WO2021088529A1 - 基于偶氮苯-量子点的荧光探针及制备方法以及其在分子开关型荧光传感器中的应用 - Google Patents

基于偶氮苯-量子点的荧光探针及制备方法以及其在分子开关型荧光传感器中的应用 Download PDF

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WO2021088529A1
WO2021088529A1 PCT/CN2020/116110 CN2020116110W WO2021088529A1 WO 2021088529 A1 WO2021088529 A1 WO 2021088529A1 CN 2020116110 W CN2020116110 W CN 2020116110W WO 2021088529 A1 WO2021088529 A1 WO 2021088529A1
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solution
azobenzene
fluorescent probe
quantum dots
concentration
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李楠
薛巍
査勇超
陈浩凌
张美莹
海戎
牟宗霞
周平
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暨南大学
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

Definitions

  • the invention belongs to the fields of chemical molecular synthesis, fluorescent probe design and biochemical detection, and particularly relates to a fluorescent probe based on azobenzene-quantum dots and a preparation method and its application in a molecular switch type fluorescent sensor.
  • Fluorescence sensing is a sensitive, fast and reliable analysis method.
  • the chemical information of the interaction between molecules is transformed into the influence on the fluorescence signal (such as fluorescence enhancement or quenching, the movement of the fluorescence characteristic peak position, the change of fluorescence lifetime and Changes in fluorescence polarization, etc.), qualitative or quantitative analysis of substances. Because of its high sensitivity, low detection limit, simple operation and low equipment, it has become a research hotspot in the field of chemical and biological analysis.
  • a fluorescent sensing probe which can be a molecule, an aggregate, a coordination polymer, a nanoparticle, and so on.
  • Semiconductor quantum dots have the advantages of adjustable luminescence wavelength, high fluorescence quantum yield, difficult photobleaching, and good chemical stability. They are a new class of excellent fluorescent sensing probes. , Optics, and life sciences have important application prospects, and their preparation and application have received extensive attention.
  • the fluorescence quantum yield (fluorescence intensity) of QDs is affected by photo-induced electron transfer (PET) when the electron-deficient or electron-rich compound acts on it. Photo-induced electron transfer (PET) is an important photophysical process.
  • a series of switched fluorescent sensors with different response behaviors to the analyte can be designed and prepared .
  • a compound with the opposite electrical properties is selected to promote the occurrence of PET.
  • the electron-deficient groups are modified on the surface of QDs, and PET occurs, which causes the quenching of fluorescence intensity.
  • the analyte is used to remove the fluorescence quenching groups on the surface of QDs. Cutting off the path of PET generation can restore the fluorescence of QDs. Changes in "off-on" reflect the analyte.
  • switch-type fluorescence sensors can be divided into “off-on” (off-on) and “on-off” (on-off) types. Off) two types. Compared with the general fluorescence sensor, the switch type fluorescence sensor has the characteristics of fast response and convenient use. In recent years, it has become a hot field of optical sensor research.
  • the primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing a fluorescent probe based on azobenzene-quantum dots.
  • Another object of the present invention is to provide a fluorescent probe based on azobenzene-quantum dots prepared by the method.
  • Another object of the present invention is to provide the application of the azobenzene-quantum dot-based fluorescent probe in a molecular switch type fluorescent sensor.
  • Another object of the present invention is to provide a method for detecting biochemical analytes by using the switch type fluorescence sensor.
  • the objective of the present invention is achieved by the following technical solution: a method for preparing a fluorescent probe based on azobenzene-quantum dots, including the following steps:
  • the quantum dots described in step (A) are CdSe/ZnS quantum dots (CdSe/ZnS are core/shell respectively); preferably purified CdSe/ZnS quantum dots; more preferably acetone (a large amount of quantum dots are added to the aqueous solution of quantum dots). Acetone) CdSe/ZnS quantum dots after purification.
  • the molar ratio of quantum dots to ethylenediamine in step (A) is preferably 1:100.
  • the concentration of the ethylenediamine solution in step (A) is 0.005-0.2 mol/L; preferably 0.01 mol/L.
  • the stirring time in step (A) is 10 to 45 minutes; preferably 30 minutes.
  • the molar ratio of 4,4'-dithiodibenzylamine, hydrochloric acid (hydrochloric acid in hydrochloric acid solution) and sodium nitrite (sodium nitrite in aqueous sodium nitrite solution) described in step (1) is 1 to 5: 5-15:0.01-0.05; preferably 2.3:10:0.01.
  • the concentration of the hydrochloric acid solution in step (1) is 0.1-1 mol/L; preferably 0.5 mol/L.
  • the concentration of the sodium nitrite aqueous solution in step (1) is 1-10 mol/L; preferably 5 mol/L.
  • the time for the stirring reaction in step (1) is 10 to 45 minutes; preferably 30 minutes.
  • the molar ratio of m-phenylenediamine to sodium hydroxide in step (2) is 2-15:2-20; preferably 3:5.
  • the concentration of the sodium hydroxide solution in step (2) is 0.5-1 mol/L; preferably 1 mol/L.
  • the molar ratio of the m-phenylenediamine to the 4,4'-dithiodibenzylamine in step (2) is 6:2.3.
  • the pH value described in step (3) is preferably 10.
  • step (3) the adjusted pH value is adjusted with NaOH solution; preferably, 1 mol/L NaOH solution is used for adjustment.
  • the stirring reaction described in step (3) is realized by the following method: first stirring under ice bath conditions for 10 to 45 minutes (preferably 30 minutes), and then stirring at room temperature for 5 to 2 hours (preferably 1 hour).
  • the washing with water in step (3) is washing with water 3 times or more.
  • the drying described in step (3) is preferably vacuum drying.
  • the molar ratio of the azobenzene compound to the quantum dots in step (B) is 200-1000:1.
  • the volume ratio of H 2 O and DMSO in the H 2 O/DMSO solution described in step (B) is 80:20.
  • step (B) The amount of 3-mercaptopropionic acid described in step (B) is calculated based on the ratio of 0.02-0.05 mol 3-mercaptopropionic acid per liter of H 2 O/DMSO solution.
  • the stirring time in step (B) is 10 to 45 minutes; preferably 30 minutes.
  • the molar ratio of 3-mercaptopropionic acid to azobenzene compound in step (B) is 7:3-9:1.
  • the reaction time in step (C) is 0.5 to 2 hours; preferably 1 hour.
  • a fluorescent probe based on azobenzene-quantum dots is prepared by the method described in any one of the above.
  • a switch type fluorescence sensor prepared by the following method:
  • the concentration of the PBS buffer in step (a) is 10-100 mmol/L; preferably 50 mmol/L.
  • an azo reductase such as sodium dithionite solution, Potassium dithionite aqueous solution, zinc dithionite aqueous solution, manganese dithionite aqueous solution,
  • the azo reductase aqueous solution is preferably an azo reductase aqueous solution containing NADH; the concentration of NADH in the azo reductase aqueous solution containing NADH is 0.01-1 ⁇ mol/L (preferably 0.1 ⁇ mol/L); The concentration of nitrogen reductase is 0.02-2 ⁇ g/mL (preferably 0.02-1.2 ⁇ g/mL).
  • the molar ratio of the NADH to the fluorescent probe is 1:100.
  • the concentration of the dithionite ion in step (b) is 0.01-1 ⁇ mol/L; preferably 0.1 ⁇ mol/L.
