response to mercury ions, and has application value for mercury ion detection in water environment or organisms.
  • the starting materials are conventional commercial products, unless otherwise stated, neither starting materials nor intermediates are used without any intermediate treatment.
  • Down-conversion test Tested with Edinburgh Fluorescence Spectrometer, excitation wavelength 500 nm.
  • a 655 nm semiconductor laser was selected as the excitation light source, and a fiber optic spectrometer was used as the signal receiving and processing equipment. All kinds of instruments and meters used in the synthesis and testing process are conventional products, and the mercury dichloride aqueous solution is used as the mercury ion solution.
  • Fig. 3(a–c) Absorption spectra, fluorescence spectra (that is, down-conversion fluorescence spectra) and up- The converted fluorescence spectra are shown in Fig. 3(a–c). It can be seen from the absorption spectrum (Fig. 3a) that the absorption peak of the probe molecule is located at ⁇ 358 nm, and a very weak absorption peak appears at 545 nm; this makes the powder solid of the probe molecule appear pale pink, while in DMSO solution The medium is a colorless solution.
  • the down-converted fluorescence spectrum of the probe solution (10 ⁇ M) was measured under light excitation at 500 nm wavelength, see Fig. 3b. It can be seen that the fluorescence peak of the probe is at 563-569 nm. It is worth noting that by comparing Figure 4, it can be clearly seen that the intensity of the fluorescence peak at 563-569 nm is very weak.
  • the upconversion fluorescence spectrum of the probe solution (100 ⁇ M) was measured under the light excitation of 655 nm wavelength, as shown in Fig. 3c. It can be seen that the upconversion fluorescence intensity of the probe molecule is very weak.
  • Preparation of an aqueous solution with a pH of 1 ⁇ 14 Prepare 0.1 M NaOH aqueous solution and 0.1 M HCl aqueous solution respectively, adjust the pH value by a pH meter, and prepare an aqueous solution with a pH of 1 ⁇ 14 in turn.
  • Preparation of the down-conversion fluorescence detection solution take 3 mL of buffer/DMSO (1/2, v/v) in a quartz cuvette, then take out 3 ⁇ L of the above mother solution and add it to the quartz cuvette, dissolve by ultrasonic, and prepare 10. ⁇ M down-conversion probe detection solution.
  • Upconversion fluorescence detection solution take 3 mL of buffer/DMSO (1/3, v/v) in a quartz cuvette, then take out 90 ⁇ L of the above mother solution and add it to a quartz cuvette, dissolve by ultrasonic, and prepare 300 ⁇ M Upconversion Probe Detection Solution (Probe 1).
  • the down-conversion fluorescence enhanced response of the probe to Hg 2+ In 17 cuvettes containing the down-conversion probe detection solution (10 ⁇ M), 16 kinds of metal cation aqueous solutions (concentration of 100 ⁇ M) were added, respectively. Yes: Hg 2+ , Cu 2+ , Mn 2+ , NH 4 + , Zn 2+ , Mg 2+ , Cd 2+ , Pb 2+ , Li + , Na + , K + , Ca 2+ , Ni 2+ , Co 2+ , Fe 2+ and Fe 3+ . Then, the down-conversion fluorescence spectrum (excitation wavelength of 500 nm) was measured, as shown in Fig. 6 (probe 1) and Fig.
  • the upconversion fluorescence response of the probe to Hg 2+ In 17 cuvettes containing the upconversion probe detection solution (300 ⁇ M probe 1), 16 kinds of metal cation aqueous solutions (100 ⁇ M) were added respectively, which were : Hg 2+ , Cu 2+ , Mn 2+ , NH 4 + , Zn 2+ , Mg 2+ , Cd 2+ , Pb 2+ , Li + , Na + , K + , Ca 2+ , Ni 2+ , Co 2+ , Fe 2+ and Fe 3+ .
  • the upconversion fluorescence spectrum (excitation wavelength: 655 nm) was then measured.
  • the down-conversion fluorescence response of the probe to the concentration of Hg 2+ Add different concentrations of Hg 2+ to the down-conversion probe detection solution (10 ⁇ M), and observe the change of the down-conversion fluorescence spectrum of the probe (excitation wavelength 500 nm), see Figure 10 (probe 1) and Figure 11 (probe 2). It can be seen that before the addition of mercury ions, the fluorescence of the probe is very weak; when 3 ⁇ L ⁇ 30 ⁇ L of Hg 2+ aqueous solution (1 mM) is added to the above probe solution (Hg 2+ concentration is equivalent to 1 ⁇ 10 ⁇ M), the , the fluorescence intensity of the probe increased continuously.
  • the detection limit of Hg 2+ by fluorescence spectrum can be calculated as 8.617 ⁇ 10 -9 M (probe 2) and 1.45 ⁇ 10 -8 M (probe 1).
  • the upconversion fluorescence response of the probe to the concentration of Hg 2+ add Hg 2+ of different concentrations to the solution of the upconversion probe detection solution (300 ⁇ M probe 1), and observe the change of the upconversion fluorescence spectrum of the probe (excitation wavelength 655 nm), see Figure 12 (probe 1). It can be seen that before the addition of mercury ions, the up-conversion fluorescence of the probe is very weak, and 6 ⁇ L ⁇ 24 ⁇ L of Hg 2+ aqueous solution (5 mM) was added to the above probe solution (the Hg 2+ concentration is equivalent to 5 ⁇ 45 ⁇ M, The fluorescence intensity of the probe increased continuously.
  • the detection limit of Hg 2+ by upconversion fluorescence spectrum can be calculated to be 1.393 ⁇ 10 -5 M.
  • the down-conversion fluorescence response time of the probe to Hg 2+ It can be seen from Figure 14 (probe 1) and Figure 15 (probe 2) that the fluorescence of the down-conversion probe detection solution (10 ⁇ M) is very weak.
