CN107793421B - Probe with aggregation-induced emission characteristic and preparation method and application thereof - Google Patents

Probe with aggregation-induced emission characteristic and preparation method and application thereof Download PDF

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CN107793421B
CN107793421B CN201710741119.8A CN201710741119A CN107793421B CN 107793421 B CN107793421 B CN 107793421B CN 201710741119 A CN201710741119 A CN 201710741119A CN 107793421 B CN107793421 B CN 107793421B
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唐本忠
陈韵聪
张卫杰
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Hong Kong University of Science and Technology HKUST
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Abstract

The invention provides a probe with aggregation-induced emission characteristics, a preparation method and an application thereof, wherein the probe comprises:
Figure DDA0001385544760000011
wherein each R is independently selected from H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; x is a chromophore bonded to a aggregation inducing emission fluorophore. The probe provided by the invention has high sensitivity and strong practicability.

Description

Probe with aggregation-induced emission characteristic and preparation method and application thereof
Technical Field
The invention relates to the field of detection materials, in particular to a proportional metering type probe with aggregation-induced emission characteristics and a preparation method and application thereof.
Background
Photoluminescence (PL) technology has made scientists fascinating for decades in numerous fields such as chemical sensing, environmental science, biological imaging and medical diagnostics due to its high sensitivity, non-invasiveness and good spatio-temporal resolution. Some of the frequently used radioactive luminophores, such as BODIPY, fluorescein and rhodamine, exhibit relatively small stokes shifts (typically less than 30nm), which may lead to practical problems including notorious internal filtering effects and interference between excitation light and emitted light. Fluorescence Resonance Energy Transfer (FRET) has been a powerful tool that can provide a design strategy for not only developing fluorescent dyes with large pseudo-stokes shifts, but also for multi-color sensing and imaging.
FRET systems comprise a donor and an acceptor that are typically connected by a flexible aliphatic spacer. The efficiency of FRET is mainly adjusted by changing three parameters: 1) distance between donor and acceptor (rDA); 2) overlap integral (J) between emission spectrum of donor and absorption spectrum of acceptor; 3) relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET has been widely used in applications such as artificial photosynthesis and light collection of solar cells, chemical sensing, DNA or protein conformational change monitoring, and enzyme activity detection.
However, to achieve large pseudo-stokes shifts, the spectral overlap between the donor emission and acceptor absorption may be attenuated, which will result in a decrease in FRET efficiency and leakage of the donor emission. Bigies and colleagues have developed a cross-bond energy transfer (TBET) mechanism that can be a valuable method to solve this paradox. In the TBET system, the donor and acceptor are linked by a rigid linker rather than the flexible aliphatic linker in the FRET system. It should be noted that although the donor and acceptor are typically connected by a conjugated group (typically a benzene ring, double bond or triple bond), the twist angle between the donor and acceptor is large, which prevents them from becoming fluorophores. The energy transfer rate in TBET systems can be 2 orders of magnitude higher than that of conventional FRET systems, making it less dependent on spectral overlap. Therefore, even with small spectral overlap, high Energy Transfer Efficiency (ETE) can be easily achieved by the TBET mechanism, which is useful for generating large pseudo-stokes shifts.
Recently, common and colleagues have proposed a new FRET system called dark state resonance energy transfer (DRET) which contains a dark state donor with low quantum yield (< 1%). Fluorescent dyes in DRET libraries show attractive properties such as single excitation and tunable emission of large pseudo-stokes shifts. Notably, due to the low quantum yield of the donor, no background effects were observed, making the dyes in DRET profiles ideal candidates for biological applications. However, the energy transfer efficiency of DRET dyes is still strongly affected by spectral overlap. Thus, the choice of donor and acceptor in the DRET system may be limited. Furthermore, when the non-radiative decay rate of the dark state donor is fast enough to be comparable to the resonant energy transfer rate, the energy transfer efficiency may be reduced. The introduction of the TBET mechanism in a dark state energy transfer system can be a more efficient strategy because TBET rates are fast relative to non-radiative decay and energy transfer efficiency is less limited by spectral overlap. Furthermore, due to the low quantum yield of the donor, on-type sensing can be achieved by the DRET mechanism. Since fluorescence intensity is significantly affected by dye concentration, excitation power intensity, and other environmental factors, it is difficult to turn on the sensor to provide quantitative information about the analyte. In this respect, ratiometric probes would be more advantageous for quantitative detection because they allow self-calibration of two wavelengths to eliminate most of the interference described above.
Aggregation Induced Emission (AIE) dyes may exhibit no or weak PL signal in solution, but exhibit strong fluorescence emission in the aggregated state, which is a completely opposite phenomenon compared to conventional dyes that typically exhibit quenching (ACQ) effects caused by unwanted aggregation. The mechanism of AIE is attributed to the limitation of intramolecular movement (RIM), and a luminescent substance having AIE properties has excellent characteristics such as high brightness in a solid state and excellent light stability. Therefore, aggregation-inducing luminophores are a new class of materials with utility in fields including OLEDs, bio-imaging, and chromatography. Therefore, combining AIE and TBET mechanisms may be a very promising direction to generate new materials. Among these AIE cores, Tetraphenylethylene (TPE) is most widely used because of its ease of synthesis, solid state luminescence, ease of modification, ability to achieve tunable emission, and other advantages.
Disclosure of Invention
The present invention relates to a novel dark state cross-bond energy transfer (DTBET) strategy based on TPE derivatives and rhodamine moieties. Due to the fact that the TBET speed is high, the energy of the TPE derivatives in the dark state is obviously transferred to the rhodamine part before non-radiative decay release, and ETE reaches up to 99%. The invention researches the DTBET process and the structure-performance relation of the DTBET box by carrying out quantum chemical calculation. In addition, due to the emission characteristics of the TPE derivatives in the solid, Hg with high selectivity and high sensitivity is designed and synthesized by the invention2+And HClO proportional metering typeAnd (3) a probe.
The technical scheme provided by the invention is as follows:
in one aspect, the present invention provides a ratiometric probe having aggregation-induced emission characteristics, the probe comprising:
Figure BDA0001385544740000031
wherein each R is independently selected from H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; x is a chromophore bonded to a aggregation inducing emission fluorophore.
Specifically, in the probe of the present invention, X is:
Figure BDA0001385544740000032
wherein each R is independently selected from H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; y is O or Si; z is a aggregation-induced emission fluorophore.
Specifically, in the probe of the present invention, X is:
Figure BDA0001385544740000041
wherein Y is O or Si; z is a aggregation-induced emission fluorophore.
Specifically, in the above probe of the present invention, the probe is:
Figure BDA0001385544740000042
wherein R is1、R2And R3Each independently selected from H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; y is O or Si; z is a aggregation-induced emission fluorophore.
Specifically, in the above-mentioned probe of the present invention,R1、R2and R3Is substituted or unsubstituted and is independently selected from CnH2n+1、C10H7、C12H9、OC6H5、OC10H7、OC12H9、CnH2nNOH、CnH2nCOOH、CnH2nNCS、CnH2nN3、CnH2nNH2、CnH2nSO3、CnH2nCl、CnH2nBr、CnH2nI and
Figure BDA0001385544740000043
wherein n is 0 to 20;
r' is from CnH2nNCS、CnH2nN3、CnH2nNH2、CnH2nCl、CnH2nBr、CnH2nI and
Figure BDA0001385544740000044
to select.