  • the concentration of hypochlorite ion in step (b) is 0.05-0.5 ⁇ mol/L; preferably 0.15 ⁇ mol/L.
  • the concentration of the azo reductase in step (b) is 0.02-2 ⁇ g/mL; preferably 0.02-1.2 ⁇ g/mL.
  • the molar ratio of the dithionite ion to the fluorescent probe in step (b) is 10-1000:1; preferably 100:1.
  • the molar ratio of the hypochlorite ion to the fluorescent probe in step (b) is 50-500:1; preferably 150:1.
  • the molar ratio of the azo reductase to the fluorescent probe in step (b) is 10 to 200:1; preferably 50:1.
  • a method for detecting biochemical analytes using the switch type fluorescence sensor includes the following steps:
  • step (ii) According to the fluorescence spectrum intensity obtained in step (i) and the concentration of the biochemical analyte, a working curve is produced to obtain a linear equation;
  • step (iii) Mix the sample to be tested with the azobenzene-quantum dot fluorescent probe and incubate, then measure the FL spectrum to obtain the fluorescence spectrum intensity, and then obtain the content of the sample to be tested according to the linear equation obtained in step (ii);
  • the incubation time of the azobenzene-quantum dot fluorescent probe solution described in steps (i) and (iii) with the dithionite ion is more than 5 minutes; the azobenzene-quantum dot fluorescent probe solution The incubation time with hypochlorite ions is more than 8 minutes; the incubation time of the fluorescent probe solution of azobenzene-quantum dots and azo reductase is more than 15 minutes.
  • an azo reductase such as sodium dithionite solution, Potassium dithionite aqueous solution, zinc dithionite aqueous solution, manganese dithionite aqueous solution, calcium dithi
  • the azo reductase aqueous solution is preferably an aqueous solution containing NADH-containing azo reductase; the NADH concentration in the NADH-containing azo reductase aqueous solution is 0.01-1 ⁇ mol/L; the concentration of azo reductase is 0.0.2 ⁇ 1.2 ⁇ g/mL.
  • the concentration of the aqueous solution of dithionite ions in step (i) is 1 to 80 nmol/L.
  • the concentration of the aqueous hypochlorite ion solution in step (i) is 4 to 150 nmol/L.
  • the concentration of the azo reductase in step (i) is 0.02-1.2 ⁇ g/mL.
  • a novel azobenzene compound is prepared, and based on this, an azobenzene-QDs composite fluorescent probe is developed, which can be used to design and prepare an off-on type fluorescent sensor.
  • DTPABDA azobenzene compound
  • DTPABDA carries an electron-withdrawing azophenyl group, which can be quenched by the photo-induced electron transfer (PET) effect
  • PET photo-induced electron transfer
  • the azobenzene-QDs fluorescent probe In order to verify the application effect of the azobenzene-QDs fluorescent probe, we removed the azophenyl group through a series of different reactions, including 1) the reduction reaction of dithionite ion; 2) the oxidation reaction of hypochlorite ion; 3 ) Enzymatic reaction of azo reductase. By monitoring the recovery changes of the fluorescence signal of quantum dots quenched by azobenzene in the above reaction, it is proved that the azobenzene-QDs fluorescent probe can be used to develop switches for detecting dithionite ions, hypochlorite ions and azo reductase. Type fluorescent sensor.
  • this sensor Compared with reported fluorescent sensors using organic dyes as probes, this sensor exhibits short response time, high sensitivity, and low detection limit. It can be widely used in monitoring industrial hydrosulfite and biomedical diagnostics. Endogenous hypochlorous acid in cells and hypoxia in tumors.
  • the present invention has the following advantages and effects:
  • a compound containing azobenzene-bis[4,4'-(dithiophenylazo-1,3-phenylenediamine] (Bis[4,4'-(dithiophenyl azo)-1,3-benzenediamine], DTPABDA), which can be modified on the surface of QDs through a ligand exchange reaction.
  • the electron-withdrawing functional group on the DTPABDA molecule, namely the azophenyl group can be photo-induced electron transfer effect (photo-induced electron transfer). Electron transfer, PET), which quenches the fluorescence of QDs.
  • the analytical sensing method proposed by the present invention exhibits the advantages of high sensitivity, wide linear range and good selectivity, and the detection limit reaches 0.5 mmol/L of dithionite ion.
  • Chlorate ion is 0.2nmol/L
  • azo reductase is 2ng/mL.
  • This new sensing method can be widely used in monitoring dithionite in the industry, endogenous hypochlorous acid in living cells in biomedical diagnosis, and hypoxia in tumors. It is used in biology, chemistry, chemical industry, food safety, The fields of environmental testing and biomedical diagnosis have important potential application value.
  • Figure 1 is the synthesis pathway of DTPABDA and the reaction pathway of DTPABDA with S 2 O 4 2- , ClO - and azo reductase; among them, a is the synthesis pathway of DTPABDA, b, c and d are DTPABDA and S 2 O, respectively 4 2- , The reaction pathway of ClO-and azo reductase.
  • Figure 2 is the solution after incubation of DTPABDA and 1mM Na 2 S 2 O 4 and the corresponding UV absorption spectrum; among them, a is the solution of DTPABDA and the solution after incubation of DTPABDA and 1mM Na 2 S 2 O 4 (I in the figure is DTPABDA solution; II is the solution after incubation of DTPABDA and 1mM Na 2 S 2 O 4 ); b is the ultraviolet absorption spectrum.
  • Figure 3 is the solution of DTPABDA incubated with 1mM NaClO and the corresponding UV absorption spectrum; where a is the solution of DTPABDA and the solution after incubation of DTPABDA with 1mM NaClO (in the figure: I is the DTPABDA solution; II is the DTPABDA and 1mM NaClO Solution after incubation); b is the ultraviolet absorption spectrum.
  • Figure 4 is the solution of DTPABDA incubated with 5 ⁇ g/mL azoreductase and the corresponding UV absorption spectrum; where a is the DTPABDA solution and the solution of DTPABDA incubated with 5 ⁇ g/mL azoreductase (Figure: I Is the DTPABDA solution; II is the solution after incubating DTPABDA with 5 ⁇ g/mL azoreductase); b is the ultraviolet absorption spectrum.
  • Figure 5 is the time-dependent UV absorption spectrum of DTPABDA in the presence of 1mM Na 2 S 2 O 4 and the time-dependent change in absorbance of the sample at a wavelength of 458 nm; where a is the time of DTPABDA in the presence of 1 mM Na 2 S 2 O 4 Relative UV absorption spectrum; b is the time-dependent change in absorbance when the sample is at a wavelength of 458nm.
  • Figure 6 is the time-dependent UV absorption spectrum of DTPABDA in the presence of 1mM NaClO, and the time-dependent absorbance change diagram of the sample at a wavelength of 458nm; where a is the time-dependent UV absorption spectrum of DTPABDA in the presence of 1mM NaClO; b is the sample at the wavelength At 458nm, the time-dependent change in absorbance.
  • Figure 7 is the time-dependent UV absorption spectrum of DTPABDA in the presence of 5 ⁇ g/mL azoreductase, and the time-dependent absorbance change of the sample at a wavelength of 458nm; where a is the time-correlation of DTPABDA in the presence of 5 ⁇ g/mL azoreductase Ultraviolet absorption spectrum; b is the time-dependent change in absorbance when the sample is at a wavelength of 458nm.