  • Hg 2+ aqueous solution (10 mM) was added to the above probe solution (Hg 2+ concentration was equivalent to 100 ⁇ M)
  • the fluorescence intensity of the probe increased sharply, and the fluorescence peak was located at 560 nm, and the change was almost instantaneous (10 within seconds).
  • the up-conversion fluorescence response time of the probe to Hg 2+ Similar to the down-conversion fluorescence, the probe’s response to Hg 2+ is almost instantaneous (within 10 seconds), see Figure 16 (probe 1), Figure 17 (Probe 2).
  • Example Two-probe test paper detects Hg 2+ .
  • Hg 2+ detection test paper was prepared. The specific operation is as follows: soak the 2cm ⁇ 1cm test paper in the solution of probe 1 (the concentration of the probe is 3 ⁇ 10 -4 M, solvent: ethanol) for 30 min, and then take it out to dry naturally. Then, use a glass rod to dip Hg 2+ aqueous solutions of different concentrations and drop them on the test paper, as shown in FIG. 18 . As the concentration of Hg 2+ increases, the test paper changes from colorless (white) to red, and the color deepens in turn. Therefore, the test paper can realize rapid naked-eye detection of high concentrations of Hg 2+ in the solution.
  • Hg 2+ detection test paper was prepared. The specific operation is as follows: soak the 2cm ⁇ 1cm test paper in the solution of probe 2 (the concentration of the probe is 1 ⁇ 10 -4 M, solvent: ethanol) for 30 min, and then take it out to dry naturally. Then, use a glass rod to dip Hg 2+ aqueous solutions of different concentrations and drop them on the test paper, as shown in FIG. 19 . With the increase of Hg 2+ concentration, the test paper changed from colorless (white) to rose red, and the color deepened in turn. The lowest response concentration was 0.01 mM, and the effect was better than that of the test paper made of tetrabromofluorescein spirolactone. it is good. Therefore, the test paper can realize rapid naked-eye detection of high concentrations of Hg 2+ in the solution.
  • the reported organic upconversion (UC) are mainly realized by two-photon absorption mechanism (TPA-UC) and triplet-triplet annihilation (TTA-UC) mechanism, and upconversion with single-photon absorption mechanism (OPA-UC) is another unique luminescence mechanism, which is rarely reported.
  • the present invention uses single-photon up-conversion (OPA-UC) fluorescence detection technology.
  • the single-photon absorption upconversion (OPA-UC) mechanism is that electrons transition from the thermally activated vibrational-rotational energy level (S t ) of the ground state to the first excited state (S 1 ), and then fall back to the ground state and emit fluorescence.