Specifically, in the above-mentioned probe of the present invention, the probe is m-TPE-RNS, that is:
Figure BDA0001385544740000051
specifically, in the above-mentioned probe of the present invention, the probe is p-TPE-RNS, that is:
Figure BDA0001385544740000052
in particular, in the above-mentioned probe of the present invention, the probe is used for detecting a trace amount of Hg in water or a living cell2+。
Specifically, in the above-mentioned probe of the present invention, the probe is used for detecting exogenous HClO or endogenous HClO and for distinguishing cancer cells from normal cells.
The invention also provides a preparation method of the probe, which comprises the following steps:
s1, dissolving p-RBr in ethanol, adding hydrazine monohydrate, and reacting to generate p-RHZ;
step S2, mixing p-RHZ, TPE-B (OH)2、Pd(PPh3)4And K2CO3Mixing, adding THF and H under nitrogen atmosphere2O, reacting to generate p-TPE-RHZ;
step S3, mixing p-TPE-RHZ, benzyl isothiocyanate and triethylamine, and reacting in a nitrogen atmosphere to generate p-TPE-RNS;
wherein the chemical formula of the p-RBr is as follows:
Figure BDA0001385544740000061
p-RHZ has the formula:
Figure BDA0001385544740000062
the chemical formula of the p-TPE-RHZ is as follows:
Figure BDA0001385544740000063
the chemical formula of the p-TPE-RNS is as follows:
Figure BDA0001385544740000071
in the preparation method of the invention, in step S1, p-RBr is dissolved in ethanol, hydrazine monohydrate is added, and the solvent is removed by stirring, refluxing and reduced pressure distillation; the residue after removal of the solvent was purified by silica gel chromatography to give p-RHZ.
In the above preparation method of the present invention, step S2 includes:
mixing p-RHZ, TPE-B (OH)2、Pd(PPh3)4And K2CO3Placing the mixture in a two-necked flask; the two-necked flask was evacuated and filledNitrogen, then THF and H were added2O, refluxing;
THF was removed under vacuum and then DCM and H were added2O, carrying out liquid separation and extraction; DCM was removed from the extract, and the residue after DCM removal was purified by means of a silica gel column chromatography to obtain p-TPE-RHZ.
In the above preparation method of the present invention, step S3 includes:
dissolving p-TPE-RHZ, benzyl isothiocyanate and triethylamine in DMF, stirring under nitrogen atmosphere, and removing solvent under vacuum; the residue obtained after removal of the solvent was purified by silica gel chromatography to obtain p-TPE-RNS.
The invention also provides a preparation method of the probe, which comprises the following steps:
s1, dissolving m-RBr in ethanol, adding hydrazine monohydrate, and reacting to generate m-RHZ;
step S2, mixing m-RHz, TPE-B (OH)2、Pd(PPh3)4And K2CO3Mixing, adding THF and H under nitrogen atmosphere2O, reacting to generate m-TPE-RHZ;
step S3, mixing m-TPE-RHZ, benzyl isothiocyanate and triethylamine, and reacting in a nitrogen atmosphere to generate m-TPE-RNS;
wherein the chemical formula of m-RBr is as follows:
Figure BDA0001385544740000081
m-RHZ has the formula:
Figure BDA0001385544740000082
the chemical formula of the m-TPE-RHZ is as follows:
Figure BDA0001385544740000083
the chemical formula of the m-TPE-RNS is as follows:
Figure BDA0001385544740000091
in the preparation method of the invention, in step S1, m-RBr is dissolved in ethanol, hydrazine monohydrate is added, and the solvent is removed by stirring, refluxing and reduced pressure distillation; the residue after removal of the solvent was purified by silica gel chromatography to give m-RHZ.
In the above preparation method of the present invention, step S2 includes:
mixing m-RHZ, TPE-B (OH)2、Pd(PPh3)4And K2CO3Placing the mixture in a two-necked flask; the two-necked flask was evacuated and purged with nitrogen, and then THF and H were added2O, refluxing;
THF was removed under vacuum and then DCM and H were added2O, carrying out liquid separation and extraction; DCM was removed from the extract, and the residue after DCM removal was purified by means of a silica gel column chromatography to obtain m-TPE-RHZ.
In the above preparation method of the present invention, step S3 includes:
dissolving m-TPE-RHZ, benzyl isothiocyanate and triethylamine in DMF, stirring under nitrogen atmosphere, and removing solvent under vacuum; the residue obtained after removal of the solvent was purified by silica gel chromatography to give m-TPE-RNS.
The invention also provides trace Hg in water or living cells2+The detection method comprises the following steps:
step S1, staining a water sample or a living cell sample by using the probe;
step S2, making photoluminescence spectra of the dyed water sample or the living cell sample; and detecting trace amount of Hg according to photoluminescence spectrum2+。
In the detection method, the probe is m-TPE-RNS or p-TPE-RNS;
step S2 includes the following steps:
calculating the ratio of the photoluminescence intensity at 595nm and the photoluminescence intensity at 480nm in the photoluminescence spectrum of the dyed water sample or the living cell sample under the excitation of a 355nm UV lamp, namely the PL intensity ratio;
hg determination from PL intensity ratio2+The concentration of (c).
The invention also provides trace Hg in living cells2+The detection method comprises the following steps:
step S1, staining the living cell sample by using the probe;
s2, manufacturing a fluorescence confocal microscopic image of the dyed living cell sample by adopting a confocal laser scanning confocal microscope; and detecting trace amount of Hg according to the fluorescence confocal microscopic image2+。
The invention also provides trace amount of ClO in water or living cells-The detection method comprises the following steps:
step S1, staining a water sample or a living cell sample by using the probe;
step S2, making photoluminescence spectra of the dyed water sample or the living cell sample; and detecting trace ClO according to photoluminescence spectrum-。
In the detection method of the invention, the probe is p-TPE-RNS;
step S2 includes the following steps:
calculating the ratio of the photoluminescence intensity at 595nm and the photoluminescence intensity at 485nm in the photoluminescence spectrum of the dyed water sample or the living cell sample under the excitation of a 355nm UV lamp, namely the PL intensity ratio;
ClO determination from PL intensity ratio-The concentration of (c).
The invention also provides a trace amount of ClO in living cells-The detection method comprises the following steps:
step S1, staining the living cell sample by using the probe;
s2, manufacturing a fluorescence confocal microscopic image of the dyed living cell sample by adopting a confocal laser scanning confocal microscope; and detecting trace ClO according to fluorescence confocal microscopic image-。
The invention also provides a method for distinguishing cancer cells from normal cells, which comprises the following steps:
step S1, staining the living sample with the probe as described above;
step S2, shooting a fluorescence image of the dyed biological sample; and distinguishing the cancer cells from the normal cells according to different fluorescence intensities of the cells displayed by the fluorescence image.
In another aspect, the invention features a ratiometric probe having aggregation-induced emission properties, the probe including one or more chromophores bonded to one or more aggregation-induced emission fluorophores; the probe comprises one or more backbone structures selected from the group consisting of:
Figure BDA0001385544740000111
wherein each R is independently selected from H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; x is one or more chromophores for bonding with one or more aggregation-inducing emissive fluorophores.
