CN108663346B - L-cysteine fluorescent probe based on conjugated polymer/metal ion compound, and preparation method and application thereof - Google Patents

L-cysteine fluorescent probe based on conjugated polymer/metal ion compound, and preparation method and application thereof Download PDF

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CN108663346B
CN108663346B CN201810640498.6A CN201810640498A CN108663346B CN 108663346 B CN108663346 B CN 108663346B CN 201810640498 A CN201810640498 A CN 201810640498A CN 108663346 B CN108663346 B CN 108663346B
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cys
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陆燕
刘丽华
王晶
张强
赵琳琳
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Tianjin University of Technology
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Abstract

Based on conjugated polymer/goldAn L-cysteine fluorescent probe belonging to an ion complex, a preparation method and application thereof. The fluorescent probe is a compound consisting of a conjugated polymer and metal ions, wherein the conjugated polymer is a polythiophene derivative (PT) with a side chain containing double carboxylate, and the structural formula is as follows:
Figure DDA0001702291690000011
PT emits strong fluorescence in aqueous solution, with metal ions (M)n+) Upon binding, a complex is formed, and the fluorescence of the PT solution is quenched due to the paramagnetism of the metal ion. When L-cysteine (Cys) is added, Cys can react with Mn+To form a more stable complex Cys-Mn+Thereby enabling PT to be driven from PT/Mn+Released from the complex, and its fluorescence is restored. The PT/Mn+The compound fluorescent probe has the advantages of simple synthesis method, good water solubility, high response speed and high selectivity, can be used for quantitative detection of Cys in an aqueous solution, and can also be used for labeling and fluorescence imaging of Cys in living cells.

Description

L-cysteine fluorescent probe based on conjugated polymer/metal ion compound, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of fluorescence sensing detection, and particularly relates to an L-cysteine fluorescent probe based on a conjugated polymer/metal ion compound, and a preparation method and application thereof.
Background
L-cysteine (Cys) is a very important sulfhydryl-containing amino acid in human body, can participate in reversible redox reaction process in human body, and has multiple important physiological functions of regulating cell homeostasis and cell metabolism in biochemical pathway. In vivo, abnormal Cys concentrations can cause many diseases. Too low a Cys content can cause growth retardation, hair color loss, lethargy, liver and skin tissue damage, fat loss and other symptoms in children; and diseases such as atherosclerosis, ischemic cerebrovascular disease, hypertension syndrome, gestational diabetes and the like are accompanied by obvious increase of Cys content. Therefore, the development of a Cys detection method with high selectivity and high sensitivity has important scientific and practical values for deep understanding of biological functions and clinical detection and treatment of related diseases.
In various analysis methods, the fluorescence technology has the characteristics of simple operation, high sensitivity, good space-time resolution performance and the like, shows unique advantages in-situ non-destructive detection of living cells, living tissues, even living bodies and the like, and has the core of a fluorescence probe. The detection principle of the currently reported Cys fluorescent probe is mainly that a thiol group in Cys reacts with a specific functional group to cause the change of system fluorescence, and the purpose of quantitatively detecting Cys is achieved by utilizing the linear relation between the change of system fluorescence intensity and the concentration of Cys. Functional groups often contained in such Cys probes include: n-substituted maleimides, aldehydes, disulfide bonds, sulfonyl esters, sulfonamides, and the like. Such Cys fluorescent probes generally require a certain reaction time and may have an influence on the detection result along with the generation of new substances. Recently, researchers have attracted attention to Cys detection methods based on a combination of a fluorescent probe and metal ions, the method utilizes metal ions as quenchers to quench the fluorescence of the fluorescent probe, and Cys can form a more stable complex with the metal ions, so that a free fluorescent probe is released, and the fluorescence of a system is recovered. The detection strategy of the replacement avoids the generation of new fluorescent substances and improves the reliability of detection. However, the currently reported fluorescent probes are usually derivatives of organic dyes, such as coumarin, fluorescein, naphthol, anthracene, bipyridine, etc., which are generally poor in solubility in water, so that detection of Cys must be performed in an organic solvent or a mixed system of organic solvent/water, which greatly limits the in-situ detection performance in a biological system.