  • the advantage of OPA-UC is that, in addition to the advantages of large penetration depth and small damage to the living body; the intensity of the excitation light source required for OPA up-conversion Small, the required up-conversion detection equipment is cheap and portable; the concentration of the required probe is small, and it can be detected in the air without isolating oxygen, so it is more practical.
  • the compounds In the OPA-UC (excitation wavelength 655 nm) fluorescence, the compounds (Luc-1, Luc-4, Luc-5) have no selective OPA-UC fluorescence enhancement to metal ions.
  • Example 3 Ion detection performance of tetraiodofluorescein spirolactone (Luc-7) in TTA-UC.
  • TTA-UC tetraiodofluorescein spirolactone as a sensitizer and a luminescent agent DPA (9,10-diphenylanthracene) to form a two-component in solution
  • DPA 9,10-diphenylanthracene
  • the sensitivity test of TTA-UC adopts the titration method.

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Abstract

公开了卤代荧光素螺环内硫酯探针及其制备方法与在金属离子检测中的应用，将探针的溶液与待测溶液混合，或者将所述探针的溶液与发光剂混合，然后再与待测溶液混合，然后用光照射，检测荧光光谱；还可以将试纸浸泡所述探针的溶液后，然后干燥得到检测试纸，将待测溶液滴在检测试纸上，根据颜色变化完成待测溶液中汞离子的检测。首次实现三通道荧光检测（Stokes荧光、OPA-UC荧光和TTA-UC荧光），结果表明：将上述两种弱光上转换检测方法同时运用在对汞离子的检测，可实现单一化合物分子同时达到高灵敏性(nM级别)和宽检测范围(mM级别)的要求，解决了使用常规的荧光检测方法始终存在着高灵敏性和宽检测范围之间的矛盾。

Description

卤代荧光素螺环内硫酯探针及其制备方法与在金属离子检测中的应用 技术领域
本发明属于上转换发光和重金属离子检测技术领域，具体涉及一种卤代荧光素螺环内硫酯探针及其制备方法与在金属离子检测中的应用。
 背景技术
在各种重金属中，汞离子更是被认为是毒性最强的金属离子之一。汞离子在极低浓度也会导致多种人类健康问题，包括视力丧失、严重的认知运动障碍、产前脑损伤以及对人类心脏、肾脏、胃等的损害，甚至会导致哺乳动物死亡。因此，开发高效灵敏的铜离子和汞离子化合物得到广泛研究。目前，测定汞离子的方法有很多，主要包括：分光光度法、荧光分析法、电化学法和原子吸收光谱法等。其中，荧光分析法具有很高的灵敏度（如可实现单分子检测）和选择性且成本低廉性价比高，同时其操作简易、适用范围广等优点。至今为止，被设计合成出来用于汞的检测的荧光探针已经有上百种，均是采用下转换荧光检测方法，其机理是在短波长光源的激发下，电子从基态的零振动级（S 0）跃迁到第一激发态（S 1），然后回落至基态并释放出长波长的荧光，可见，下转换荧光的光谱特征是“短波（长）激发，长波（长）发射”。
技术问题
本发明的目的是提供一种卤代荧光素螺环内硫酯探针及其制备方法与在金属离子检测中的应用，具体为利用荧光转换检测溶液中汞离子的方法；利用的探针分子可通过上/下转换荧光快速增强响应来检测汞离子，且对细胞伤害性小，具有潜在的活体细胞应用价值。
 技术解决方案
为达上述发明目的，本发明采用的技术方案是：一种卤代荧光素螺环内硫酯探针，具有如下化学结构式： 
Figure 68173dest_path_image001
。
本发明公开了上述卤代荧光素螺环内硫酯探针的制备方法，以四卤代荧光素、三氯氧磷、硫化钠为原料，反应制备卤代荧光素螺环内硫酯探针；所述四卤代荧光素为四溴代荧光素或者四碘代荧光素。
本发明公开了上述卤代荧光素螺环内硫酯探针在生物成像中的应用；或者本发明公开了上述卤代荧光素螺环内硫酯探针在制备生物成像试剂中的应用。优选的，生物成像为细胞成像。
本发明公开了一种汞离子检测试纸，由试纸与上述卤代荧光素螺环内硫酯探针组成；其制备方法为，将试纸浸泡所述卤碘代荧光素螺环内硫酯探针的溶液后，干燥得到汞离子检测试纸。
本发明公开了一种利用荧光转换检测溶液中汞离子的方法，包括以下步骤：（1）以四卤代荧光素、三氯氧磷、硫化钠为原料，反应制备探针。
（2）将所述探针的溶液与待测溶液混合，然后用光照射，检测荧光光谱；根据荧光光谱完成待测溶液中汞离子的检测。
或者。
（3）将所述探针的溶液与发光剂混合，然后再与待测溶液混合，然后用光照射，检测荧光光谱；根据荧光光谱完成待测溶液中汞离子的检测。
或者。
（4）将试纸浸泡所述探针的溶液后，然后干燥得到检测试纸；将待测溶液滴在所述检测试纸上，根据颜色变化完成待测溶液中汞离子的检测。
上述步骤（2）、步骤（3）、步骤（4）为并列方案，具体如下。
一种利用荧光转换检测溶液中汞离子的方法，包括以下步骤。
（1）以四卤代荧光素、三氯氧磷、硫化钠为原料，反应制备探针。
（2）将所述探针的溶液与待测溶液混合，然后用光照射，检测荧光光谱；根据荧光光谱完成待测溶液中汞离子的检测。