In the above probe of the present invention, the skeleton structure is selected from:
Figure BDA0001385544740000112
wherein R is1、R2And R3Each independently selected from H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; y is O or Si.
In the above-mentioned probe of the invention, R1、R2And R3Is substituted or unsubstituted and is independently selected from CnH2n+1、C10H7、C12H9、OC6H5、OC10H7、OC12H9、CnH2nNOH、CnH2nCOOH、CnH2nNCS、CnH2nN3、CnH2nNH2、CnH2nSO3、CnH2nCl、CnH2nBr、CnH2nI and
Figure BDA0001385544740000113
wherein n is 0 to 20;
r' is from CnH2nNCS、CnH2nN3、CnH2nNH2、CnH2nCl、CnH2nBr、CnH2nI and
Figure BDA0001385544740000114
to select.
In the above-mentioned probe of the present invention, the probe is m-TPE-RNS, that is:
Figure BDA0001385544740000121
in the above-mentioned probe of the present invention, the probe is p-TPE-RNS, that is:
Figure BDA0001385544740000122
among the above-mentioned probes of the present invention, the probe is used for detecting a trace amount of Hg in water or in living cells2+。
In the above-described probes of the present invention, the probes are used for detecting exogenous HClO or endogenous HClO and for distinguishing cancer cells from normal cells.
The present invention relates to a just proposed dark state cross-bond energy transfer (DTBET) mechanism and its proportional metering type Hg at high performance2+The application of the probe and the HClO sensor in the development. More specifically, the present invention relates to a light-emitting body comprising a tetraphenylethylene derivative having Aggregation Induced Emission (AIE) characteristics and serving as an energy donor, wherein a rhodamine derivative is used as an energy acceptor. Also, the mechanism of the present invention can be applied to a transmission having a large pseudo-stokes shift and a high signal-to-noise ratioResearch and development of light and substance, and trace pollutant Hg2+And imaging of endogenous HClO.
Drawings
The invention will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 shows the chemical formula of p-TPE-RNS;
FIG. 2 shows the chemical formula of m-TPE-RNS;
FIG. 3 shows CH with different moisture content 310 μ M p-TPE-RNS (A) PL spectra under excitation of 355nm UV lamp in CN-water mixture;
FIG. 4 shows CH with different moisture content3PL intensity profile at 485nm under excitation by 355nm UV lamp of 10. mu.M p-TPE-RNS (B) in CN-water mixture;
FIG. 5 shows CH with different moisture content 310 μ M M-TPE-RNS (C) PL spectra under excitation of 355nm UV lamp in CN-water mixture;
FIG. 6 shows CH with different moisture content3PL intensity profile at 480nm under excitation by 355nm UV lamp of 10 μ M M-TPE-RNS (D) in CN-water mixture;
FIG. 7 shows in the absence of 2 equivalents of Hg2+(lines 1 and 2) and the presence of 2 equivalents of Hg2+(lines 3 and 4) with different moisture contents fw% CH3Photographs of 10 μ M M-TPE-RNS in CN-water mixture under excitation by ambient light and 365nm UV lamp, respectively;
FIG. 8 shows Hg at various contents2+In the presence of CH 310 μ M p-TPE-RNS (A) in CN-water mixture (with 60% moisture) PL spectrum under excitation of UV lamp at 355 nm;
FIG. 9 shows Hg at various contents2+In the presence of CH3PL spectrum under excitation of UV lamp at 355nm of 10 μ M-TPE-rns (b) in CN-water mixture (with 60% moisture);
FIG. 10 shows K at different metal ions (1 mM)+、Ca2+、Na+、Mg2+) In the presence of CH3CN-Water mixture (with 60% moisture)PL spectrum of 10. mu.M p-TPE-RNS (A);
FIG. 11 shows the reaction in CH in the presence of other metal ions (20. mu.M)3PL spectra of 10 μ M M-TPE-RNS (C) in CN-water mixture (with 60% moisture);
FIG. 12 shows a view at CH3PL Strength ratio (I) of 10. mu.M p-TPE-RNS (B) in CN-Water mixture (60% moisture)595/I480) A schematic diagram; wherein the white bars indicate: blank or presence of different metal ions; black bars indicate: treatment with labeled metal ions followed by addition of 20. mu.M Hg2+；
FIG. 13 shows a channel in CH3PL Strength ratio (I) of 10 μ M M-TPE-RNS (D) in CN-Water mixture (60% moisture)595/I480) A schematic diagram; wherein the white bars indicate: blank or presence of different metal ions; black bars indicate: treatment with labeled metal ions followed by addition of 20. mu.M Hg2+；
FIG. 14 shows Hg at 0-10ppb2+CH with 70% moisture in the Presence3Fluorescence spectrum of 0.1. mu.M M-TPE-RNS (A) in CN-water mixture;
FIG. 15 shows Hg at 0-3ppb2+A line fit plot of m-TPE-RNS (B) shown in FIG. 14 in the presence;
FIG. 16 shows Hg at 0-10ppb2+CH with 70% moisture in the Presence3Fluorescence spectrum of 0.1. mu.M p-TPE-RNS (C) in CN-water mixture;
FIG. 17 shows Hg at 0-3ppb2+A linear fit plot of p-TPE-RNS (D) shown in FIG. 16 in the presence;
FIG. 18 shows Hg for p (m) -TPE-RNS2+A schematic diagram of a sensing mechanism;
FIG. 19 shows the chemical formula of TPE;
FIG. 20 shows the chemical formula of RNO;
FIG. 21 shows the chemical formula of p-TPE-RNO;
FIG. 22 shows the chemical formula of m-TPE-RNO;
FIG. 23 shows 10 μ M TPE, RNO, M-TPE-RNO and p-TPE-RNO in CH3Absorption in CN-Water (v/v, 1: 1) mixturesSpectrum (a);
FIG. 24 shows a signal at CH3PL spectra (B) of 10. mu.M RNO, M-TPE-RNO and p-TPE-RNO in CN-water (v/v, 1: 1) mixture under 530nm light excitation;
FIG. 25 shows a signal at CH3PL spectra (C) of 10. mu.M TPE, RNO, M-TPE-RNO and p-TPE-RNO in CN-water (v/v, 1: 1) mixture under 355nm light excitation;
FIG. 26 shows PL intensity profiles (D) at 595nm for the RNO, m-TPE-RNO and p-TPE-RNO shown in FIG. 24 and FIG. 25;
FIG. 27 shows the theoretically optimized molecular geometry of the RNO and its calculated orientation lines of the donor transition dipole moment (arrows) associated with the linker axis (black dashes);
FIG. 28 shows the calculated orientation lines of the theoretically optimized molecular geometry of p-TPE-RNO and its donor transition dipole moment (arrow) associated with the linker axis (black dashes);
FIG. 29 shows the theoretically optimized molecular geometry of m-TPE-RNO and its calculated orientation lines of the donor transition dipole moment (arrows) associated with the linker axis (black dashes);
FIG. 30 shows graphs evaluating the toxicity of p-TYPE-RNS to HeLa cells by MTT assay;
FIG. 31 shows the absence of Hg2+(A-D) and the presence of 2. mu.M Hg2+(E-H) fluorescence confocal microscopy images of HeLa cells stained with 20. mu.M p-TPE-RNS for 40min at 30 min; wherein A, E is a bright field image; B. f is a blue channel image (420nm-520 nm); C. g is a red channel image (550nm-650 nm); D. h is an R/B ratio image under the excitation of 405nm light;