Disclosure of Invention
The invention aims to solve the defects of the current Cys fluorescent probe, designs and synthesizes a water-soluble polythiophene derivative with a side chain containing double carboxylate, and the water-soluble polythiophene derivative and metal ions form a compound fluorescent probe, can detect the content of Cys in aqueous solution at high selectivity and high sensitivity and quickly, and can also be used as a fluorescent dye for marking and carrying out fluorescence imaging on Cys in living cells.
The technical scheme adopted by the invention is as follows:
the L-cysteine (Cys) fluorescent probe based on the conjugated polymer/metal ion compound is a compound consisting of the conjugated polymer and metal ions, wherein the conjugated polymer is a polythiophene derivative (PT) with a side chain containing double carboxylate groups, and the metal ions are Cu2+，Fe2+，Fe3+，Co3+，Pd2+，Eu3+，Ni2+，Hg2+，Ag+，Ca2+，Cd2+Or Zn2+An ion which is paramagnetic and capable of binding to the conjugated polymer. The detection principle of the conjugated polymer/metal ion complex fluorescent probe is shown in figure 1: the conjugated polymer is dissolved in water, and the solution has strong fluorescence under the excitation of light with the wavelength of 400 nm. An appropriate amount of metal ions is added to the solution, which quenches the fluorescence of the conjugated polymer once the conjugated polymer forms a complex with the metal ions due to the paramagnetism of the metal ions. When Cys is gradually added into the conjugated polymer/metal ion compound solution, the Cys can be combined with metal ions through an M-S bond to form a more stable complex Cys-Mn+. Therefore, as the content of Cys in the solution increases, the metal ions gradually separate from the conjugated polymer/metal ion complex, and the fluorescence of the conjugated polymer is recovered again. According to the linear relation between the fluorescence intensity of the conjugated polymer and the concentration of Cys, the content of Cys can be rapidly detected in water with high selectivity and high sensitivity, and the Cys can be labeled and subjected to fluorescence imaging in living cells.
The conjugated polymer is a polythiophene derivative with a side chain containing double carboxylate, and the structural formula of the conjugated polymer is as follows:
Figure BDA0001702291670000021
wherein R is hydrogen, methyl, ethyl, propyl or a saturated alkyl chain; x is a carbon, oxygen, sulfur, selenium or tellurium atom; m is univalent lithium, sodium, potassium, silver or ammonium.
The invention also provides a preparation method of the L-cysteine fluorescent probe based on the conjugated polymer/metal ion compound, which comprises the following steps:
step 1, preparation of conjugated Polymer-Polythiophene derivative having Bicarboxylate in the side chain
3-bromo-thiophene with hydrogen, methyl, ethyl, propyl or saturated alkyl chain at the 4-position is taken as an initial raw material, and a side chain functionalized monomer is obtained through etherification reaction and nucleophilic substitution in sequence; dissolving the functionalized monomer in chloroform and adding FeCl3Oxidizing and polymerizing for 48 hours at 50 ℃ under the protection of nitrogen, stopping reaction, cooling to room temperature, removing the solvent by rotary evaporation, dissolving the obtained solid in methanol, adding a few drops of hydrazine hydrate, stirring for reacting overnight, then performing suction filtration, and performing rotary drying on the filtrate to obtain a crude product; performing alkaline hydrolysis on the obtained crude product, transferring into a dialysis bag with molecular weight cutoff of 3500, and dialyzing for 3 days; and then, freeze drying to obtain polymer solid powder, namely the polythiophene derivative with the side chain containing the double carboxylate.