一种利用荧光转换检测溶液中汞离子的方法，包括以下步骤。
（1）以四卤代荧光素、三氯氧磷、硫化钠为原料，反应制备探针。
（2）将所述探针的溶液与发光剂混合，然后再与待测溶液混合，然后用光照射，检测荧光光谱；根据荧光光谱完成待测溶液中汞离子的检测。
一种利用荧光转换检测溶液中汞离子的方法，包括以下步骤：（1）以四卤代荧光素、三氯氧磷、硫化钠为原料，反应制备探针。
（2）将试纸浸泡所述探针的溶液后，干燥得到检测试纸；将待测溶液滴在所述检测试纸上，根据颜色变化完成待测溶液中汞离子的检测。
上述技术方案中，先将四卤代荧光素与三氯氧磷反应，得到四卤代荧光素酰氯；再将四卤代荧光素酰氯与硫化钠反应，得到探针。优选的，四卤代荧光素、三氯氧磷、硫化钠的用量比例为1 mmol∶（0.8～1.2 mL）∶1 0mmol。优选的，四卤代荧光素与三氯氧磷的反应为回流反应6～10小时；四卤代荧光素酰氯与硫化钠的反应为氮气下回流反应20～30小时。 优选的，四卤代荧光素与三氯氧磷反应的溶剂为二氯乙烷，反应结束旋蒸，得到的中间产物无需提纯，直接与硫化钠在氮气氛中回流反应，反应结束后，旋转蒸发除去溶剂，通过柱层析和真空干燥，得到探针。
本发明中，发光剂为蒽或者蒽衍生物，比如DPA。
本发明使用的是反斯托克斯发射，即“长波长激发，短波长发射”的上转换检测方法。由于以长波长光为激发的光源，可加深激发光源在介质中的穿透力，检测深度广；同时，因其所需激发能量较低，可有效地消除生物体背景荧光从而提高检测分辨率；另外，又因其所需激发能量较低，对生物活体细胞的检测杀伤力小，使其在生物成像和细胞环境检测中具有潜在应用价值。因而，相对于斯托克斯发射的荧光（即下转换荧光）检测技术，上转换检测技术则具有更诱人的应用价值。
本发明中，可以采用荧光光谱检测待测溶液中的汞离子；也可以采用检测试纸肉眼观察，检测待测溶液中的汞离子。
上述技术方案中，采用荧光光谱检测待测溶液中的汞离子时，荧光光谱为下转换荧光光谱或者上转换荧光光谱。优选的，荧光光谱为下转换荧光光谱时，探针的溶液中，探针的浓度为1～50 μM。荧光光谱为上转换荧光光谱时，上转换为OPA-UC上转换或者TTA-UC上转换。具体的，本发明将所述探针的溶液与待测溶液混合，采用OPA-UC检测，此时探针的溶液中，探针的浓度为50～150μM；将所述探针的溶液与发光剂混合，然后再与待测溶液混合，采用TTA-UC上转换，此时探针的溶液中，探针的浓度为1～20μM，发光剂的浓度为0.1～10 mM，优选0.5～5 mM。进一步的，荧光光谱为下转换荧光光谱时，激发光波长为500 nm；荧光光谱为上转换荧光光谱时，激发光波长为655 nm。
上述技术方案中，采用检测试纸检测待测溶液中的汞离子时，探针的溶液中，探针的浓度为0.1×10 -4～10×10 -4 M。颜色变化为，肉眼观察下，检测试纸的颜色由白色变为玫瑰红色或者红色时，待测溶液中含有汞离子，反之，不含汞离子。
有益效果
本发明的技术方案具有如下技术效果和优点：本发明的方法中，探针的检测体系为水/DMSO（1/2，v/v）中性介质，实用性强；首次运用同一化合物实现三通道荧光检测（Stokes荧光、OPA-UC荧光和TTA-UC荧光），对汞离子的传统的的Stokes荧光的检测限和量程分别为8.617×10 -9 M和0~10 μM，而TTA-UC荧光检测限在nM级别（1.48×10 -9 M），而OPA-UC荧光检测量程在mM级别（0~0.5 mM），结果表明：将上述两种弱光上转换检测方法同时运用在对汞离子的检测，可实现单一化合物分子同时达到高灵敏性（nM级别）和宽检测范围（mM级别）的要求，解决了使用常规的荧光检测方法始终存在着高灵敏性和宽检测范围之间的矛盾，具有鲜明的特色和创新性；探针试纸可用于高浓度的汞离子的裸眼检测，且探针试纸遇汞均可快速变色，方便快捷；OPA-UC检测所用仪器为小型半导体激光器和光纤光谱仪，无需常规的大型荧光光谱仪器，因此，OPA-UC检测显示出更加经济和便携。从而使得上转换检测技术更具实际应用价值。
附图说明
图1 为四卤代荧光素螺环内硫酯的核磁氢谱（氘代DMSO）。
图2为四卤代荧光素螺环内硫酯的质谱图。
图3为探针1和2吸收光谱（a）、下转换荧光光谱（b）和上转换荧光光谱（c）（探针浓度：（a,b）10 μM，（c）100 μM，溶剂：DMSO)。
图4为在不同pH值下，探针1荧光光谱（a）和最大荧光峰位强度的变化（b）（其中，探针浓度10 μM，水/DMSO：1/2，v/v，激发波长：500 nm）。
图5为在不同pH值下，探针2荧光光谱（a）和最大荧光峰位强度的变化（b）（其中，探针浓度10 μM，水/DMSO：1/2，v/v，激发波长：500 nm）。
图6为加入16种金属阳离子后，探针1的下转换荧光增强光谱（a）和相应的荧光响应变化柱状图（b，纵坐标F/F 0为加入Hg 2+前后探针在560 nm荧光强度）（其中，阳离子浓度100 μM，探针浓度10 μM）。
图7为加入16种金属阳离子后，探针2的下转换荧光增强光谱（a）和相应的荧光响应变化柱状图（b，纵坐标F/F 0为加入Hg 2+前后探针在577 nm荧光强度）（其中，阳离子浓度100 μM，探针浓度10 μM）。
图8为加入16种金属阳离子后，探针1的上转换荧光增强光谱（a）和相应的上转换增强响应柱状图（b，纵坐标UCPL/UCPL 0为加入Hg 2+前后探针在576 nm上转换荧光强度）（其中，阳离子浓度300 μM，探针浓度300 μM）。
图9为加入16种金属阳离子后，探针2的上转换荧光增强光谱（a）和相应的上转换增强响应柱状图（b，纵坐标UCPL/UCPL 0为加入Hg 2+前后探针在592 nm上转换荧光强度）（其中，阳离子浓度100 μM，探针浓度100 μM）。
图10为加入不同浓度的汞离子（0~10 μM），探针1下转换荧光光谱（a）与相应的工作曲线（b，纵坐标F/F 0为加入Hg 2+前后探针在561 nm荧光强度）（其中，探针浓度10 μM，激发波长500 nm）。
图11为加入不同浓度的汞离子（0~10 μM），探针2下转换荧光光谱（a）与相应的工作曲线（b，纵坐标F/F 0为加入Hg 2+前后探针在575 nm荧光强度）（其中，探针浓度10 μM，激发波长500 nm）。