  • Figure 8 is a schematic diagram of the thiol-disulfide exchange reaction between MPA and DTPABDA, and a schematic diagram of the ligand exchange mediated by EDA on the QD surface using MPA and MPABDA as ligands; where a is the thiol of MPA and DTPABDA- Schematic diagram of the disulfide exchange reaction; b is a schematic diagram of EDA-mediated ligand exchange on the QD surface using MPA and MPABDA as ligands.
  • Figure 9 is a photograph of QDs prepared by EDA-mediated ligand exchange method under UV irradiation with different ratios of MPA/DTPABDA (molar ratios are 100/0 (I), 90/10 (II), 80/ 20(III), 70/30(IV)).
  • Figure 10 is the FL spectrum of the QDs sample in Figure 9 at 560 nm.
  • FIG. 11 is a graph of FL intensity at 560 nm of the QDs sample in FIG. 9.
  • Figure 12 is an XPS measurement spectrum of QDs prepared with MPA/DTPABDA in a ratio of 70/30 as ligands.
  • Figure 13 is the nuclear energy level spectrum of QDs N 1s (the solid line is the data fitting).
  • a is a schematic diagram of removing the quencher
  • b is a schematic diagram of fluorescence recovery by removing the quencher.
  • Figure 15 is a time-dependent FL change diagram of QDs in the presence of 100 nM Na 2 S 2 O 4.
  • Figure 16 is a graph showing changes in FL intensity with reaction time in the presence of 100 nM Na 2 S 2 O 4.
  • Figure 17 is the fluorescence spectra of QDs after incubation with different concentrations of Na 2 S 2 O 4.
  • Figure 18 is a comparison diagram of the fluorescence intensity of Na 2 S 2 O 4 at different concentrations at 560 nm.
  • Figure 19 is a fluorescence response graph of 0.1 ⁇ M Na 2 S 2 O 4 and 2 ⁇ M other sulfur-containing compounds.
  • Figure 20 is the time-dependent FL change diagram of QDs in the presence of 150 nM NaClO.
  • Figure 21 is a graph showing changes in FL intensity with reaction time in the presence of 150 nM NaClO.
  • Figure 22 shows the FL spectra of QDs after incubation with different concentrations of NaClO.
  • Figure 23 is a comparison diagram of FL intensity at 560 nm of different concentrations of NaClO.
  • Figure 24 is the result of FL reaction of 0.15 ⁇ M NaClO and 2 ⁇ M other ROS/RNS.
  • Figure 25 is the time-dependent FL change diagram of QDs in the presence of 1.2 ⁇ g/mL azo reductase and 0.1 ⁇ M NADH.
  • Figure 26 is a graph showing changes in FL intensity with reaction time in the presence of 1.2 ⁇ g/mL azo reductase and 0.1 ⁇ M NADH.
  • Figure 27 shows the FL spectra of QDs after incubation with different concentrations of azoreductase.
  • Figure 28 is a comparison diagram of FL intensity at 560 nm of different concentrations of azo reductase.
  • the present invention will be further described in detail below in conjunction with examples, but the implementation of the present invention is not limited thereto.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the test methods that do not indicate specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions recommended by the manufacturer.
  • the reagents and raw materials used in the present invention can be obtained from commercial sources.
  • This embodiment provides a method for synthesizing DTPABDA.
  • DTPABDA bis[4,4'-(dithiophenylazo)-1,3-phenylenediamine](Bis[4,4'- (dithiophenyl azo)-1, 3-benzenediamine]).
  • 1 H NMR spectrum 600MHz, DMSO-d6, room temperature: ⁇ 7.56 (d, 2H), 7.30 (d, 1H), 6.74 (d, 2H), 6.60 (s, 2H) ).
  • DTPABDA is synthesized by azo coupling reaction,
  • Figure 1a is a schematic diagram of the synthesis of DTPABDA. The structural formula of DTPABDA is shown as follows:
  • This embodiment provides a method for DTPABDA to respond to multiple substances.
  • DMSO dimethyl sulfoxide
  • the reaction between DTPABDA and azo reductase is as follows: Mix 100 ⁇ L 0.1mmol/L DTPABDA aqueous solution and 100 ⁇ L 5mmol/L nicotinamide adenine dinucleotide (NADH) aqueous solution in 1mL DMSO/PBS buffer (volume ratio 20/80) Mix in the liquid, and then add 10 ⁇ L of 500 ⁇ g/mL azo reductase (purchased from Sigma Orange (China) Company) aqueous solution, mix well and measure the corresponding UV absorption spectrum.
  • NADH nicotinamide adenine dinucleotide
  • DTPABDA can react with three types of substances including: 1) S 2 O 4 2- with reducing properties; 2) ClO- with oxidizing properties; 3) Azo reductase.
  • Figures 1b to 1d show the reaction path diagrams of DTPABDA with the above three types of substances. These substances can specifically break the azo bond in the DTPABDA molecule through chemical or enzymatic reactions.
  • This embodiment provides a method for synthesizing DTPABDA-modified CdSe/ZnS QDs.
  • DTPABDA and MPA were used as ligands to prepare DTPABDA-modified CdSe/ZnS QDs.
  • MPA has two important functions: 1) The thiol group of MPA can cleave the disulfide bond of DTPABDA into 4-mercaptophenylazo-1,3-phenylenediamine (MPABDA) with thiol group ( Figure 8a) ), its binding ability to the QD surface is higher than the disulfide bond; 2) Unreacted MPA and MPABDA are adsorbed on the surface of QDs as stabilizers, and the electrostatic repulsion of negatively charged MPA at physiological pH can significantly improve the stability of the resulting QDs Sex ( Figure 8b).
  • Figure 9 is a picture of QDs aqueous solutions prepared with different ratios of MPA/DTPABDA under UV irradiation, the ratios are 100/0(I), 90/10(II), 80/20(III), 70/30(IV) .
  • QDs cover 100% MPA, it appears green; when DTPABDA/MPA mixed ligand covers the surface of QDs, the brightness decreases.
  • Figure 10 shows the corresponding fluorescence spectra of QDs.
  • the QDs covered by MPA showed a strong FL emission peak at 560nm.
  • the DTPABDA/MPA mixed ligand covers the surface of QDs, the peak does not show any shift, but the intensity is reduced.
  • the FL intensity of QDs was reduced to 18.7% compared with MPA-modified QDs (Figure 11).
  • the XPS data in Figure 12 represents the elements Cd, Se, Zn, and S, indicating the presence of QDs.
  • the present embodiment provides a S 2 O 4 2- “on” sensor, ClO - “on” and Azoreductase sensor “on” mode detecting sensor.
  • FL sensing for S 2 O 4 2- FL intensity gradually increased and reached equilibrium after 5 minutes, indicating a response time of 5 minutes. After incubating with different concentrations of Na 2 S 2 O 4 for 5 minutes (concentrations are 0, 2, 4, 8, 10, 20, 40, 60, 80, 100, 150 nmol/L), the intensity of QDs FL at 560 nm varies with It increases as the concentration of S 2 O 4 2- increases ( Figure 17). There is a good linear relationship between 1 and 80nmol/L, and the calculated limit of detection (LOD) is 0.05nmol/L ( Figure 18).
  • FIG 24 is a 2uL 1mmol ROS / RNS (OH ⁇ , O 2 -, 1 O 2, H 2 O 2, NO, ONOO -1) and in response FL 10uL 10 ⁇ mol / L QDs solution was incubated in 1mL of buffer solution, which are ClO - the main interference in sensing. There was no obvious change in FL intensity after incubation with these substances, indicating the excellent selectivity of the sensor to ClO -.