Step 2, preparation of fluorescent probe based on conjugated polymer/metal ion complex
Step 2.1, dissolving a proper amount of the conjugated polymer prepared in the step 1 in deionized water to prepare the conjugated polymer with the concentration of 5 × 10-5The solution of M is called solution a.
Step 2.2, taking a proper amount of metal chloride or metal nitrate to dissolve in deionized water to prepare the metal chloride or metal nitrate with the concentration of 5 × 10-3The solution of M is called solution b.
And 2.3, gradually adding the metal ion solution b obtained in the step 2.2 into the conjugated polymer solution a until the fluorescence of the conjugated polymer is completely quenched to obtain a conjugated polymer/metal ion compound fluorescent probe solution c.
The application of the L-cysteine fluorescent probe based on the conjugated polymer/metal ion complex can realize the quantitative detection of L-cysteine in an aqueous solution sample; the compound fluorescent probe has low toxicity and good biocompatibility, and can be used as a fluorescent dye to label and perform fluorescence imaging on L-cysteine in living cells.
The application comprises the following specific steps:
applications ofFirstly, Cys is dissolved in deionized water to prepare the solution with the concentration of 5 × 10-3A solution of M. And (3) gradually adding a Cys solution into the conjugated polymer/metal ion complex fluorescent probe solution c, exciting by using light with the wavelength of 400nm, and measuring the emission spectrum of the solution at the wavelength of 520nm to obtain a linear relation standard curve of the fluorescence intensity of the conjugated polymer and the concentration of Cys. According to the standard curve, the content of Cys in the aqueous solution sample can be quantitatively detected.
And secondly, co-culturing the conjugated polymer/metal ion combined fluorescent probe solution c and Hela cells, exciting by light with the wavelength of 400nm, and realizing the marking and fluorescent imaging of Cys in living cells by using a confocal laser microscope.
The invention has the advantages and beneficial effects that:
1. the raw materials for synthesizing the fluorescent probe are easy to obtain, and the preparation method is simple;
2. the fluorescent probe has good water solubility, high selectivity to Cys and high detection speed, and can be used for quantitative detection of Cys in an aqueous solution sample;
3. the fluorescent probe has low toxicity and good biocompatibility, and can be used for labeling Cys in living cells and fluorescence imaging.
Drawings
FIG. 1 is a schematic diagram of Cys detection by a conjugated polymer/metal ion complex fluorescent probe.
FIG. 2 is a fluorescence spectrum of Cys titrated continuously by a conjugated polymer/metal ion complex fluorescent probe.
FIG. 3 is a linear relationship curve of fluorescence emission intensity of conjugated polymer/metal ion complex fluorescent probe and Cys concentration.
Detailed Description
The invention will be further described with reference to specific embodiments, which will aid in the understanding of the invention. It is not intended that the scope of the invention be limited thereby, but rather that the invention be defined by the claims appended hereto.
Preparation of fluorescent probe based on conjugated polymer/metal ion complex
Example 1:
1.1: synthesis of 3-methoxy-4-methylthiophene