FIG. 32 shows ClO at various levels-In the presence of 10. mu.M p-TPE-RNS in CH3PL spectra via 355nm light excitation in CN-water mixture (with 60% moisture);
FIG. 33 shows 10. mu.M p-TPE-RNS ClO at 0-1.2. mu.M-A linear fit plot over a range of (a);
FIG. 34 shows a process for forming a semiconductor device including Cu2+Cysteine, Fe3+Glutathione, hydrogen peroxide, HOCl,KO2Various biologically relevant analytes (50. mu.M) of NO, OH, ROO and TBHP in the presence of CH3PL Strength ratio (I) of 10. mu.M p-TPE-RNS in CN-Water mixture (60% moisture)590/I480) A drawing;
FIG. 35 shows PL intensity ratio (I) of p-TPE-RNS monitored at 355nm excitation at consecutive intervals in the presence of 50 μ M NaOCl590/I480) A schematic diagram of (a);
FIG. 36 shows fluorescence images of p-TPE-RNS treated in different ways for HeLa cells; wherein, A-C is a confocal image of HeLa cells incubated with p-TPE-RNS for 0.5 h; D-F is a confocal image of HeLa cells pretreated with NaClO (10. mu.M, 0.5h) and then incubated with TPE-RNS for 0.5 h; G-I is a confocal image of p-TPE-RNS stained cells pretreated with 10. mu.M NAC (HClO scavenger) for 30min, then incubated with 10. mu.M NaClO at 37 ℃ for 30 min; wherein A, D, G is a blue channel image (420-530 nm); B. f, H is a red channel image (570-680 nm); C. f, I is a ratio image obtained by the fluorescence intensity ratio of red channel/blue channel; meanwhile, the excitation wavelength is 405 nm; the scale bar is 20 μm; the amount of p-TPE-RNS is 5 mu M;
fig. 37 shows a fluorescence image of endogenous HOCl of live RAW264.7 macrophages; wherein, A-C is the condition that RAW264.7 macrophage and a probe p-TPE-RNS are incubated together for 30 min; D-F is 1 mu g/mL for RAW264.7 macrophage-1LPS (Dexps. RTM.) was pretreated for 24 hours with 1. mu.gmL-1PMA pretreatment is carried out for 1h, and then the mixture is incubated with a probe p-TPE-RNS for 30 min; G-I is 1 mu G/mL for RAW264.7 macrophage-1LPS (Dexps. RTM.) was pretreated for 24 hours with 1. mu.gmL-1PMA pretreatment for 1h, also pretreatment with 10 μ M NAC for 30min, and then incubation with probe p-TPE-RNS for 30 min; wherein A, D, G is a blue channel image; B. f, H red channel images (570nm-680 nm); C. f, I is an image covering the blue, red channel and bright field images; meanwhile, the excitation wavelength is 405 nm; the scale bar is 20 μm; the amount of p-TPE-RNS is 5 mu M;
fig. 38 shows fluorescence images of endogenous HOCl in RAW264.7 macrophages of living organisms caused by bacterial infection; wherein, A-C is the condition that RAW264.7 macrophage and a probe p-TPE-RNS are incubated together for 30 min; D-F is the condition that RAW264.7 macrophage is pre-infected by escherichia coli for 4h and then is incubated with the probe p-TPE-RNS for 30 min; A. d is a blue channel image; B. f is a red channel image (570-680 nm); C. f is an image covering blue, red channel and bright field images; meanwhile, the excitation wavelength is 405 nm; the scale bar is 20 μm; the amount of p-TPE-RNS is 5 mu M;
FIG. 39 shows fluorescence images of endogenous HOCl in live COS-7 and HeLa cells stained with 10 μ M p-TPE-RNS for 30 min; wherein A is a bright field image; b is a fluorescence image under the excitation of light of 510nm-550 nm; c is a fluorescence image under the excitation of 330nm-385nm light; scale bar: 30 mu m;
FIG. 40 is a fluorescence image of endogenous HOCl in live COS-7 and MCF-7 cells stained with 10. mu.M p-TPE-RNS for 30 min; wherein A is a bright field image; b is a fluorescence image under the excitation of light of 510nm-550 nm; scale bar: 30 mu m;
FIG. 41 is a flow chart of a method for synthesizing m/p-TPE-RNS;
FIG. 42 is a flow chart of a method for synthesizing RNO.
Detailed Description
In one embodiment, FIG. 1 shows the chemical formula of p-TPE-RNS; FIG. 2 shows the chemical formula of m-TPE-RNS. The PL intensity of p-TPE-RNS is quite low when the moisture is 0% -50%, and aggregation starts from 55% moisture; the strong emission peak centered at 485nm reached a maximum at 95% moisture as shown in fig. 3 and 4. The PL spectrum of m-TPE-RNS showed similar weak PL signal when the moisture was below 50%; the PL spectra of m-TPE-RNS begin to aggregate when the moisture is above 55%; but the PL intensity at 480nm reached a maximum at 60% moisture and then decreased with increasing moisture as shown in fig. 5 and 6. The absolute quantum yields of p-TPE-RNS and m-TPE-RNS were 4.1% and 15.2% at a moisture of 60% as determined by integrating sphere method, respectively. Water content of 0-60%, Hg2+The addition of (a) resulted in a dramatic fluorescence emission color change from blue-green to orange-red, as shown in fig. 7. Therefore, 60% moisture was selected as the optimal detection condition.
In one embodiment, with Hg2+The emission intensity of p-TPE-RNS at 485nm disappears and the new peak at 595nm increases significantly with increasing concentration. A distinct isoemission point was observed at 564nm with a large emission difference in the 110nm range. I is595/I485From the absence of Hg2+In the case of (2) to 0.13 in the presence of 2 equivalents of Hg2+462.9 where the scale enhancement factor exceeds 3500, as shown in fig. 8. In one embodiment, m-TPE-RNS is coupled to Hg2+Response of (2) with p-TPE-RNS to Hg2+The response was similar, with a red shift at 115nm and a distinct isoemission point at 572nm being observed, as shown in FIG. 9. I is595/I485Intensity ratio of (2) never in the presence of Hg2+In the case of (2) to 0.17 to 2 equivalents of Hg2+1038.6 where the scale enhancement factor exceeds 6100 times! Good separation peaks indicate that two stoichiometric fluorescent probes are directed to Hg2+Is very sensitive.
PL spectra of DTBET system in the presence of different metal ions in CH with 60% moisture3CN-H2The O mixture was collected as shown in FIGS. 10-13. For p-TPE-RNS, it is only in the presence of 2 equivalents Hg2+In this case, a significant emission change from 485nm to 595nm is observed, which is in turn caused by 2 equivalents of other transition metal ions or 100 equivalents of K+、Ca2+、Na+、Mg2+The triggered emission changes can be ignored. In addition, Hg is present in the presence of other metal ions2+Initiated I595/I485The sharp increase in intensity ratio is hardly affected. m-TPE-RNS in other metal ions to Hg2+The selectivity of the catalyst is similar to that of p-TPE-RNS on Hg in other metal ions2+Selectivity of (2). The results show that both DTBET systems exhibit ratiometric Hg with excellent selectivity even in the presence of other competing metal ions2+Sensing capability.