图12为加入不同浓度的汞离子（0~100 μM），探针1上转换荧光光谱（a）与相应的工作曲线（b，纵坐标UCPL/UCPL 0为加入Hg 2+前后探针在572 nm荧光强度）（其中，探针浓度300 μM，激发波长655 nm）。
图13为加入不同浓度的汞离子（0~60 μM），探针2上转换荧光光谱（a）与相应的工作曲线（b，纵坐标UCPL/UCPL 0为加入Hg 2+前后探针在588 nm荧光强度）（其中，探针浓度100 μM，激发波长655 nm）。
图14为探针1加入Hg 2+后随时间变化荧光图（a）及其在562 nm处荧光强度的工作曲线（b）（其中，探针浓度为10 μM，Hg 2+浓度=100 μM，激发波长500 nm）。
图15为探针2加入Hg 2+后随时间变化荧光图（a）及其在575 nm处荧光强度的工作曲线（b）（其中，探针浓度为10 μM，Hg 2+浓度=100 μM，激发波长500 nm）。
图16为探针1加入Hg 2+后随时间变化荧光图（a）及其在576 nm处荧光强度的工作曲线（b）（其中，探针浓度为300 μM，Hg 2+浓度=300 μM，激发波长655 nm）。
图17为探针2加入Hg 2+后随时间变化荧光图（a）及其在588 nm处荧光强度的工作曲线（b）（其中，探针浓度为100 μM，Hg 2+浓度=100 μM，激发波长655 nm）。
图18为探针1加入Hg 2+的Job’s plot荧光图（a）及其工作曲线（b）（其中，[探针+Hg 2+]=20 μM，激发波长500 nm）。
图19为探针2试纸条检测Hg 2+的颜色变化（Hg 2+浓度从左到右依次为：0，0.1，0.5，1，10，单位：mM）。
图20为Luc-1对金属离子的下转换荧光增强响应图。
图21为Luc-4对金属离子的下转换荧光增强响应图。
图22为Luc-5对金属离子的下转换荧光增强响应图。
图23为 Luc-7对阳离子的的TTA-UC荧光选择性（Luc-7/DPA：10 μM/1 mM，离子：10 μM）。
图24为加入不同浓度汞离子后的Luc-7的TTA-UC荧光光谱及其工作曲线（Luc-7/DPA：10 μM/1 mM，DMSO）。
 本发明的实施方式
本发明公开的四碘代荧光素螺环内硫酯类荧光探针分子，对汞离子具有“上/下转换荧光”快速增强响应特性，对水环境或生物体中汞离子检测具有应用价值。原料为常规市售产品，除非另外说明，原料或中间体均不经任何中间处理使用。下转换测试：用爱丁堡荧光光谱仪器测试，激发波长为500 nm。上转换测试，选用655 nm半导体激光器作为激发光源，光纤光谱仪作为信号接收和处理设备。合成以及测试过程中所用到的各类仪器仪表都为常规产品，以二氯化汞水溶液为汞离子溶液。
制备例：
Figure 205893dest_path_image002
。
四碘荧光素螺环内硫酯（Luc-7，探针2）的制备。
在50 mL的三口烧瓶中加入四碘荧光素（1 mmol）、三氯氧磷（1 mL）和10 mL二氯乙烷，常规超声将其溶解；90℃回流反应8h，反应过程点板跟踪，展开剂为二氯甲烷：石油醚（1/1，v/v），停止反应，冷却至室温；减压蒸馏除去二氯乙烷和三氯氧磷，得到土黄色固体中间产物（四碘荧光素酰氯）。在不提纯的情况下，直接将上述中间产物溶于无水四氢呋喃中，加入无水硫化钠（10 mM, 0.78 g），常规超声使其溶解；在氮气氛下，70℃回流反应24h；反应过程点板跟踪，展开剂为二氯甲烷：石油醚（1/1，v/v）。停止反应，冷却至室温；加圧蒸留除去四氢呋喃得到固体混合物，通过柱层析方法提纯产物，展开剂为乙酸乙酯/二氯甲烷（8/1，v/v）。得到0.09g淡粉色的探针（产率：10 %）。 1H NMR (400 MHz, DMSO-d6)：δ 10.04 (s, 2H，Ar-OH)，7.89 (d, J = 7.6 Hz, 1H，Ar-H), 7.78 (t, J = 7.5 Hz, 1H，Ar-H), 7.70 (t, J = 7.4 Hz, 1H，Ar-H), 7.37 (d, J = 7.8 Hz, 1H，Ar-H), 7.09 (s, 2H，Ar-H)； 13C NMR (101 MHz, DMSO- d 6) δ 207.25, 170.01, 157.58, 151.71, 148.28, 137.81, 136.36, 134.84, 130.35, 128.06, 123.72, 121.68, 117.43, 99.33 (d, J = 23.7 Hz), 84.07, 81.89, 78.10。MS，计算值：[M+H +] =852.63948，测试值：[M+Na +] = 852.64029；见附图1、附图2。
四溴荧光素螺环内硫酯（Luc-6，探针1）的制备。
在50 mL的三口烧瓶中加入四溴荧光素（0.647 g，1 mmol）、三氯氧磷（1 mL）和10 mL二氯乙烷，常规超声将其溶解；90℃回流反应8h，反应过程点板跟踪，展开剂为二氯甲烷：石油醚（1/1，v/v），停止反应，冷却至室温；减压蒸馏除去二氯乙烷和三氯氧磷，得到土黄色固体中间产物（四溴荧光素酰氯）。在不提纯的情况下，直接将上述中间产物溶于无水四氢呋喃中，加入无水硫化钠（10 mM, 0.78 g），常规超声使其溶解；在氮气氛下，70℃回流反应24h；反应过程点板跟踪，展开剂为二氯甲烷：石油醚（1/1，v/v）。停止反应，冷却至室温；加圧蒸留除去四氢呋喃得到固体混合物，通过柱层析方法提纯产物，展开剂为乙酸乙酯/二氯甲烷（8/1，v/v）。得到0.1g淡粉色的产物（产率：12 %）。 1H NMR (400 MHz, DMSO- d 6)：δ 10.70 (s, 2H, Ar-OH), 7.89 (s, 1H, Ar-H), 7.78 (s, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.48 (d, 1H, Ar-H), 7.00 (s, 2H, Ar-H)。MS，计算值：[M+Na +] =686.67336，测试值：[M+Na +] = 686.67181；见附图1、附图2。
探针溶液的吸收光谱和荧光光谱。