3-bromo-4-methylthiophene (2.5g,14mmol) was dissolved in 3.8mL of NMP, and after complete dissolution, it was added to a three-necked flask containing 20mL of 35% sodium methoxide in methanol, and cuprous bromide (1.5g,10mmol) was added to the reaction, and the reaction was refluxed for 24h under nitrogen protection. Cool to room temperature, filter the solids, spin dry the filtrate, dissolve in dichloromethane, and extract with water. The resulting organic phase was dried over anhydrous magnesium sulfate. Filtering, spin-drying, and purifying the crude product by column chromatography (silica gel and n-hexane) to obtain 1.3g of 3-methoxy-4-methylthiophene with a yield of 72.2%.1HNMR(400MHz,CDCl3):6.74(s,1H),6.08(d,1H),3.74(s,3H),2.01(s,3H)。
1.2: synthesis of 3- (3-bromo) propoxy-4-methylthiophene
3-methoxy-4-methylthiophene (1.5g,11.7mmol), 3-bromo-1-propanol (3.6g,25.9mmol) and NaHSO4(300mg,2.5mmol) was added to 20mL of anhydrous toluene and the reaction refluxed for 10h under nitrogen. Cooled to room temperature, extracted three times with water, and the organic phase dried over anhydrous magnesium sulfate, filtered and spin-dried. The crude product was purified by column chromatography (silica gel, n-hexane) to obtain 2.2g of 3- (3-bromo) propoxy-4-methylthiophene in 80% yield.1H NMR(400MHz,CDCl3):6.90-6.81(m,1H),6.20(d,1H),4.15-4.04(m,2H),3.61(t,2H),2.44–2.26(m,2H),2.10(d,3H)。13CNMR(100MHz,CDCl3):156.1,129.3,120.0,96.5,67.2,32.4,30.4,12.8。
1.3: synthesis of 3- (3-N, N' -diethyl diacetate) propoxy-4-methylthiophene
3- (3-bromo) propoxy-4-methylthiophene (0.95g,4.04mmol), diethyl iminodiacetate (1.09g,5.75mmol), K2CO3(0.80g,5.80mmol) and KI (0.16g,0.98mmol) were added to 30mL of a mixed solution of tetrahydrofuran and acetonitrile (1:1, v/v) and reacted at 85 ℃ for 48 hours under a nitrogen atmosphere. Cooling to room temperature and spin-drying the solvent to obtain the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate: N-hexane ═ 1:6) to give 0.8g of 3- (3-N, N' -diethyl diacetate) propoxy-4-methylthiophene in 57.6% yield.1H NMR(400MHz,CDCl3):6.79(d,1H),6.14(d,1H),4.15(q,4H),4.00(t,2H),3.57(s,4H),2.91(t,2H),2.07(s,3H),1.99–1.89(m,2H),1.25(t,6H)。13C NMR(100MHz,CDCl3):171.2,156.0,129.1,119.9,96.0,67.7,60.6,55.2,51.4,28.0,14.1,12.7。
1.4: synthesis of poly (3- (3-N, N' -diethyl diacetate) propoxy-4-methylthiophene)
3- (3-N, N' -diethyl diacetate) propoxy-4-methylthiophene (800mg,2.32mmol), 15mL chloroform was added to a 50mL three-necked flask, and FeCl was added to 5mL chloroform3(2.15g,13.2mmol) and added dropwise to the mixture and reacted at 50 ℃ for 72h under nitrogen protection. Cool to room temperature and spin dry the solvent to give a brown solid. The brown solid was dissolved in methanol, a small amount of hydrazine hydrate was added dropwise, stirred well, filtered to give a yellowish brown solid, which was washed several times with methanol to give poly (3- (3-N, N' -diethyl diacetate) propoxy-4-methylthiophene) 550mg in 68.7% yield.
1.5: synthesis of poly (3- (3-N, N' -sodium diacetate) propoxy-4-methylthiophene)
Poly (3- (3-N, N' -diethyl diacetate) propoxy-4-methylthiophene) (550mg, 1.60mmol) was placed in 100mL of water dissolved with NaOH (500mg,12.5mmol), heated to 50 ℃ and stirred for 48 h. After cooling to room temperature, the liquid was packed in a dialysis bag of 3500, dialyzed for 3 days, and freeze-dried to obtain 300mg of poly (3- (3-N, N' -sodium diacetate) propoxy-4-methylthiophene) in a yield of 54.5%.
1.6: poly (3- (3-N, N' -sodium diacetate) propoxy-4-methylthiophene)/Cu2+Preparation of Complex fluorescent Probe
Weighing a plurality of poly (3- (3-N, N '-sodium diacetate) propoxy-4-methylthiophene), dissolving the poly (3- (N, N' -sodium diacetate) propoxy-4-methylthiophene) in water to prepare the poly (5 × 10)-5M to which 5 × 10 was gradually added-3CuCl of M2And (5) monitoring the solution by using a fluorescence spectrum until the fluorescence of the system is completely quenched, thus obtaining the fluorescent probe.