  • azo reductase is a feature of various diseases, including cancer, heart disease, ischemia, and vascular disease.
  • Azoreductase is a physiological hypoxia marker, which can be used to detect the degree of hypoxia in tumors. Based on "turning on” the FL sensing mechanism, azoreductase can reduce the azo group from NADH to amine through an enzyme-catalyzed reaction, thereby removing the azo group, thereby turning on the FL of the DTPABDA-modified QDs.
  • Figures 25 and 26 show that in the presence of 1.2 ⁇ g/mL azo reductase and 0.1 ⁇ mol/L NADH, the FL intensity of QDs gradually increases and reaches a plateau within 15 minutes, that is, the response time is 15 minutes.
  • azo reductase with different concentrations (0, 0.02, 0.04, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.5, 2 ⁇ g/mL) with DTPABDA-modified QDs for 15 minutes.
  • the FL intensity gradually increases ( Figures 27 and 28), and there is a good linear relationship between 0.02 and 1.2 ⁇ g/mL.
  • the calculated LOD is 2ng/mL.

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Abstract

一种基于偶氮苯-量子点的荧光探针及制备方法以及其在分子开关型荧光传感器中的应用。该荧光探针的制备方法包括如下步骤：(A)将量子点溶于CHCl 3溶液中，然后加入乙二胺溶液混合均匀，得到混合溶液II；(B)将偶氮苯化合物与3-巯基丙酸加入到H 2O/DMSO溶液中混合均匀，得到混合溶液III；(C)将混合溶液II和III混合后进行反应，获得所述的荧光探针。基于该荧光探针与连二亚硫酸离子引起的还原反应、与次氯酸离子引起的氧化反应以及与偶氮还原酶引起的酶促反应，开发了分子开关型荧光传感器，能够灵敏准确地检测连二亚硫酸离子、次氯酸离子和偶氮还原酶，可用于检测多种化学生物分析物。

Description

基于偶氮苯-量子点的荧光探针及制备方法以及其在分子开关型荧光传感器中的应用 技术领域
本发明属于化学分子合成、荧光探针设计和生物化学检测领域，特别涉及一种基于偶氮苯-量子点的荧光探针及制备方法以及其在分子开关型荧光传感器中的应用。
背景技术
荧光传感是一种灵敏、快速且可靠的分析手段，根据分子间相互作用的化学信息转变为对荧光信号的影响(如荧光增强或者猝灭、荧光特征峰位置的移动、荧光寿命的变更和荧光偏振的改变等)，对物质进行定性或定量分析。因其具有灵敏度高、检测限低、操作简便、设备低廉等特点而成为近期化学和生物分析领域的研究热点。
荧光传感中执行这种转变过程的物质被称为荧光传感探针，其可以是分子，也可以是聚集体、配位聚合物、纳米粒子等。半导体量子点(semiconductor quantum dots，QDs)由于具有发光波长可调，荧光量子产率高，不易光漂白和化学稳定性好等优点，是新兴的一类优良荧光传感探针，在纳米电子学、光学，和生命科学等方面有着重要的应用前景，其制备和应用受到人们广泛重视。QDs作为荧光探针，其荧光量子产率(荧光强度)受缺电子性或富电子性较强化合物作用时产生光致电子转移(photo-induced electron transfer，PET)的影响。光诱导电子转移(PET)是一种重要的光物理过程，基于荧光发射强度对PET过程的依赖性以及对PET过程的调控，可以设计制备一系列对分析物具有不同响应行为的开关型荧光传感器。例如，在设计时根据荧光基团的电性，选择与之电性相反的化合物作用以促进PET发生。在QDs表面上修饰上缺电子性基团，发生PET从而引起荧光强度的淬灭，利用分析物作用去除QDs表面上的荧光淬灭基团，切断PET发生途径能够使得QDs的荧光恢复，通过荧光“关-开”(off-on)的变化反应分析物。依据以有荧光(开)还是以无荧光(关)方式显示被检测物质的存在,可将开关型荧光传感器分为“关-开”(off-on)型和“开-关”(on-off)型两大类。相比于一般的荧光传感器，开关型荧光传感器具有响应快速、使用方便等特点，近年来其已经成为光学传感器研究的一个热门领域。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种基于偶氮苯-量子点的荧光探针的制备方法。
本发明的另一目的在于提供所述方法制备得到的基于偶氮苯-量子点的荧光探针。
本发明的又一目的在于提供所述基于偶氮苯-量子点的荧光探针在分子开关型荧光传感器中的应用。
本发明的再一目的在于提供一种利用所述的开关型荧光传感器检测生化分析物的方法。
本发明的目的通过下述技术方案实现：一种基于偶氮苯-量子点的荧光探针的制备方法，包括如下步骤：
(A)将量子点溶于CHCl 3溶液中，然后加入乙二胺(EDA)溶液，搅拌混合均匀，得到混合溶液II；
(B)将偶氮苯化合物与3-巯基丙酸(MPA)加入到H 2O/DMSO溶液中，搅拌混合均匀，得到混合溶液III；其中，偶氮苯化合物为双[4,4'-(二硫代苯基偶氮)-1,3-苯二胺](Bis[4,4’-(dithiophenyl azo)-1,3-benzenediamine，DTPABDA]；
(C)将混合溶液III加入到混合溶液II中，剧烈震荡反应，待反应结束后收集上层水溶液，并加入丙酮沉淀离心，得到DTPABDA修饰的量子点，即所述基于偶氮苯-量子点的荧光探针。
步骤(A)中所述的量子点为CdSe/ZnS量子点(CdSe/ZnS分别为核/壳)；优选为纯化后的CdSe/ZnS量子点；更优选为采用丙酮(量子点水溶液中加入大量丙酮)纯化后的CdSe/ZnS量子点。
步骤(A)中所述的量子点与乙二胺的摩尔比优选为1:100。
步骤(A)中所述的乙二胺溶液的浓度为0.005～0.2mol/L；优选为0.01mol/L。