四溴荧光素螺环内硫酯荧光探针（探针1）、四碘荧光素螺环内硫酯荧光探针（探针2）的吸收光谱、荧光光谱（即下转换荧光光谱）和上转换荧光光谱见图3（a~c）所示。由吸收光谱（附图3a）可见，探针分子的吸收峰位于~358 nm处，在545 nm处则出现非常微弱的吸收峰；这使得探针分子的粉末固体呈现淡粉红，而在DMSO溶液中则为均无色溶液。
在500 nm波长的光激发下测得探针溶液（10 μM）的下转换荧光光谱，见附图3b。可见，探针的荧光峰位在563 ~569 nm处。值得注意的是，通过对比图4，可以明显看出，在563~569 nm处的荧光峰的强度是非常弱的。
在655 nm波长的光激发下，测得探针溶液（100 μM）的上转换荧光光谱，见附图3c。可以看出，探针分子的上转换荧光强度非常之弱。
pH值对探针的荧光光谱的影响。
pH为1~14的水溶液配制：分别配制0.1 M NaOH水溶液和0.1 M HCl水溶液，通过pH计调整pH值，依次配制为pH为1~14的水溶液。
然后，分别取出2 mL pH为1~14的水溶液，加入到14个比色皿中；再分别加入0.003 mL的探针溶液（10 μM）于上述14个比色皿中，用DMSO定容至3 mL，得到pH为1~14的探针溶液。测试在不同pH值下，探针溶液的荧光光谱和最大荧光峰位强度的变化，见附图4（探针1）、附图5（探针2）。可见，在pH ≤11时，探针分子不出现荧光。由于本发明反应型的荧光探针检测为荧光增强响应；故在检测之前，探针分子不呈现荧光是一个好的优点，而且中性环境适合生物体的检测。
实施例一 汞离子检测。
取0.0426 g（0.001 mol）四碘荧光素螺环内硫酯于容量瓶中，加入5 mL DMSO，超声振荡使其溶解，配制得到浓度为10 mM的母液，避光保存。
取0.0332 g（0.001 mol）四溴荧光素螺环内硫酯于容量瓶中，加入5 mL DMSO，超声振荡使其溶解，配制得到浓度为10 mM的母液，避光保存。
下转换荧光检测溶液配制：取3 mL缓冲液/DMSO（1/2，v/v）于石英比色皿中，再取出3 μL 上述母液加入石英比色皿中，超声溶解，即配得10 μM的下转换探针检测液。
上转换荧光检测溶液配制：取3 mL缓冲液/DMSO（1/3，v/v）于石英比色皿中，再取出90 μL 上述母液加入石英比色皿中，超声溶解，即配得300 μM的上转换探针检测液（探针1）。
取3 mL缓冲液/DMSO（1/3，v/v）于石英比色皿中，再取出30 μL 上述母液加入石英比色皿中，超声溶解，即配得100 μM的上转换探针检测液（探针2）。
上述缓冲液（pH=7）的配制如下：称取1.2114 g（0.01 mol）的三羟甲基氨基甲烷（Tris）于100 mL去离子水中，溶解后得到三羟甲基氨基甲烷（Tris）水溶液（0.1 M）；再取出8.3 mL的浓盐酸（HCl，36%）稀释至1000 mL，配得HCl稀溶液（0.1 M）；然后，分别取出50 mL的Tris溶液和45.7 mL HCl稀溶液，用pH计微调至pH=7，定容至100 mL，即配得0.05 M的三羟甲基氨基甲烷/盐酸缓冲液（Tris-HCl，pH=7）。
探针对Hg 2+的下转换荧光增强响应： 在17个盛有下转换探针检测液（10 μM）的比色皿中，分别加入16种金属阳离子水溶液（浓度为100 μM），它们分别是：Hg 2+、Cu 2+、Mn 2+、NH 4 +、Zn 2+、Mg 2+、Cd 2+、Pb 2+、Li +、Na +、K +、Ca 2+、Ni 2+、Co 2+、Fe 2+和Fe 3+。然后测定下转换荧光光谱（激发波长500 nm），见附图6（探针1）、附图7（探针2）所示。可见，Hg 2+的加入使探针的荧光强度增强了120～160倍，其余金属阳离子几乎不变化。显示出探针对Hg 2+具有高度的选择性荧光响应。
探针对Hg 2+的上转换荧光响应：在17个盛有上转换探针检测液（300 μM探针1）的比色皿中，分别加入16种金属阳离子水溶液（100 μM），它们是：Hg 2+、Cu 2+、Mn 2+、NH 4 +、Zn 2+、Mg 2+、Cd 2+、Pb 2+、Li +、Na +、K +、Ca 2+、Ni 2+、Co 2+、Fe 2+和Fe 3+。然后测定上转换荧光光谱（激发波长：655 nm）。
将上转换探针检测液更换为100 μM探针2进行同样的测试。
见附图8（探针1）、附图9（探针2）所示。可见，除了Hg 2+外，加入其他15种金属阳离子后，探针溶液在短波长处并不出现上转换荧光峰形；而Hg 2+的加入使得探针溶液的上转换荧光强度增强到原来的5倍，而加入其余15种金属阳离子，探针溶液并不出现上转换荧光峰形。值得注意的是：在下转换荧光检测中起干扰作用的Cu 2+，在上转换荧光检测中不再发生干扰。这说明，探针只对Hg 2+具有高度的选择性上转换荧光增强响应。
探针对Hg 2+浓度的下转换荧光响应：在下转换探针检测液（10 μM）中加入不同浓度的Hg 2+，观察探针的下转换荧光光谱的变化（激发波长500 nm），见附图10（探针1）、附图11（探针2）所示。可见，未加入汞离子之前，探针的荧光很弱；分别加入3 μL~30 μL Hg 2+水溶液（1 mM）于上述探针溶液中（Hg 2+浓度折合为1~10 μM），时，探针荧光强度不断地增强。由附图可见，在Hg 2+浓度0~10 μM范围内，其荧光强度与Hg 2+浓度呈现出很好的线性关系，相关系数R 2=0.99001、R 2=0.99482。根据公式“检测限=3δ/k”可计算出利用荧光光谱检测Hg 2+的检测限为8.617×10 -9 M（探针2）、1.45×10 -8 M（探针1）。
探针对Hg 2+浓度的上转换荧光响应：在上转换探针检测液（300 μM探针1）溶液中加入不同浓度的Hg 2+，观察探针上转换荧光光谱的变化（激发波长655 nm），见附图12（探针1）所示。可见，未加入汞离子之前，探针的上转换荧光很弱，分别加入6 μL~24 μL Hg 2+水溶液（5 mM）于上述探针溶液中（Hg 2+浓度折合为5~45 μM，探针荧光强度不断地增强。由附图可见，在Hg 2+浓度0~50 μM范围内，其上转换荧光强度与Hg 2+浓度呈现出很好的线性关系，相关系数R 2=0.99206。根据公式“检测限=3δ/k”可计算出利用上转换荧光光谱检测Hg 2+的检测限为1.393×10 -5 M。