Measurement of detection limit:
the probe (0.1. mu.M) was dissolved in an acetonitrile-water (3:7, volume: volume) mixed solution, and the measurement was conductedThe spectrum was emitted 10 times to determine the background noise σ. Then increasing Hg gradually2+The concentration is from 0 to 10.0ppb, each time Hg is added2+Fluorescence spectra were collected after 30 seconds of mixing. Then according to Hg2+The slope S of the curve was obtained by linear fitting of data ranging in concentration from 0 to 3.0 ppb. Accordingly, the detection limits (3. sigma./S) of m-TPE-RNS and p-TPE-RNS were calculated and determined to be 0.3ppb and 1.2ppb, respectively.
For p-TPE-RNS, the detection limit of the two DTBET sensors is determined to be 1.2 ppb; for m-TPE-RNS, the detection limit of the two DTBET sensors was determined to be 0.3ppb, as shown in FIGS. 14-17, both below Hg in U.S. EPA-standard drinking water2+Maximum allowable concentration (2 ppb). Hg based on AIE mechanism2+The selectivity of the DTBET sensor is significantly improved compared to the sensor. Hg is a mercury vapor2+The addition of (c) has two effects: 1) (ii) production of a rhodamine core; due to the rapid and effective TBET process, the generation of rhodamine nucleus leads to the increase of PL intensity at 595nm, and the PL intensity of TPE part is reduced; 2) the concentration of the DTBET sensor is reduced and the clustering of the sensor is reduced, further reducing the PL strength of the TPE unit. Thus, Hg of DTBET2+The sensor shows extraordinary proportional increments and very low detection limits. Combining the AIE and TBET mechanisms, the DTBET mechanism may be a practical design strategy to develop high performance sensors.
Proportional metering type Hg2+The working mechanism of the sensor is summarized in fig. 18. In the absence of Hg2+In the case of (2), the sensor is hydrophobic and tends to aggregate in aqueous solutions. Due to the non-emissive caprolactam form of rhodamine, only blue emission of polymerized TPE can be expected. With Hg2+After treatment, a positively charged rhodamine fluorophore is produced and its solubility in water is increased. Thus, due to the DTBET process and non-radiative decay, TPE emission is not observable, while strong emission of rhodamine should be expected. Thus, a proportional metering type Hg can be achieved by this rational design strategy2+The sensor.
Next, it was confirmed by photophysical spectroscopy that the energy transfer of TPE derivatives (dark state donors) to rhodamine moieties (acceptors) was 72% for p-TPE-RNS and 72% for m-TPE-RNS, as shown in FIGS. 19-26. In the solution state, no emission of the donor moiety is observed, and a large pseudo-stokes shift of up to 280nm is achieved, which would be advantageous for bioimaging with low background.
Theoretical calculations were performed to better understand the structure-property relationship of the DTBET process and the DTBET cassette. The dipole moment of the donor transition in the p-TPE-RNO is oriented at an angle of about 76.1 to the joint axis, while the dipole moment of the donor transition in the m-TPE-RNO is oriented at an angle of only 29.2 to the joint axis, as shown in FIGS. 27-29. The energy transfer rate is faster when the donor's transition dipole moment is parallel to the joint axis than when the donor's transition dipole moment is perpendicular to the joint axis. Therefore, the ETE rate of m-TPE-RNO should be faster than p-TPE-RNO, consistent with the fact that ETE in m-TPE-RNO is higher than that in p-TPE-RNO.
Confocal fluorescence imaging
At 5% CO2And HeLa cells were cultured in minimum essential medium MEM containing 10% fetal bovine serum at 37 ℃. HeLa cells were ratiometrically imaged using a laser scanning confocal fluorescence cell microscope (Zeiss LSM7 DUO). The blue channel 420-520nm, the red channel 550-650nm, and the excitation wavelength 405 nm. Cells were washed three times with phosphate buffered saline (PBS, pH 7.4) solution prior to imaging. The scaled images were processed using an image analysis program MATLAB.
Excellent Hg of DTBET cassette in solution2+The sensing performance enabled us to evaluate it in biological Hg2+Potential application in imaging. p-TPE-RNS is more easily penetrated into living cells than m-TPE-RNS due to its relatively large polarity and better solubility in water. Meanwhile, it was found that when the concentration of p-TPE-RNS was as high as 100. mu.M, no significant cytotoxicity was observed when MTT assay was performed, as shown in FIG. 30. Therefore, the use of confocal laser scanning confocal microscopy in HeLa cells can be compared with the measurement of Hg2+Imaging of (2). In the absence of Hg2+In case of (2) HeLa cells stained for 20min with 20. mu.M p-TPE-RNS showed suitability in the blue channel (420-520nm)Intensity of emission, while showing weak fluorescence in the red channel (550-650nm) using only blue scale images, indicating intracellular Hg2+The levels are very low as shown in fig. 31A-31D. Further incubation with 2. mu.M Hg2+After 30min, a decrease in intensity in the blue channel and a significant increase in intensity in the red channel can be observed, while the proportional image changes from green to orange, indicating Hg2+After incubation, intracellular Hg2+The levels were significantly increased as shown in fig. 31E-31H. The results demonstrate that p-TPE-RNS can be used as a reagent for biological Hg2+Good ratiometric imaging agents tested.
Cell viability assay:
cells were seeded in 96-well plates at a density of 5000 cells per well. After overnight incubation, the cultures were replaced in each well with fresh medium containing different concentrations of p-TPE-RNS. After 24 hours of treatment, 10. mu.l of MTT solution (5 mg/mL phosphate buffer) was added to each well). After incubation for 4 hours at 37 deg.C, 100. mu.L DSS-HCl solution (10% SDS, 0.01M HCl) was added. After 6 hours incubation at 37 ℃ by Perkin-Elmer Victor3TMThe instrument records the absorbance at 570nm for each concentration.
In one embodiment, with ClO-The emission intensity of p-TPE-RNS at 485nm decreased significantly with increasing concentration, and the new peak at 595nm increased significantly. I is595/I485In the presence of 5 equivalents of Hg2+The increase is 120 times or more in the case of (2), as shown in fig. 32. The well separated peaks indicate that the two stoichiometric fluorescent probes may be used for HClO-Is very sensitive. The detection limit for p-TPE-RNS was determined to be 50nM, as shown in FIG. 33. The addition of NaClO produces two effects: 1) (ii) production of a rhodamine core; due to the rapid and effective TBET process, the generation of rhodamine nucleus leads to the increase of PL intensity at 595nm, and the PL intensity of TPE part is reduced; 2) reducing the concentration of the sensor and reducing the clustering of the sensor, further reducing the PL strength of the TPE units. Thus, the DTBET HClO sensor shows extraordinary proportional gain and very low detection limits. Combining the AIE and TBET mechanisms, the DTBET mechanism may be one that can exploit the variety of vectors with high performancePractical design strategy of the sensor.