步骤(A)中所述的搅拌的时间为10～45分钟；优选为30分钟。
步骤(B)中所述的偶氮苯化合物的结构式如式I所示：
Figure PCTCN2020116110-appb-000001
步骤(B)中所述的双[4,4'-(二硫代苯基偶氮)-1,3-苯二胺](Bis[4,4’-(dithiophenylazo)-1,3-benzenediamine，DTPABDA]优选为通过如下方法制备得到：
(1)将4,4'-二硫代二苄胺(4,4’-dithiodibenzylamine，DTDBA)加入到盐酸溶液中，冰浴冷却后，逐滴加入亚硝酸钠水溶液，在冰浴条件下搅拌反应，得到4,4'-二硫代二苯基重氮；
(2)将间苯二胺溶解于氢氧化钠溶液中，冰水冷却，得到混合溶液I；
(3)将4,4'-二硫代二苯基重氮加入到混合溶液I中，并调节pH值至9～11，冰浴和/或常温条件下搅拌反应，收集沉淀物，水洗，干燥，得到所述的偶氮苯化合物(DTPABDA)。
步骤(1)中所述的4,4'-二硫代二苄胺、盐酸(盐酸溶液中盐酸)和亚硝酸钠(亚硝酸钠 水溶液中的亚硝酸钠)的摩尔比为1～5:5～15:0.01～0.05；优选为2.3:10:0.01。
步骤(1)中所述的盐酸溶液的浓度为0.1～1mol/L；优选为0.5mol/L。
步骤(1)中所述的亚硝酸钠水溶液的浓度为1～10mol/L；优选为5mol/L。
步骤(1)中所述的搅拌反应的时间为10～45分钟；优选为30分钟。
步骤(2)中所述的间苯二胺与氢氧化钠的摩尔比为2～15：2～20；优选为3:5。
步骤(2)中所述的氢氧化钠溶液的浓度为0.5～1mol/L；优选为1mol/L。
步骤(2)中所述的间苯二胺与所述4,4'-二硫代二苄胺的摩尔比为6:2.3。
步骤(3)中所述的pH值优选为10。
步骤(3)中所述的调节pH值为采用NaOH溶液进行调节；优选为采用1mol/L NaOH溶液进行调节。
步骤(3)中所述的搅拌反应为通过如下方式实现：先在冰浴条件下搅拌10～45分钟(优选为30分钟)，然后室温下搅拌5～2小时(优选为1小时)。
步骤(3)中所述的水洗为用水洗涤3次以上。
步骤(3)中所述的干燥优选为真空干燥。
步骤(B)中所述的偶氮苯化合物与所述量子点的摩尔比为200～1000：1。
步骤(B)中所述的H 2O/DMSO溶液中H 2O和DMSO的体积比为80：20。
步骤(B)中所述的3-巯基丙酸的用量为按每升H 2O/DMSO溶液配比0.02～0.05mol 3-巯基丙酸计算。
步骤(B)中所述的搅拌的时间为10～45分钟；优选为30分钟。
步骤(B)中所述的3-巯基丙酸与偶氮苯化合物的摩尔比为7:3～9:1。
步骤(C)中所述的反应的时间为0.5～2小时；优选为1小时。
一种基于偶氮苯-量子点的荧光探针，通过上述任一项所述的方法制备得到。
所述的基于偶氮苯-量子点的荧光探针在制备开关型荧光传感器中的应用。
一种开关型荧光传感器，通过如下方法制备得到：
(a)将上述基于偶氮苯-量子点的荧光探针溶于PBS缓冲液中，得到荧光探针溶液；
(b)将生化分析物水溶液与荧光探针溶液混合后进行孵育，得到开关型荧光传感器；其中，生化分析物为连二亚硫酸离子(S 2O 4 2-)、次氯酸离子(ClO -)或偶氮还原酶。
步骤(a)中所述的PBS缓冲液的浓度为10～100mmol/L；优选为50mmol/L。
步骤(b)中所述的生化分析物水溶液为含有连二亚硫酸离子(S 2O 4 2-)、次氯酸离子(ClO -)或偶氮还原酶的水溶液，如连二亚硫酸钠水溶液，连二亚硫酸钾水溶液，连二亚硫酸锌水溶液，连二亚硫酸锰水溶液，连二亚硫酸钙水溶液，次氯酸钠水溶液、氯酸钾水溶液，次氯酸钙水溶液，次氯酸锂水溶液，次氯酸锌水溶液，偶氮还原酶水溶液等。
所述的偶氮还原酶水溶液优选为含有NADH的偶氮还原酶水溶液；所述的含有NADH的偶氮还原酶水溶液中NADH的浓度为0.01～1μmol/L(优选为0.1μmol/L)；偶氮还原酶的浓度为0.02～2μg/mL(优选为0.02～1.2μg/mL)。
所述的NADH与所述荧光探针的摩尔比为1：100。
步骤(b)中所述的连二亚硫酸离子的浓度为0.01～1μmol/L；优选为0.1μmol/L。
步骤(b)中所述的次氯酸离子的浓度为0.05～0.5μmol/L；优选为0.15μmol/L。
步骤(b)中所述的偶氮还原酶的浓度为0.02～2μg/mL；优选为0.02～1.2μg/mL。
步骤(b)中所述的连二亚硫酸离子与所述荧光探针的摩尔比为10～1000：1；优选为100:1。
步骤(b)中所述的次氯酸离子与所述荧光探针的摩尔比为50～500：1；优选为150:1。
步骤(b)中所述的偶氮还原酶与所述荧光探针的摩尔比为10～200：1；优选为50:1。
所述的基于偶氮苯-量子点的荧光探针，和/或所述的开关型荧光传感器在化学化工，食品安全，环境检测以及生物医学(非疾病诊断领域)领域中的应用。
一种利用所述的开关型荧光传感器检测生化分析物的方法，包括如下步骤：
(i)将至少3个浓度的生化分析物水溶液分别与基于偶氮苯-量子点的荧光探针溶液混合后孵育，然后测量FL光谱，得到荧光光谱强度；其中，生化分析物为连二亚硫酸离子、次氯酸离子或偶氮还原酶；
(ii)根据步骤(i)得到的荧光光谱强度与生化分析物的浓度，制作工作曲线，得到线性方程；
(iii)将待测样品与偶氮苯-量子点的荧光探针混合后孵育，然后测量FL光谱，获得荧光光谱强度，再根据步骤(ii)得到的线性方程，获得待测样品的含量；
其中，步骤(i)和(iii)中所述的偶氮苯-量子点的荧光探针溶液与连二亚硫酸离子孵育的时间为5分钟以上；偶氮苯-量子点的荧光探针溶液与次氯酸离子孵育的时间为8分钟以上；偶氮苯-量子点的荧光探针溶液与偶氮还原酶孵育的时间为15分钟以上。
步骤(i)中所述的生化分析物水溶液为含有连二亚硫酸离子(S 2O 4 2-)、次氯酸离子(ClO -)或偶氮还原酶的水溶液，如连二亚硫酸钠水溶液，连二亚硫酸钾水溶液，连二亚硫酸锌水溶液，连二亚硫酸锰水溶液，连二亚硫酸钙水溶液，次氯酸钠水溶液、氯酸钾水溶液，次氯酸钙水溶液，次氯酸锂水溶液，次氯酸锌水溶液，偶氮还原酶水溶液等。
所述的偶氮还原酶水溶液优选为含有NADH的偶氮还原酶的水溶液；所述的含有NADH的偶氮还原酶的水溶液中NADH的浓度为0.01～1μmol/L；偶氮还原酶的浓度为0.0.2～1.2μg/mL。
步骤(i)中所述的连二亚硫酸离子水溶液的浓度为1～80nmol/L。
步骤(i)中所述的次氯酸离子水溶液的浓度为4～150nmol/L。
步骤(i)中所述的偶氮还原酶的浓度为0.02～1.2μg/mL。