在上转换探针检测液（100 μM探针2）溶液中加入不同浓度的Hg 2+，观察探针上转换荧光光谱的变化（激发波长655 nm），见附图13所示。可见，未加入汞离子之前，探针的上转换荧光很弱，分别加入6μL~24 μL Hg 2+水溶液（5 mM）于上述探针溶液中（Hg 2+浓度折合为5~45 μM，探针荧光强度不断地增强。由附图可见，在Hg 2+浓度0~50 μM范围内，其上转换荧光强度与Hg 2+浓度呈现出很好的线性关系，相关系数R 2=0.99052。根据公式“检测限=3δ/k”可计算出利用上转换荧光光谱检测Hg 2+的检测限为1.01×10 -5 M。
探针对Hg 2+的下转换荧光响应时间：由附图14（探针1）、附图15（探针2）可见，下转换探针检测液（10 μM）的荧光很弱，当加入30 μL Hg 2+水溶液（10 mM）于上述探针溶液中（Hg 2+浓度折合为100 μM）时，探针的荧光强度急剧增强，荧光峰位位于560 nm，其变化几乎是瞬时（10秒以内）的。
探针对Hg 2+的上转换荧光响应时间：与下转换荧光情况类似，探针对于Hg 2+的响应也几乎是瞬时（10秒以内）完成的，见附图16（探针1）、附图17（探针2）。
实施例二 探针试纸检测Hg 2+。
制备了Hg 2+检测试纸。具体操作为：将2cm×1cm 的试纸置于探针1的溶液（探针的浓度为 3×10 -4 M，溶剂：乙醇）中浸泡30 min，取出自然晾干备用。然后，用玻璃棒蘸取不同浓度的Hg 2+水溶液滴在试纸上，如附图18所示。随着Hg 2+浓度的增加，试纸由无色（白色）变成红色，且颜色依次加深。因此，该试纸可以对溶液中高浓度的Hg 2+实现快速裸眼检测。
制备了Hg 2+检测试纸。具体操作为：将2cm×1cm 的试纸置于探针2的溶液（探针的浓度为 1×10 -4 M，溶剂：乙醇）中浸泡30 min，取出自然晾干备用。然后，用玻璃棒蘸取不同浓度的Hg 2+水溶液滴在试纸上，如附图19所示。随着Hg 2+浓度的增加，试纸由无色（白色）变成玫瑰红色，且颜色依次加深，最低响应浓度为0.01 mM，且效果较四溴代荧光素螺环内硫酯所制试纸更好。因此，该试纸可以对溶液中高浓度的Hg 2+实现快速裸眼检测。
迄今为止，报道的有机上转换发光（upconversion, UC）主要有双光子吸收机制（TPA-UC）和三线态-三线态湮灭（TTA-UC）机制来实现的，具有单光子吸收机制的上转换（OPA-UC）则是另一种独特的发光机制，鲜见报道。本发明使用的是单光子上转换（OPA-UC）荧光探测技术。单光子吸收上转换（OPA-UC）机理是电子从基态的热活化振动-转动能级（S t）跃迁到第一激发态（S 1）, 然后回落至基态并发出荧光。与上述的两种上转换（即TPA-UC和TTA-UC）相比，OPA-UC的优势在于，除了具有穿透深度大和对活体伤害小等优势外；OPA上转换所需的激发光源强度较小，所需上转换检测设备价格低廉便携；所需探针的浓度较小，无需隔绝氧气，空气中即可检测，因而实用性更强。
对比例。
称取2.5 mmol的荧光素、3.5mmol水合肼加入100 mL三口烧瓶中，加入30 mL乙醇加热回流24小时后，旋蒸除去溶剂，加入去离子水进行重沉淀得到粗产物，分别通过水/乙醇加热挥发结晶法得到最终产物化合物Luc-1：MS，计算值：347.102633，测量值：347.102992； 1H NMR (400 MHz, DMSO- d 6) δ 10.05 – 9.49 (m, 2H), 7.86 – 7.73 (m, 1H), 7.49 (dd, J = 6.3, 2.9 Hz, 2H), 7.07 – 6.93 (m, 1H), 6.60 (d, J = 2.3 Hz, 2H), 6.53 – 6.32 (m, 4H), 4.40 (s, 2H)。
称取2.5 mmol四溴荧光素、3.5mmol水合肼加入100 mL三口烧瓶中，加入30 mL乙醇加热回流24小时后，旋蒸除去溶剂，加入去离子水进行重沉淀得到粗产物，分别通过水/乙醇加热挥发结晶法得到最终产物化合物Luc-4：MS，计算值：593.88，测量值：685.43075（593+4Na +）； 1H NMR (400 MHz, DMSO- d 6) δ 10.52 (s, 2H), 7.85 (td, J = 6.3, 3.0 Hz, 1H), 7.58 (ddt, J = 11.0, 8.1, 4.9 Hz, 2H), 7.23 (dd, J = 5.9, 2.7 Hz, 1H), 6.67 (s, 2H), 4.76 (s, 2H)。
称取2.5 mmol四碘荧光素、3.5mmol水合肼加入100 mL三口烧瓶中，加入30 mL乙醇加热回流24小时后，旋蒸除去溶剂，加入去离子水进行重沉淀得到粗产物，分别通过水/乙醇加热挥发结晶法得到最终产物化合物Luc-5：MS，计算值：849.68193，测量值：850.69090； 1H NMR (400 MHz, DMSO- d 6) δ 10.34 (d, J = 393.8 Hz, 2H), 7.82 (d, J = 8.6 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.03 (d, J = 13.1 Hz, 1H), 6.78 (dd, J = 20.3, 13.2 Hz, 2H), 6.63 – 6.34 (m, 1H), 4.63 (s, 2H)； 13C NMR (101 MHz, DMSO- d 6) δ 165.92, 158.77, 152.79, 136.70, 129.73, 129.46, 129.16, 128.38, 123.05 (d, J = 14.8 Hz), 79.41, 79.05, 78.16。
上述对比例合成路线以及产物结构如下。
Figure 792732dest_path_image003
。
探针（Luc-1）对金属离子的下转换荧光增强响应：在17个盛有探针溶液（10 μM，DMF/缓冲液，pH=7.0, v/v，1/2）的比色皿中，分别加入16种金属阳离子水溶液（浓度为100μM），它们分别是：Hg 2+、Cu 2+、Mn 2+、NH 4 +、Zn 2+、Mg 2+、Cd 2+、Pb 2+、Li +、Na +、K +、Ca 2+、Ni 2+、Co 2+、Fe 2+和Fe 3+。然后测定下转换荧光光谱（激发波长500 nm），见附图20，加入一定量的不同阳离子后，化合物Luc-1仅对Mg 2+有微弱的响应且干扰离子较多。