Example 2:
2.1: synthesis of 3-methylthio-4-methylthiothiophene:
3-bromo-4-methylthiophene (2.5g,14mmol) was dissolved in 3.8mL NMP and, after complete dissolution, added to a solution containing 20mL of 35% sodium thiomethoxideIn a three-neck flask containing the solution of methyl mercaptan, cuprous bromide (1.5g,10mmol) and butyl lithium (60mg, 9.3mmol) were added to the reaction and the reaction was refluxed for 24h under nitrogen. Cool to room temperature, filter the solids, spin dry the filtrate, dissolve in dichloromethane, and extract with water. The resulting organic phase was dried over anhydrous magnesium sulfate. After filtration and spin-drying, the crude product was purified by column chromatography (silica gel, n-hexane) to obtain 1.05g of 3-methylthio-4-methylthiothiophene in 51.7% yield.1HNMR(400MHz,CDCl3):6.73(s,1H),6.71(d,1H),2.74(s,3H),2.21(s,3H)。
2.2: synthesis of 3- (3-bromo) propylthio-4-methylthiophene
3-methylthio-4-methylthiothiophene (1.05g, 7.3mmol), 3-bromo-1-propanol (3.0g, 21.6mmol) and NaHSO4(300mg,2.5mmol) was added to 20mL of anhydrous toluene and the reaction refluxed for 10h under nitrogen. Cooled to room temperature, extracted three times with water, and the organic phase dried over anhydrous magnesium sulfate, filtered and spin-dried. The crude product was purified by column chromatography (silica gel, n-hexane) to obtain 1.2g of 3- (3-bromo) propylthio-4-methylthiophene in 70% yield.1H NMR(400MHz,CDCl3):6.73(s,1H),6.72(s,1H),3.30(m,2H),2.93(t,2H),2.21(m,2H),2.19(d,3H)。
2.3: synthesis of 3- (3-N, N' -diethyl diacetate) propylthio-4-methylthiophene
3- (3-bromo) propylthio-4-methylthiophene (1.2g,4.78mmol), diethyl iminodiacetate (1.09g,5.75mmol), K2CO3(0.80g,5.80mmol) and KI (0.16g,0.98mmol) were added to 30mL of a mixed solution of tetrahydrofuran and acetonitrile (1:1, v/v) and reacted at 85 ℃ for 48 hours under a nitrogen atmosphere. Cooling to room temperature and spin-drying the solvent to obtain the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate: N-hexane ═ 1:6) to give 1.1g of 3- (3-N, N' -diethyl diacetate) propylthio-4-methylthiophene in 64.7% yield.1H NMR(400MHz,CDCl3):6.73(d,1H),6.72(d,1H),4.12(q,4H),3.35(s,4H),2.93(m,2H),2.36(t,4H),2.21(s,3H),1.79(m,2H),1.30(t,6H)。
2.4: synthesis of poly (3- (3-N, N' -diethyl diacetate) propylthio-4-methylthiophene)
Reacting 3- (3-N, N' -diacetic acid diethyl esterEster) propylthio-4-methylthiophene (900mg,2.50mmol), 15mL of chloroform was added to a 50mL three-necked flask, and FeCl was added to 5mL of chloroform3(2.15g,13.2mmol) and added dropwise to the mixture and reacted at 50 ℃ for 48h under nitrogen protection. Cooling to room temperature, spin-drying the solvent to obtain a brown solid, dissolving with methanol, dropwise adding a small amount of hydrazine hydrate, stirring thoroughly, filtering to obtain a yellowish-brown solid, and washing with methanol several times to obtain 650mg of poly (3- (3-N, N' -diethyl diacetate) propylthio-4-methylthiophene) in a yield of 72.2%.