PL spectra of DTBET system in the Presence of different biological species in CH with 60% moisture3CN-H2The O mixture was collected as shown in fig. 34. Only in the presence of 5 equivalents of NaClO, a significant emission change from 485nm to 595nm can be observed, corresponding to 5 equivalents of other biologically relevant species or 5 equivalents of Cu2+Cysteine, Fe3 +Glutathione, hydrogen peroxide, HOCl, KO2Emission changes triggered by NO,. OH, ROO and TBHP can be ignored. The results show that both DTBET systems have ratiometric HOCl sensing capability with excellent selectivity. The response time of the p-TPE-RNS to HClO was determined to be 2min, as shown in FIG. 35, which is faster than many of the HClO sensors found.
In one embodiment, p-TPE-RNS is permeable to living cells, and thus, can be used for comparative metric imaging of exogenous HClO in HeLa cells using confocal laser scanning confocal microscopy. HeLa cells stained with 5 μ M p-TPE-RNS for 30min in the absence of NaClO showed moderate emission intensity in the blue channel (420-530nm) and weak fluorescence in the red channel (570-680nm) with a weak scale of image, indicating very low levels of HClO inside the cells, as shown in FIGS. 36A-14C. When HeLa cells were pretreated with 10. mu.M NaClO for 30min and then incubated with 5. mu.M p-TPE-RNS for 30min, a decrease in emission intensity in the blue channel and a significant increase in emission intensity in the red channel were observed, as shown in FIGS. 36D-14F. The increased scale image indicates a significant increase in intracellular levels of HClO after NaClO incubation. However, when HeLa cells were pretreated with 10. mu.M NaClO and 10. mu.M NAC (HClO scavenger) for 30min and then incubated with 5. mu.M p-TPE-RNS for 30min, intensity reductions in the red channel and scale images were observed, as shown in FIG. 36G-FIG. 36I.
In one embodiment, the good sensing performance of p-TPE-RNS on exogenous HClO in solution prompted us to evaluate its potential application in ratiometric imaging of endogenous HClO. RAW264.7 macrophages stained with 5 μ M p-TPE-RNS in the absence of NaClO showed moderate emission intensity in the blue channel (420-530nm) and weak fluorescence in the red channel (570-680nm), indicating only very low levels of HClO within the cells, as shown in FIGS. 37A-37C. When RAW264.7 macrophages were pretreated with 1. mu.g/mL LPS for 24h and 1. mu.g/mL PMA for 1h, and then incubated with probe p-TPE-RNS for 30min, it was observed that the emission intensity in the blue channel decreased and the emission intensity in the red cell channel significantly increased, as shown in FIGS. 37D-37F; this indicates a significant increase in intracellular levels of HClO following LPS and PMA incubation to induce inflammation. However, when HeLa cells were pretreated with 1. mu.g/mL LPS for 24h and 1. mu.g/mL PMA for 1h, and also with 10. mu.M NAC (HClO scavenger) for 30min, followed by incubation with 5. mu.M p-TPE-RNS for 30min, only a small magnitude of enhancement in emission intensity was observed in the red channel and ratio images, as shown in FIGS. 37G-37I. These results demonstrate that p-TPE-RNS can be a good ratiometric imaging agent for exogenous and endogenous HClO.
Next, we further tested the ratiometric imaging capability of endogenous HClO caused by bacterial infection. Incubation of RAW264.7 macrophages with 5. mu.M p-TPE-RNS for 30min, moderate emission intensity in the blue channel (420-530nm) was observed, and weak fluorescence in the red channel (570-680nm) was seen, indicating very low intracellular HClO levels, as shown in FIGS. 38A-38C. However, when RAW264.7 macrophages were preinfected with E.coli for 4h and then incubated with 5. mu.M p-TPE-RNS probe for 30min, a decrease in emission intensity in the blue channel and a significant increase in emission intensity in the red channel was observed, as shown in FIG. 38D-as shown in FIG. 38F, indicating that E.coli infection promotes a significant increase in intracellular HClO levels.
Cancer cells are reported to produce more reactive oxygen species than normal cells. We envision that the use of p-TPE-RNS allows differentiation between cancer cells and normal cells. Thus, we co-cultured COS-7 cells (normal cell type) and HeLa cells (cancer cells) and stained with 10. mu.M p-TPE-RNS, and then, the strong fluorescence intensity corresponding to HeLa cells in the red channel was observed, as shown in FIG. 39. When MCF-7 cells were co-cultured with normal COS-7 cells, a high fluorescence intensity pattern similar to that of MCF-7 cells (cancer cell type) was observed, as shown in FIG. 40.
Ultraviolet absorption spectroscopy was performed on a Milton Roy spectral 3000 array spectrophotometer. Fluorescence spectra were recorded on a Perkin-Elmer LS 55 spectrometer. Quantum efficiency measurements were performed on a Huffman C11347Quantaurus-QY integrating sphere. High resolution mass spectra were obtained on a GCT Premier CAB 048 mass spectrometer. The size of the nano-aggregated particles was determined using a ZETA-Plus potential analyzer.
The invention also provides a method for synthesizing the m/p-TPE-RNS and the RNO, which is shown in figure 41 and figure 42.
Synthesis of p-RHZ:
compound p-RBr (0.535g, 1.0mmol) was dissolved in 20mL ethanol and 2mL hydrazine monohydrate was added. The reaction mixture was refluxed for 6 hours with stirring, and then the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (n-hexane/ethyl acetate, 5: 1 to 3: 1, v/v) to give p-RHZ (0.35g) as a white powder in 64% yield; 1H NMR (400MHz, CDCl3):7.82(d, J ═ 8.0Hz,1H),7.60(dd, J ═ 1.6Hz, J ═ 8.0Hz,1H),7.26(d, J ═ 1.6Hz,1H),6.48(d, J ═ 8.8Hz,2H),6.44(d, J ═ 1.6Hz,2H),6.32(dd, J ═ 1.6Hz, J ═ 8.8Hz,2H),3.62(s,2H),3.37(q, J ═ 7.2Hz,8H),1.20(t, J ═ 7.2Hz, 12H); 13C NMR (100MHz, CDCl3) 165.3,153.8,153.3,149.1,131.7,128.8,128.0,127.2,124.6,108.1,103.6,98.0,65.8,44.4, 12.6; HRMS calculated value: [ M + H ]]+535.1703, actual value: 535.1729.
synthesis of m-RHZ:
the synthetic procedure was analogous to p-RHZ, using m-RBr as starting material, yield, 68%. 1H NMR (400MHz, CDCl3):8.09(d, J ═ 2.0Hz,1H),7.59(dd, J ═ 1.0Hz, J ═ 7.6Hz,1H),7.01(d, J ═ 7.6Hz,1H),6.47(d, J ═ 8.4Hz,2H),6.43(d, J ═ 2.4Hz,2H),6.32(dd, J ═ 2.4Hz, J ═ 8.4Hz,2H),3.63(s,2H),3.36(q, J ═ 7.2Hz,8H),1.19(t, J ═ 7.2Hz, 12H); 13C NMR (100MHz, CDCl3) 164.6,153.8,150.2,149.0,135.5,132.0,128.0,126.1,125.6,122.2,108.1,103.7,98.0,66.0,44.4, 12.6; HRMS calculated value: [ M ]534.1630, actual value: 534.1635.