探针（Luc-4）对金属离子的下转换荧光增强响应：在17个盛有探针溶液（10 μM，DMF/缓冲液，pH=7.0, v/v，1/2）的比色皿中，分别加入16种金属阳离子水溶液（浓度为100μM），它们分别是：Hg 2+、Cu 2+、Mn 2+、NH 4 +、Zn 2+、Mg 2+、Cd 2+、Pb 2+、Li +、Na +、K +、Ca 2+、Ni 2+、Co 2+、Fe 2+和Fe 3+。然后测定下转换荧光光谱（激发波长500 nm），见附图21，加入一定量的不同阳离子后，化合物Luc-4对汞离子无响应。
探针（Luc-5）对金属离子的下转换荧光增强响应：在17个盛有探针溶液（10 μM，DMF/缓冲液，pH=7.0, v/v，1/2）的比色皿中，分别加入16种金属阳离子水溶液（浓度为100μM），它们分别是：Hg 2+、Cu 2+、Mn 2+、NH 4 +、Zn 2+、Mg 2+、Cd 2+、Pb 2+、Li +、Na +、K +、Ca 2+、Ni 2+、Co 2+、Fe 2+和Fe 3+。然后测定下转换荧光光谱（激发波长500 nm），见附图22，加入一定量的不同阳离子后，化合物Luc-5对汞离子无响应。
   而在OPA-UC（激发波长655 nm）荧光中，化合物（Luc-1、Luc-4、Luc-5）对金属离子都未有选择性OPA-UC荧光增强现象。
实施例三 四碘代荧光素螺环内硫酯（Luc-7）在TTA-UC的离子探测性能。
 以四碘代荧光素螺环内硫酯作为敏化剂与发光剂DPA（9,10-二苯基蒽）在溶液中组成双组份，产生TTA-UC，TTA-UC探测性能研究中均采用纯溶剂DMSO。
  在17个盛有双组份溶液（Luc-7/DPA：10 μM/1 mM，DMSO）的比色皿中，分别加入16种金属阳离子水溶液（浓度为10μM），它们分别是：Hg 2+、Cu 2+、Mn 2+、NH 4 +、Zn 2+、Mg 2+、Cd 2+、Pb 2+、Li +、Na +、K +、Ca 2+、Ni 2+、Co 2+、Fe 2+和Fe 3+，除氧后，测试其TTA-UC荧光光谱（激发波长655 nm）。见图23，发现在加入Hg 2+后，溶液的上转换荧光明显增强，而加入其它金属离子则无明显的TTA-UC荧光增强，说明在TTA-UC光谱检测中，化合物Luc-7也能选择性的识别Hg 2+。
作为对比，将Luc-7更换为四溴代荧光素螺环内硫酯、Luc-1或者Luc-5，采用同样的双组份以及测试方法，对所有离子都没有TTA-UC响应。
TTA-UC的灵敏度测试采用滴定法。向10 μM/1 mM的Luc-7/DPA的DMSO溶液依次滴加1 μL的0.3 mM的汞离子水溶液，除氧后，依次测试其TTA-UC的荧光强度变化（见图24a）。发现依次滴加定量的汞离子后其荧光强度依次增强，由于TTA-UC发光过程对水及其敏感，在汞离子的浓度0~0.8 μM时，荧光强度的增加具有良好的相关性，R 2为0.993332，斜率k为11.13683（见图24b），通过公式“检测限=3δ/k”可算得Luc-7在TTA-UC荧光检测通道中对Hg 2+的检测限为1.48×10 -9 M，其中δ=4.53×10 -1。可以看出，Luc-7对汞离子的TTA-UC的检测限极高，极为灵敏。

Claims (10)

  1. 一种卤代荧光素螺环内硫酯探针，具有如下化学结构式：
     
    Figure 979864dest_path_image001
    、
    Figure 43635dest_path_image002
    。
  2. 权利要求1所述卤代荧光素螺环内硫酯探针在生物成像中的应用；或者在制备生物成像试剂中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述生物成像为细胞成像。
  4. 权利要求1所述卤代荧光素螺环内硫酯探针作为金属离子荧光探针在金属离子检测中的应用。
  5. 权利要求1所述卤代荧光素螺环内硫酯探针的制备方法，其特征在于，以四卤代荧光素、三氯氧磷、硫化钠为原料，反应制备卤代荧光素螺环内硫酯探针；所述四卤代荧光素为四溴代荧光素或者四碘代荧光素。
  6. 根据权利要求5所述卤代荧光素螺环内硫酯探针的制备方法，其特征在于，四卤代荧光素、三氯氧磷、硫化钠的用量比例为1 mmol∶（0.8～1.2 mL）∶1 0mmol。
  7. 一种汞离子检测试纸，由试纸与权利要求1所述卤代荧光素螺环内硫酯探针组成。
  8. 一种利用荧光转换检测溶液中汞离子的方法，其特征在于，包括以下步骤：
    （1）以四卤代荧光素、三氯氧磷、硫化钠为原料，反应制备探针；
    （2）将所述探针的溶液与待测溶液混合，然后用光照射，检测荧光光谱；根据荧光光谱完成待测溶液中汞离子的检测；
    或者
    （3）将所述探针的溶液与发光剂混合，然后再与待测溶液混合，然后用光照射，检测荧光光谱；根据荧光光谱完成待测溶液中汞离子的检测；
    或者
    （4）将试纸浸泡所述探针的溶液后，然后干燥得到检测试纸；将待测溶液滴在所述检测试纸上，根据颜色变化完成待测溶液中汞离子的检测。
  9. 根据权利要求8所述利用荧光转换检测溶液中汞离子的方法，其特征在于，采用荧光光谱检测待测溶液中的汞离子时，荧光光谱为下转换荧光光谱或者上转换荧光光谱。
  10. 根据权利要求8所述利用荧光转换检测溶液中汞离子的方法，其特征在于，上转换为OPA-UC上转换时，探针的溶液中，探针的浓度为50～150μM；上转换为TTA-UC上转换时，探针的溶液中，探针的浓度为1～20μM，发光剂的浓度为0.1～10 mM。
PCT/CN2021/098271 2021-04-21 2021-06-04 卤代荧光素螺环内硫酯探针及其制备方法与在金属离子检测中的应用 WO2022222237A1 (zh)

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