偶氮苯是由两个通过N＝N键连接的苯环组成的化学分子，被广泛用于染料和颜料、食品添加剂、比色指示剂和治疗剂。特别是由于其偶氮部分在紫外光照下发生可逆的顺式反式异构化，已被用于荧光传感探针中的有效荧光淬灭剂。本发明中制备了一种新型结构的偶氮苯化合物，基于此开发了一种偶氮苯-QDs复合荧光探针，可用于设计制备“关-开”(off-on)型荧光传感器。我们首先通过偶氮偶合反应合成了该偶氮苯化合物DTPABDA，其通过配体交换反应修饰于QDs表面；DTPABDA携带吸电子的偶氮苯基团，通过光诱导电子转移(PET)效应可淬灭QDs的荧光，使得QDs荧光“关”；当通过化学反应特异性移除修饰于量子点表面的吸电子偶氮苯基团，QDs的荧光强度能够有效恢复，实现QDs荧光“开”。为了验证该偶氮苯-QDs荧光探针的应用效果，我们通过一系列不同反应去除偶氮苯基团，包括1)连二亚硫酸离子的还原反应；2)次氯酸离子氧化反应；3)偶氮还原酶的酶促反应。通过监测上述反应中量子点荧光受偶氮苯淬灭荧光信号的恢复变化，证明偶氮苯-QDs荧光探针可用于开发检测连二亚硫酸离子，次氯酸离子和偶氮还原酶的开关型荧光传感器。与报道的使用有机染料作为探针的荧光传感器相比，该传感器表现出响应时间短、灵敏度高及检测限低的特点，可广泛应用于监测工业中的连二亚硫酸、生物医学诊断中活细胞内源性次氯酸及肿瘤内的低氧度。
本发明相对于现有技术具有如下的优点及效果：
1、本发明中，首先合成了含有偶氮苯的化合物—双[4,4'-(二硫代苯基偶氮-1,3-苯二胺](Bis[4,4’-(dithiophenyl azo)-1,3-benzenediamine]，DTPABDA)，通过配体交换反应可将其修饰于QDs表面。DTPABDA分子上的吸电子官能团即偶氮苯基团可通过光致电子转移效应(photo-induced electron transfer,PET)，使得QDs的荧光猝灭。我们发现并提出，通过三种不同类型反应，包括连二亚硫酸离子引起的还原反应、次氯酸离子引起的氧化反应和偶氮还原酶引起的酶促反应，可有效去除QDs表面的偶氮苯基团，从而恢复QDs的荧光。基于不同反应促使的QDs荧光强度恢复，我们开发新型分子开关型荧光传感器，能够灵敏准确地分别检测连二亚硫酸离子、次氯酸离子和偶氮还原酶，可用于检测多种化学生物分析物。
2、与现有的方法相比，本发明提出的分析传感方法展现出高灵敏度、宽线性范围和良好的选择性等优点，检出限分别达到连二亚硫酸离子0.5mmol/L，次氯酸离子0.2nmol/L，偶氮还原酶2ng/mL。这一传感新方法可广泛应用于监测工业中的连二亚硫酸、生物医学诊断中活细胞中的内源性次氯酸及肿瘤内的低氧度，在生物、化学化工、食品安全、环境检测及生物医学诊断等领域具有重要的潜在应用价值。
附图说明
图1是DTPABDA的合成途径、以及DTPABDA与S 2O 4 2-,ClO -和偶氮还原酶的反应途径；其中，a为DTPABDA的合成途径，b、c和d分别为DTPABDA与S 2O 4 2-,ClO -和偶氮还原酶的反应途径。
图2是DTPABDA与1mM Na 2S 2O 4孵育后的溶液以及对应的紫外吸收光谱图；其中，a为DTPABDA溶液、以及DTPABDA与1mM Na 2S 2O 4孵育后的溶液(图中I为DTPABDA溶液；II为DTPABDA与1mM Na 2S 2O 4孵育后的溶液)；b为紫外吸收光谱。
图3是DTPABDA与1mM NaClO孵育后的溶液以及对应的紫外吸收光谱图；其中，a为DTPABDA溶液、以及DTPABDA与1mM NaClO孵育后的溶液(图中：I为DTPABDA溶液；II为DTPABDA与1mM NaClO孵育后的溶液)；b为紫外吸收光谱。
图4是DTPABDA与5μg/mL偶氮还原酶孵育后的溶液以及对应的紫外吸收光谱图；其中，a为DTPABDA溶液、以及DTPABDA与5μg/mL偶氮还原酶孵育后的溶液(图中：I为DTPABDA溶液；II为DTPABDA与5μg/mL偶氮还原酶孵育后的溶液)；b为紫外吸收光谱。
图5是1mM Na 2S 2O 4存在时DTPABDA的时间相关紫外吸收光谱，以及样品在波长458nm时，时间相关的吸光度变化图；其中，a为1mM Na 2S 2O 4存在时DTPABDA的时间相关紫外吸收光谱；b为样品在波长458nm时，时间相关的吸光度变化。
图6是1mM NaClO存在时DTPABDA的时间相关紫外吸收光谱，以及样品在波长458nm时，时间相关的吸光度变化图；其中，a为1mM NaClO存在时DTPABDA的时间相关紫外吸收光谱；b为样品在波长458nm时，时间相关的吸光度变化。
图7是5μg/mL偶氮还原酶存在时DTPABDA的时间相关紫外吸收光谱，以及样品在波长458nm时，时间相关的吸光度变化；其中，a为5μg/mL偶氮还原酶存在时DTPABDA的时间相关紫外吸收光谱；b为样品在波长458nm时，时间相关的吸光度变化。
图8是MPA和DTPABDA的硫醇－二硫化物交换反应示意图，以及使用MPA和MPABDA作为配体，在QD表面上EDA介导的配体交换示意图；其中，a为MPA和DTPABDA的硫醇－二硫化物交换反应示意图；b为使用MPA和MPABDA作为配体，在QD表面上EDA介导的配体交换示意图。
图9是通过EDA介导的配体交换方法在UV照射下使用不同比例的MPA/DTPABDA制备的QDs的照片图(摩尔比分别为100/0(I)、90/10(II)、80/20(III)、70/30(IV))。
图10是图9中的QDs样品在560nm处的FL光谱图。
图11是图9中的QDs样品在560nm处的FL强度图。
图12是用比例为70/30的MPA/DTPABDA制备QDs作为配体的XPS测量光谱图。
图13是QDs N 1s的核能级谱图(实线为数据拟合)。
图14是移除淬灭剂的示意图以及荧光恢复的示意图；其中，a为移除淬灭剂的示意图；b为通过移除淬灭剂，荧光恢复的示意图。
图15是100nM Na 2S 2O 4存在下，QDs的时间相关FL变化图。
图16是100nM Na 2S 2O 4存在下，FL强度随反应时间的变化图。
图17是与不同浓度的Na 2S 2O 4孵育后的QDs的荧光光谱图。
图18是不同浓度的Na 2S 2O 4在560nm处荧光强度对比图。
图19是0.1μM Na 2S 2O 4和2μM其他含硫化合物的荧光响应图。
图20是150nM NaClO存在下，QDs的时间相关FL变化图。
图21是150nM NaClO存在下，FL强度随反应时间的变化图。
图22是与不同浓度的NaClO孵育后QDs的FL光谱图。
图23是不同浓度的NaClO在560nm处的FL强度对比图。
图24是0.15μM NaClO和2μM其他ROS/RNS的FL反应结果图。
图25是1.2μg/mL偶氮还原酶和0.1μM NADH存在下，QDs时间相关的FL变化图。
图26是1.2μg/mL偶氮还原酶和0.1μM NADH存在下，FL强度随反应时间的变化图。
图27是与不同浓度的偶氮还原酶孵育后QDs的FL光谱图。
图28是不同浓度的偶氮还原酶在560nm处的FL强度对比图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件或按照制造厂所建议的实验条件。除非特别说明，本发明所用试剂和原材料均可可从商业渠道获得。
实施例1
本实施例提供一种DTPABDA的合成方法。