synthesis of p-TPE-RHZ Compound:
mixing p-RHZ (268mg, 0.5mmol), TPE-B (OH)2(188mg，0.5mmol)，Pd(PPh3)4(20mg, 0.017mmol) and K2CO3(138mg, 1.0mmol) was placed in a 100mL two-necked flask. After evacuation and nitrogen charging for 3 times, 25mL of THF and 10mL of H were added2And O. The reaction mixture was refluxed overnight. The THF solvent was removed in vacuo, with DCM and H2And O, separating and extracting. After removal of DCM, the resulting residue was purified by silica gel column chromatography (hexane/CH 2Cl 2/ethyl acetate, 2:1:1, v/v/v) to give p-TPE-RHZ (324mg, 82%).1H NMR(400MHz,CDCl3) 7.95(d, J ═ 8.0Hz,1H),7.64(dd, J ═ 1.6Hz, J ═ 8.0Hz,1H),7.25(d, J ═ 1.6Hz,1H),6.98-7.07(m,19H),6.49(d, J ═ 8.8Hz,2H),6.42(d, J ═ 2.4Hz,2H),6.30(dd, J ═ 2.4Hz, J ═ 8.8Hz,2H),3.61(s,2H),3.35(q, J ═ 6.8Hz,8H),1.17(t, J ═ 6.8Hz, 12H); 13C NMR (100MHz, CDCl3) 166.0,153.8,152.2,148.9,145.1,143.6,141.3,140.2,137.8,131.8,131.4,131.3,131.2,128.9,128.2,127.8,127.7,127.6,127.0,126.6,126.5,123.3,122.1,108.1,104.5,97.9,66.1,44.4, 12.6; HRMS calculated value: [ M ] A]786.3934, actual value: 786.3986.
synthesis of m-TPE-RHZ compound:
the procedure was similar to p-TPE-RHZ, using m-RHZ as starting material, yield, 85%.1H NMR(400MHz,CDCl3) 8.12(d, J ═ 2.4Hz,1H),7.65(dd, J ═ 2.4Hz, J ═ 8.0Hz,1H),7.39(d, J ═ 8.0Hz,2H),7.04-7.11(m,19H),6.50(d, J ═ 8.8Hz,2H),6.43(d, J ═ 2.4Hz,2H),6.30(dd, J ═ 2.4Hz, J ═ 8.8Hz,2H),3.63(s,2H),3.35(q, J ═ 6.8Hz,8H),1.17(t, J ═ 6.8Hz, 12H); 13CNMR (100MHz, CDCl3) 166.1,153.9,150.3,148.9,143.7,143.6,143.5,143.3,141.4,141.0,140.4,137.9,131.9,131.4,131.3,131.2,130.6,128.2,127.8,127.7,127.6,127.0,126.6,126.5,126.4,126.3,124.1,121.2,108.1,104.5,98.0,65.9,44.4, 12.6; HRMS calculated value: [ M ] A]786.3934, actual value: 786.3918.
synthesis of p-TPE-RNS:
benzyl isothiocyanate (135mg, 1.0mmol), p-TPE-RHZ (197mg, 0.25mmol) and triethylamine (0.1mL) were dissolved in 10mL DMF and the reaction mixture was stirred at room temperature for 8h under protection of N2. The solvent is removed in vacuo, toThe resulting residue was purified by silica gel chromatography (hexane/CH 2Cl 2/ethyl acetate, 2:1:1v/v/v) to give p-TPE-RNS (210mg, 91%).1H NMR(400MHz,CDCl3):8.02(d,J＝8.0Hz,1H),7.77(dd,J＝1.6Hz,J＝8.0Hz,1H),7.53(s,1H),7.42(d,J＝1.6Hz,1H),7.30(d,J＝8.0Hz,2H),7.19(t,J＝3.6Hz,2H),6.97-7.10(m,21H),6.53(d,J＝8.8Hz,2H),6.45(d,J＝2.8Hz,2H),6.30(dd,J＝2.8Hz,J＝8.8Hz,2H),3.35(q,J＝6.8Hz,8H),1.17(t,J＝6.8Hz,12H)；13C NMR(100MHz,CDCl3) 182.8,167.1,154.3,150.9,149.4,147.0,144.2,143.5,143.4,141.6,140.1,137.7,137.3,132.0,131.4,131.3,128.3,127.9,127.7,127.6,126.6,126.5,126.1,125.2,124.2,122.8,108.4,104.2,98.3,67.3,44.4, 12.6; HRMS calculated value: [ M ] A]921.4076, actual value: 921.4098.
and (3) synthesizing an m-TPE-RNS compound:
the synthetic procedure was similar to p-TPE-RNS, using m-TPE-RHZ as starting material, yield, 92%.1H NMR(400MHz,CDCl3) 8.20(s,1H),7.85(d, J ═ 8.0Hz,1H),7.53(s,1H),7.45(d, J ═ 8.0Hz,2H),7.31(d, J ═ 8.0Hz,2H),7.06-7.21(m,22H),6.98(s,1H),6.54(d, J ═ 8.8Hz,2H),6.47(d, J ═ 2.0Hz,2H),6.31(dd, J ═ 2.0Hz, J ═ 8.8Hz,2H),3.36(q, J ═ 7.2Hz,8H),1.19(t, J ═ 7.2Hz, 12H); 13C NMR (100MHz, CDCl3) 182.7,167.2,154.3,149.4,148.8,143.8,143.6,143.5,142.0,141.6,140.2,137.7,137.3,133.0,132.1,131.4,131.3,131.2,129.7,129.6,128.3,127.9,127.8,127.7,127.6,126.7,126.6,126.4,126.1,125.8,125.1,125.0,124.2,121.9,108.4,104.2,98.4,67.2,44.4, 12.6; HRMS calculated value: [ M ] A]921.4076, actual value: 921.4036.
and (3) synthesis of p-TPE-RNO:
the compound p-TPE-RNS (92mg, 0.1mmol) was dissolved in 5mLCH3Adding HgCl into CN2(54mg, 0.2mmol) and the mixture was stirred at room temperature for 6 hours. After removal of the solvent, the residue was purified by silica gel chromatography (DCM/MeOH, 20:1, v/v) to give p-TPE-RNO (80mg, 90%).1H NMR(400MHz,CD2Cl2):11.20(br,1H),8.30(s,J＝8.0Hz,1H),7.88(d,J＝8.4Hz,1H),7.73(d,J＝8.4Hz,2H),7.48(s,1H),7.42(d,J＝8.0Hz,2H),7.02-7.21(m,21H),6.89(t,J＝7.6Hz,2H),6.76-6.78(m,4H),3.56(q,J＝7.2Hz,8H),1.28(t,J＝7.2Hz,12H) (ii) a 13C NMR (100MHz, CDCl3) 160.5,158.1,157.7,156.0,155.6,144.4,143.6,143.5,143.4,142.1,141.8,140.1,139.0,136.3,132.0,131.4,131.2,131.1,131.0,130.5,129.2,128.6,128.0,127.8,127.7,127.6,126.6,126.2,122.2,121.7,117.7,114.1,113.9,96.3,46.0, 12.3; HRMS calculated value: [ M ] A]888.4272, actual value: 888.4253.
and (3) synthesis of m-TPE-RNO:
the synthesis method is similar to the p-TPE-RNO, and m-TPE-RNS is used as a starting material, so that the yield is 95%.1H NMR(400MHz,CDCl3) 10.99(br,1H),8.49(s,1H),7.79(d, J ═ 8.0Hz,1H),7.73(d, J ═ 8.0Hz,2H),7.54(d, J ═ 8.0Hz,2H),7.27(d, J ═ 8.0Hz,1H),7.06-7.20(m,21H),6.86(t, J ═ 7.2Hz,1H),6.79(d, J ═ 9.6Hz,2H),6.72(s,2H),3.52(q, J ═ 6.8Hz,8H),1.28(t, J ═ 6.8Hz, 12H); 13C NMR (100MHz, CDCl3) 160.7,157.9,157.4,156.1,155.4,144.4,143.6,143.4,143.0,141.6,140.3,138.8,136.2,132.2,131.5,131.4,131.3,131.2,130.9,128.6,128.5,127.9,127.8,127.7,126.8,126.7,126.6,126.5,124.2,121.7,118.0,114.2,113.8,96.5,46.1, 12.7; HRMS calculated value: [ M ] A]888.4272, actual value: 888.4244.
it will be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings and all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.