2.5: synthesis of poly (3- (3-N, N' -sodium diacetate) propylthio-4-methylthiophene)
Poly (3- (3-N, N' -diethyl diacetate) propylthio-4-methylthiophene) (650mg,1.81mmol) was placed in 100mL of water dissolved with NaOH (500mg,12.5mmol), heated to 50 ℃ and stirred for 48 h. After cooling to room temperature, the liquid was packed in a dialysis bag of 3500, dialyzed for 3 days, and freeze-dried to obtain 350mg of poly (3- (3-N, N' -disodium diacetate) propylthio-4-methylthiophene) in a yield of 58.7%.
2.6: poly (3- (3-N, N' -sodium diacetate) propylthio-4-methylthiophene)/Fe2+Preparation of Complex fluorescent Probe
Weighing a plurality of poly (3- (3-N, N '-sodium diacetate) propylthio-4-methylthiophene), dissolving the poly (3- (N, N' -sodium diacetate) propylthio-4-methylthiophene) in water to prepare a solution with the concentration of 5 × 10-5M to which 5 × 10 was gradually added-3Fe (NO) of M3)2And (5) monitoring the solution by using a fluorescence spectrum until the fluorescence of the system is completely quenched, thus obtaining the fluorescent probe.
Example 3:
3.1: synthesis of 3- (3-bromo) propylthiophene
3- (3-thienyl) -1-propanol (1.5g,10mmol) and phosphorus tribromide (2.7g, 10mmol) were added to a three-necked flask containing 30mL of chloroform solution, and the reaction was stirred well under nitrogen for 24 h. Suction filtration is carried out, the filtrate is dried by spinning, and the crude product is purified by column chromatography (silica gel, normal hexane) to obtain 1.5g of 3- (3-bromine) propyl thiophene with the yield of 75.3 percent.1HNMR(400MHz,CDCl3):7.06(d,1H),6.75(d,1H),6.73(s,1H),3.63(m,2H),3.05(m,2H)。
3.2: synthesis of 3- (3-N, N' -diethyl diacetate) propyl thiophene
3- (3-bromo) propylthiophene (1.5g,7.85mmol), diethyl iminodiacetate (1.85g,9.75mmol), K2CO3(0.80g,5.80mmol) and KI (0.16g,0.98mmol) were added to 30mL of a mixed solution of tetrahydrofuran and acetonitrile (1:1, v/v) and reacted at 85 ℃ for 48 hours under a nitrogen atmosphere. Cooling to room temperature and spin-drying the solvent to obtain the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate: N-hexane ═ 1:6) to give 1.7g of 3- (3-N, N' -diethyl diacetate) propylthiophene in 73.9% yield.1HNMR(400MHz,CDCl3):7.06(d,1H),6.75(d,1H),6.74(d,1H),4.12(q,4H),3.32(s,4H),2.55(m,2H),2.36(t,4H),1.62(m,2H),1.39(m,2H),1.30(t,6H)。
3.3: synthesis of poly (3- (3-N, N' -diethyl diacetate) propylthiophene)
3- (3-N, N' -diethyl diacetate) propylthiophene (1.5g,4.58mmol), 20mL chloroform was added to a 50mL three-necked flask and FeCl was added to 5mL chloroform3(3.15g,19.3mmol) and added dropwise to the mixture and reacted at 50 ℃ for 48h under nitrogen protection. Cooling to room temperature, spin-drying the solvent to obtain a brown solid, dissolving with methanol, adding a small amount of hydrazine hydrate dropwise, stirring thoroughly, filtering to obtain a yellowish-brown solid, and washing with methanol several times to obtain 850mg of poly (3- (3-N, N' -diethyl diacetate) propylthiophene) with a yield of 60.7%.
3.4: synthesis of poly (3- (3-N, N' -sodium diacetate) propylthiophene)
Poly (3- (3-N, N' -diethyl diacetate) propylthiophene) (850mg,2.60mmol) was placed in 100mL of water dissolved with NaOH (500mg,12.5mmol), heated to 50 ℃ and stirred for 48 h. After cooling to room temperature, the liquid was packed in a dialysis bag of 3500, dialyzed for 3 days, and freeze-dried to obtain 450mg of poly (3- (3-N, N' -sodium diacetate) propylthiophene) in 54.9% yield.