2.3mmol 4,4'-二硫代二苄胺(4,4’-dithiodibenzylamine，DTDBA)(阿拉丁试剂公司)加入到一个含有20mL 0.5mol/L盐酸(HCl)水溶液的烧瓶，冰浴冷却后，逐滴加入2mL溶于冰水中的5.0mmol亚硝酸钠(NaNO 2)水溶液，混合物冰浴搅拌30分钟后得到4,4'-二硫代二苯基重氮(4,4’-dithiodiphenyldiazonium，DTDPDA)(溶液1)。在另一个烧瓶中，6.0mmol间苯二胺溶解于10mL 1mol/L氢氧化钠(NaOH)水溶液中，冰水冷却(溶液2)。在剧烈搅拌下，将溶液1滴加到溶液2中，用1mol/L NaOH水溶液将所得混合溶液pH值调至10。混合物在冰浴条件下搅拌30分钟，随后室温下搅拌1小时后，收集沉淀物并用水洗涤3次。真空干燥后，红色沉淀即为DTPABDA(双[4,4'-(二硫代苯基偶氮)-1,3-苯二胺](Bis[4,4’- (dithiophenyl azo)-1,3-benzenediamine])。 1H核磁共振氢谱(600MHz,DMSO-d6，室温):σ7.56(d,2H),7.30(d,1H),6.74(d,2H),6.60(s,2H)。DTPABDA通过偶氮偶合反应合成，图1a为DTPABDA的合成示意图。DTPABDA的结构式如下所示：
Figure PCTCN2020116110-appb-000002
实施例2
本实施例提供一种DTPABDA响应多种物质的方法。
(1)DTPABDA与S 2O 4 2-，ClO -的反应
DTPABDA与S 2O 4 2-和ClO -的反应如下：将DTPABDA溶于二甲基亚砜(DMSO)/磷酸盐缓冲溶液(50mmol/L,pH＝7.4，体积比为20/80)，制备1mL 0.1mmol/L的溶液，随后加入100μL 1mmol/L S 2O 4 2-或ClO -水溶液，拍摄照片并测量相应的紫外吸收光谱(Varian Cary60分光光度计)。
(2)DTPABDA与偶氮还原酶的反应
DTPABDA与偶氮还原酶的反应如下：将100μL 0.1mmol/L DTPABDA水溶液与100μL5mmol/L烟酰胺腺嘌呤二核苷酸(NADH)水溶液在1mL DMSO/PBS缓冲液(体积比为20/80)混合液中混合，随后加入10μL 500μg/mL偶氮还原酶(购买自西格玛奥里奇(中国)公司)水溶液，混合均匀后测量相应的紫外吸收光谱。
DTPABDA可与三类物质反应包括：1)具有还原性的S 2O 4 2-；2)具有氧化性的ClO-，；3)偶氮还原酶。图1b～1d为DTPABDA与以上三类物质的反应路径图。这些物质能够通过化学或酶反应，特异性使得DTPABDA分子中偶氮键断裂。
(3)DTPABDA的反应活性测试
为了测试DTPABDA的反应活性，将0.1mmol/L DTPABDA溶液与1mmol/L Na 2S 2O 4，1mmol/L NaClO或含有1mmol/L NADH的5μg/mL偶氮还原酶按体积比1:1混合后一起温育。DTPABDA溶液呈黄色，与Na 2S 2O 4混合后该溶液变为无色(图2a)。由于偶氮苯的特征吸收，DTPABDA溶液的紫外吸收光谱在458nm处显示出强烈的峰(图2b)。当S 2O 4 2-存在下，该吸收峰的消失表示偶氮部分的已移除。用NaClO孵育DTPABDA溶液后，溶液变为淡黄色(图3a)，并且其相应的偶氮苯部分的UV吸收峰显着降低(图3b)。向DTPABDA溶液中加入偶氮还原酶和NADH诱导其缓慢的颜色变为浅黄色(图4a)，并且在458nm处的UV吸收峰也显示出显着的降低(图4b)。300至400nm之间的强吸收是NADH的紫外吸收。
接下来测量了DTPABDA溶液在这些化合物存在下的时间依赖性紫外吸收光谱。加入连二亚硫酸盐后，DTPABDA的UV吸收峰立即降低，并且1分钟内反应几乎完成，1分钟后没 有可见的UV吸光度变化(图5a和5b)。次氯酸盐具有与DTPABDA相似的反应性，并且反应在1分钟内几乎达到平衡(图6a和6b)。然而偶氮还原酶的反应相对较慢，并且随着反应的进行UV吸收峰缓慢下降(图7a)，8分钟后458nm处的吸光度达到平台(图7b)。
实施例3
本实施例提供一种DTPABDA修饰的CdSe/ZnS QDs的合成方法。
通过配体交换法合成DTPABDA修饰的QDs(半导体量子点)，过程如下：0.5mL0.1mmol/L QDs(CdSe/ZnS分别为核/壳；购自南京牧科纳米科技有限公司)水溶液中加入大量丙酮，通过丙酮诱导的聚集和离心，将离心后的沉淀溶于1mL CHCl 3溶液中。在搅拌的条件下，将0.5mL 0.01mol/L乙二胺(ethylenediamine，EDA)溶液加入到QDs(量子点与乙二胺的摩尔比为1:100)，混合溶液搅拌30分钟后得到溶液3。在另一个小瓶中，将3-巯基丙酸(3-mercaptopropionic acid，MPA)和DTPABDA以不同摩尔比混合(分别为100/0,90/10,80/20,70/30)于1mL H 2O/DMSO(体积比为80/20)溶液中，搅拌30分钟后得到溶液4；其中，3-巯基丙酸的终浓度为0.01mol/L。将溶液4快速加入溶液3中，剧烈震荡1小时，收集上层水溶液并利用丙酮沉淀离心所得QDs，随后溶于50mmol/L PBS缓冲液中，得到QDs溶液，备用。
利用EDA介导的配体交换方法，以DTPABDA和MPA为配体制备DTPABDA修饰的CdSe/ZnS QDs。MPA有两个重要作用：1)MPA的硫醇基团可以将DTPABDA的二硫键裂解成具有硫醇基团的4-巯基苯偶氮-1,3-苯二胺(MPABDA)(图8a)，其对QD表面的结合能力高于二硫键；2)未反应的MPA和MPABDA作为稳定剂共同吸附在QDs表面，在生理pH下负电荷MPA的静电排斥可以显着提高所得QDs的稳定性(图8b)。
图9为在UV照射下用不同比例的MPA/DTPABDA制备的QDs水溶液图片，比例分别为100/0(I)、90/10(II)、80/20(III)、70/30(IV)。QDs覆盖100％MPA时呈现绿色；当DTPABDA/MPA混合配体覆盖在QDs表面时，亮度减弱。图10为对应QDs的荧光光谱。MPA覆盖的QDs在560nm处显示出强烈的FL发射峰。当DTPABDA/MPA混合配体覆盖在QDs表面时，峰值没有显示任何偏移，但是强度有所降低。当DTPABDA/MPA比率高达30/70时，与MPA修饰的QDs相比，QDs的FL强度降低至18.7％(图11)。图12中的XPS数据表示元素Cd、Se、Zn和S，表明有QDs存在。XPS数据N1s精细谱(图13)中N 1s峰可以被拆分为N＝N(399.3eV)和N＝C(402.1eV)，表明偶氮苯分子被成功修饰于QDs表面。
实施例4
本实施例提供一种S 2O 4 2-“开启”传感器、ClO -“开启”传感器和偶氮还原酶“开启”传感器 的检测方式。