Claims (15)

1. A ratiometric probe with aggregation-induced emission characteristics is characterized in that the probe is
Figure FDA0002688997130000011
2. A preparation method of a probe is characterized by comprising the following steps:
s1, dissolving p-RBr in ethanol, adding hydrazine monohydrate, and reacting to generate p-RHZ;
step S2, mixing p-RHZ, TPE-B (OH)2、Pd(PPh3)4And K2CO3Mixing, and thenTHF and H were added under a nitrogen atmosphere2O, reacting to generate p-TPE-RHZ;
s3, mixing p-TPE-RHZ, phenyl isothiocyanate and triethylamine, and reacting in a nitrogen atmosphere to generate p-TPE-RNS;
wherein the chemical formula of the p-RBr is as follows:
Figure FDA0002688997130000012
p-RHZ has the formula:
Figure FDA0002688997130000013
TPE-B(OH)2the chemical formula of (1) is;
Figure FDA0002688997130000021
the chemical formula of the p-TPE-RHZ is as follows:
Figure FDA0002688997130000022
the chemical formula of the p-TPE-RNS is as follows:
Figure FDA0002688997130000023
3. the method according to claim 2, wherein in step S1, the p-RBr is dissolved in ethanol, and after adding hydrazine monohydrate, the solvent is removed by stirring, refluxing, and distillation under reduced pressure; the residue after removal of the solvent was purified by silica gel chromatography to give p-RHZ.
4. The method according to claim 2, wherein step S2 includes:
mixing p-RHZ, TPE-B (OH)2、Pd(PPh3)4And K2CO3Placing the mixture in a two-necked flask; the two-necked flask was evacuated and purged with nitrogen, and then THF and H were added2O, refluxing;
THF was removed under vacuum and then DCM and H were added2O, carrying out liquid separation and extraction; DCM was removed from the extract, and the residue after DCM removal was purified by means of a silica gel column chromatography to obtain p-TPE-RHZ.
5. The method according to claim 2, wherein step S3 includes:
dissolving p-TPE-RHZ, phenyl isothiocyanate and triethylamine in DMF, stirring under nitrogen atmosphere, and removing the solvent in vacuum; the residue obtained after removal of the solvent was purified by silica gel chromatography to obtain p-TPE-RNS.
6. A preparation method of a probe is characterized by comprising the following steps:
s1, dissolving m-RBr in ethanol, adding hydrazine monohydrate, and reacting to generate m-RHZ;
step S2, mixing m-RHz, TPE-B (OH)2、Pd(PPh3)4And K2CO3Mixing, adding THF and H under nitrogen atmosphere2O, reacting to generate m-TPE-RHZ;
s3, mixing m-TPE-RHZ, phenyl isothiocyanate and triethylamine, and reacting in a nitrogen atmosphere to generate m-TPE-RNS;
wherein the chemical formula of m-RBr is as follows:
Figure FDA0002688997130000031
m-RHZ has the formula:
Figure FDA0002688997130000032
TPE-B(OH)2the chemical formula of (1) is;
Figure FDA0002688997130000033
the chemical formula of the m-TPE-RHZ is as follows:
Figure FDA0002688997130000041
the chemical formula of the m-TPE-RNS is as follows:
Figure FDA0002688997130000042
7. the method according to claim 6, wherein in step S1, m-RBr is dissolved in ethanol, and after hydrazine monohydrate is added, the solvent is removed by stirring, refluxing, and distillation under reduced pressure; the residue after removal of the solvent was purified by silica gel chromatography to give m-RHZ.
8. The method according to claim 6, wherein step S2 includes:
mixing m-RHZ, TPE-B (OH)2、Pd(PPh3)4And K2CO3Placing the mixture in a two-necked flask; the two-necked flask was evacuated and purged with nitrogen, and then THF and H were added2O, refluxing;
THF was removed under vacuum and then DCM and H were added2O, carrying out liquid separation and extraction; DCM was removed from the extract, and the residue after DCM removal was purified by means of a silica gel column chromatography to obtain m-TPE-RHZ.
9. The method according to claim 6, wherein step S3 includes:
dissolving m-TPE-RHZ, phenyl isothiocyanate and triethylamine in DMF, stirring in a nitrogen atmosphere, and removing the solvent in vacuum; the residue obtained after removal of the solvent was purified by silica gel chromatography to give m-TPE-RNS.
10. Water or living bodyTrace amount of Hg in cells2+Is used for non-disease diagnosis, comprising the steps of:
step S1, staining a water sample or a living cell sample with the probe of claim 1;
step S2, making photoluminescence spectra of the dyed water sample or the living cell sample; and detecting trace amount of Hg according to photoluminescence spectrum2+。
11. The detection method according to claim 10,
step S2 includes the following steps:
calculating the ratio of the photoluminescence intensity at 595nm and the photoluminescence intensity at 480nm in the photoluminescence spectrum of the dyed water sample or the living cell sample under the excitation of a 355nm UV lamp, namely the PL intensity ratio;
hg determination from PL intensity ratio2+The concentration of (c).
12. Trace Hg in living cells2+Is used for non-disease diagnosis, comprising the steps of:
step S1, staining a sample of living cells with the probe of claim 1;
s2, manufacturing a fluorescence confocal microscopic image of the dyed living cell sample by adopting a laser scanning confocal microscope; and detecting trace amount of Hg according to the fluorescence confocal microscopic image2+。
13. Trace amount of ClO in water or living cells-Is used for non-disease diagnosis, comprising the steps of:
step S1, staining a water sample or a living cell sample with the probe of claim 1;
step S2, making photoluminescence spectra of the dyed water sample or the living cell sample; and detecting trace ClO according to photoluminescence spectrum-。
14. The detection method according to claim 13,
step S2 includes the following steps:
calculating the ratio of the photoluminescence intensity at 595nm and the photoluminescence intensity at 485nm in the photoluminescence spectrum of the dyed water sample or the living cell sample under the excitation of a 355nm UV lamp, namely the PL intensity ratio;
ClO determination from PL intensity ratio-The concentration of (c).
15. Trace amount of ClO in living cells-Is used for non-disease diagnosis, comprising the steps of:
step S1, staining a sample of living cells with the probe of claim 1;
s2, manufacturing a fluorescence confocal microscopic image of the dyed living cell sample by adopting a confocal laser scanning confocal microscope; and detecting trace ClO according to fluorescence confocal microscopic image-。
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