3.5: poly (3- (3-N, N' -sodium diacetate) propylthiophene)/Ag+Preparation of Complex fluorescent Probe
Weighing a plurality of poly (3- (3-N, N '-sodium diacetate) propyl thiophene), dissolving the poly (3- (N, N' -sodium diacetate) propyl thiophene) in water to prepare the poly (5 × 10)-5M to which 5 × 10 was gradually added-3AgNO of M3Solution, monitoring by fluorescence spectroscopy untilThe fluorescence of the system is completely quenched, and the fluorescent probe is obtained.
Application of fluorescent probe based on conjugated polymer/metal ion complex
Example 4
The method comprises placing a certain volume of conjugated polymer/metal ion complex solution (such as example 1.6, example 2.6 or example 3.5) in a quartz cuvette, dissolving Cys in water to obtain a solution with a concentration of 5 × 10-3A solution of M. The Cys solution was gradually added to the fluorescent probe solution, mixed well, and then excited with light of 400nm wavelength, and the emission spectrum of the solution at 520nm wavelength was measured, as shown in FIG. 2. And taking the concentration of Cys as an abscissa and the corresponding fluorescence intensity at 520nm as an ordinate, and performing linear fitting to obtain a linear relation standard curve of the fluorescence intensity of the conjugated polymer and the concentration of Cys, as shown in FIG. 3. According to the standard curve, the content of Cys in the aqueous solution can be quantitatively detected.
Example 5
An application of the conjugated polymer/metal ion complex-based fluorescent probe. The method comprises the following steps: a certain volume of conjugated polymer/metal ion complex solution (as in example 1.6, example 2.6 or example 3.5) was co-cultured with Hela cells, excited by light with a wavelength of 400nm, and labeled with Cys in living cells and imaged by fluorescence using a confocal laser microscope.
Preparation of standard working curve:
2.5mL of the conjugated polymer/metal ion complex fluorescent probe solution prepared in 1.6 of example 1 was placed in a quartz cuvette, Cys was gradually added thereto and mixed uniformly, and the fluorescence spectrum was measured by excitation with an excitation wavelength of 400nm, as shown in FIG. 2. The change in fluorescence emission intensity at 520nm was then plotted against the concentration of Cys to obtain a standard working curve, as shown in FIG. 3.
FIG. 2 is a fluorescence spectrum of Cys serially titrated by a conjugated polymer/metal ion complex fluorescent probe. The figure shows that: under the excitation of light with the wavelength of 400nm, the fluorescence intensity of the conjugated polymer/metal ion compound is weak, and with the addition of Cys, the fluorescence intensity at 520nm is gradually enhanced.
FIG. 3 is a linear relationship curve of fluorescence emission intensity of conjugated polymer/metal ion complex fluorescent probe and Cys concentration. The figure shows that: the change in fluorescence emission intensity at 520nm showed a good linear relationship with the concentration of Cys.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various simple derivations or modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Such deductions or improvements are intended to fall within the scope of the claims appended to the present application.

Claims (5)

1. An L-cysteine fluorescent probe based on a conjugated polymer/metal ion complex, which is characterized in that: the fluorescent probe is a compound consisting of a conjugated polymer and metal ions, wherein the conjugated polymer is a polythiophene derivative with a side chain containing double carboxylate; the detection principle of the compound fluorescent probe is as follows: the water solution of the conjugated polymer PT emits strong fluorescence under the excitation of light with the wavelength of 400nm when the PT and the metal ion Mn+After complex formation, the fluorescence of PT is quenched; when L-cysteine (Cys) is added into the compound fluorescent probe water solution, the Cys and M are combinedn+Can form more stable Cys-M through M-S bondn+Complexing to get PT from PT/Mn+Releasing the complex, and recovering the fluorescence of the system;
the structural formula of the polythiophene derivative with the side chain containing the double carboxylate is as follows:
Figure FDA0002579166710000011
wherein R is hydrogen, methyl, ethyl, propyl or a saturated alkyl chain; x is a carbon, oxygen, sulfur, selenium or tellurium atom; m is positive univalent ion of lithium, sodium, potassium, silver or ammonium;
the metal ions are as follows: cu2+，Fe2+，Fe3+，Co2+，Pd2+，Eu3+，Ni2+，Hg2+，Ag+，Ca2+，Cd2+Or Zn2+Ions that are paramagnetic and capable of binding to the bis-carboxylate groups on the polythiophene side chains form a conjugated polymer/metal ion complex, quenching the fluorescence of the conjugated polymer.
2. The method for preparing the conjugated polymer/metal ion complex-based L-cysteine fluorescent probe of claim 1, comprising the following steps:
step 1 preparation of conjugated Polymer-Polythiophene derivative having Bicarboxylate in the side chain
3-bromo-thiophene with hydrogen, methyl, ethyl, propyl or saturated alkyl chain at the 4-position is taken as an initial raw material, and a side chain functionalized monomer is obtained through etherification reaction and nucleophilic substitution in sequence; fully dissolving the functionalized monomer in chloroform, and adding FeCl with 5 times of molar equivalent3Oxidizing an oxidant, carrying out oxidation polymerization reaction for 48 hours at 50 ℃ under the protection of nitrogen, stopping the reaction, cooling to room temperature, carrying out rotary evaporation to remove a solvent, dissolving the obtained solid in methanol, adding a few drops of hydrazine hydrate, stirring for reaction overnight, then carrying out suction filtration, and carrying out rotary drying on the filtrate to obtain a crude product; performing alkaline hydrolysis on the obtained crude product, transferring into a dialysis bag with molecular weight cutoff of 3500, and dialyzing for 3 days; then, freeze drying to obtain polymer solid powder, namely, the polythiophene derivative with the side chain containing the double carboxylate;
step 2, preparation of fluorescent probe based on conjugated polymer/metal ion complex
Step 2.1 dissolving the conjugated polymer obtained in step 1 in water to give a solution having a concentration of 5 × 10-5A solution of M, referred to as solution a;
step 2.2, dissolving the metal chloride or the metal nitrate in water to prepare the solution with the concentration of 5 × 10-3A solution of M, referred to as solution b;
and 2.3, gradually adding the metal ion solution b obtained in the step 2.2 into the conjugated polymer solution a until the fluorescence of the conjugated polymer is completely quenched to obtain a conjugated polymer/metal ion compound fluorescent probe solution, namely a solution c.
3. The use of the conjugated polymer/metal ion complex-based L-cysteine fluorescent probe of claim 1, wherein: the method can be used for quantitative detection of the content of L-cysteine in an aqueous solution sample; the compound fluorescent probe has low toxicity and good biocompatibility, and can be used for labeling L-cysteine in living cells and fluorescence imaging.
4. The use of claim 3, wherein the L-cysteine (Cys) is dissolved in water and formulated at a concentration of 5 × 10-3A solution of M; gradually adding the Cys solution into the conjugated polymer/metal ion compound fluorescent probe solution, exciting with light with the wavelength of 400nm, and measuring the emission spectrum of the solution at the wavelength of 520nm to obtain a linear relation curve of the fluorescence intensity of the conjugated polymer and the concentration of Cys, namely a standard curve; according to the standard curve, the content of Cys in the aqueous solution sample can be quantitatively detected.
5. Use according to claim 3, characterized in that: and co-culturing the conjugated polymer/metal ion compound fluorescent probe solution and living cells, exciting by light with the wavelength of 400nm, and labeling and carrying out fluorescence imaging on Cys in the living cells by using a confocal laser microscope.
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