将10uL 10μmol/L QDs溶液(即实施例3制备的DTPABDA修饰的CdSe/ZnS QDs)分别与100μL 0.1μmol/L Na 2S 2O 4水溶液、100μL 0.15μmol/LNaClO水溶液或含有0.1μmol/L NaDH水溶液的100μL 1.2μg/mL偶氮还原酶水溶液(0.05μmol/L)混合，每隔一段时间测量它们的与时间相关的FL光谱(Na 2S 2O 4和NaClO间隔半分钟，偶氮还原酶间隔1分钟)。为了定量测定S 2O 4 2-、ClO -和偶氮还原酶这三种物质，分用将不同浓度的Na 2S 2O 4、NaClO或含有NADH的偶氮还原酶与QDs溶液孵育，随后检测对应的FL光谱，根据荧光光谱强度和浓度的关系制作工作曲线，计算线性范围和检出限。基于FL淬灭机理，去除偶氮苯部分即可恢复其FL。因此，具有裂解偶氮苯部分能力的分析物可以通过恢复的FL来分析。其传感机制如图14所示。图15和16为时间相关的FL光谱。
(1)S 2O 4 2-“开启”传感器
对S 2O 4 2-的FL传感：FL强度逐渐增加并在5分钟后达到平衡，表明响应时间为5分钟。用不同浓度的Na 2S 2O 4孵育5分钟后(浓度分别为0、2、4、8、10、20、40、60、80、100、150nmol/L)，560nm处的QDs FL强度随着S 2O 4 2-浓度的增加而增加(图17)。1和80nmol/L之间有良好线性关系，计算所得检测限(LOD)为0.05nmol/L(图18)。
为了验证该传感器对S 2O 4 2-的选择性，用2uL 1mmol/L Na 2SO 4,Na 2SO 3,Na 2S或NaHS溶液与10uL 10μmol/L的QDs溶液在1mL缓冲液(二甲基亚砜(DMSO)/磷酸盐缓冲溶液(50mmol/L,pH＝7.4，体积比为20/80))中孵育5分钟，结果如图19所示，在用2μmol/L Na 2SO 4,Na 2SO 3,Na 2S和NaHS溶液孵育QDs后，FL强度没有显着变化，表明该传感器对S 2O 4 2-具有良好的选择性。
(2)ClO -“开启”传感器
对ClO -的FL传感：与检测S 2O 4 2-的FL反应类似，在1mL缓冲液(二甲基亚砜(DMSO)/磷酸盐缓冲溶液(50mmol/L,pH＝7.4，体积比为20/80))中加入10uL 10μmol/L DTPABDA修饰的QDs溶液，再加入NaClO(浓度为0、4、8、10、20、40、60、80、100、150、200、250nmol/L)逐渐诱导其FL恢复(图20和21)。FL强度在8分钟后达到其最大水平，即响应时间为8分钟。图22为用不同浓度(0、4、8、10、20、40、60、80、100、150、200、250nmol/L)的ClO -孵育8分钟后QDs的FL光谱。浓度相关的FL强度增强，并且当ClO -浓度为4到150nmol/L时，560nm处的FL强度显示线性增加(图23)。计算LOD为0.2nmol/L。
图24为2uL 1mmol ROS/RNS(OH·,O 2 -, 1O 2,H 2O 2,NO,ONOO -1)和10uL 10μmol/L QDs溶液在1mL缓冲液中孵育的FL响应，它们是ClO -传感中的主要干扰物。与这些物质一起孵育后没有明显的FL强度变化，表明该传感器对ClO -的优异选择性。
(3)偶氮还原酶“开启”传感器
对偶氮还原酶的FL传感：缺氧是各种疾病的特征，包括癌症、心脏病、缺血和血管疾病。偶氮还原酶是生理缺氧标记物，可用于检测肿瘤的缺氧程度。基于“开启”FL传感机制，偶氮还原酶能够通过酶催化反应使得偶氮基团从NADH接受氢还原生成胺，从而去除偶氮基团，从而开启DTPABDA修饰的QDs的FL。图25和26为1.2μg/mL偶氮还原酶和0.1μmol/L NADH存在下，QDs的FL强度逐渐增加，在15分钟内达到平台，即响应时间为15分钟。将不同浓度(0、0.02、0.04、0.08、0.1、0.2、0.4、0.6、0.8、1、1.2、1.5、2μg/mL)的偶氮还原酶与DTPABDA修饰的QDs孵育15分钟后，随着偶氮还原酶浓度的增加，其FL强度逐渐增加(图27和28)，在0.02和1.2μg/mL之间有良好的线性关系。计算得LOD为2ng/mL。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种基于偶氮苯-量子点的荧光探针的制备方法，其特征在于，包括如下步骤：
    (A)将量子点溶于CHCl 3溶液中，然后加入乙二胺溶液，搅拌混合均匀，得到混合溶液II；
    (B)将偶氮苯化合物与3-巯基丙酸加入到H 2O/DMSO溶液中，搅拌混合均匀，得到混合溶液III；其中，偶氮苯化合物为双[4,4'-(二硫代苯基偶氮)-1,3-苯二胺]；
    (C)将混合溶液III加入到混合溶液II中，剧烈震荡反应，待反应结束后收集上层水溶液，并加入丙酮沉淀离心，得到基于偶氮苯-量子点的荧光探针。
  2. 根据权利要求1所述的基于偶氮苯-量子点的荧光探针的制备方法，其特征在于，步骤(B)中所述的双[4,4'-(二硫代苯基偶氮)-1,3-苯二胺]通过如下方法制备得到：
    (1)将4,4'-二硫代二苄胺加入到盐酸溶液中，冰浴冷却后，逐滴加入亚硝酸钠水溶液，在冰浴条件下搅拌反应，得到4,4'-二硫代二苯基重氮；
    (2)将间苯二胺溶解于氢氧化钠溶液中，冰水冷却，得到混合溶液I；
    (3)将4,4'-二硫代二苯基重氮加入到混合溶液I中，并调节pH值至9～11，冰浴和/或常温条件下搅拌反应，收集沉淀物，水洗，干燥，得到所述的偶氮苯化合物。
  3. 根据权利要求2所述的基于偶氮苯-量子点的荧光探针的制备方法，其特征在于：
    步骤(1)中所述的4,4'-二硫代二苄胺、盐酸和亚硝酸钠的摩尔比为1～5:5～15:0.01～0.05；
    步骤(1)中所述的盐酸溶液的浓度为0.1～1mol/L；
    步骤(1)中所述的亚硝酸钠水溶液的浓度为1～10mol/L；
    步骤(1)中所述的搅拌反应的时间为10～45分钟；
    步骤(2)中所述的间苯二胺与氢氧化钠的摩尔比为2～15：2～20；
    步骤(2)中所述的间苯二胺与所述4,4'-二硫代二苄胺的摩尔比为6:2.3；
    步骤(2)中所述的氢氧化钠溶液的浓度为0.5～1mol/L；
    步骤(3)中所述的调节pH值为采用NaOH溶液进行调节；
    步骤(3)中所述的搅拌反应为通过如下方式实现：先在冰浴条件下搅拌10～45分钟，然后室温下搅拌5～2小时；
    步骤(3)中所述的水洗为用水洗涤3次以上；
    步骤(3)中所述的干燥为真空干燥。
  4. 根据权利要求1所述的基于偶氮苯-量子点的荧光探针的制备方法，其特征在于：
    步骤(A)中所述的量子点为CdSe/ZnS量子点；
    步骤(A)中所述的量子点与乙二胺的摩尔比为1:100；
    步骤(A)中所述的乙二胺溶液的浓度为0.005～0.2mol/L；
    步骤(B)中所述的偶氮苯化合物与所述量子点的摩尔比为200～1000：1
    步骤(B)中所述的3-巯基丙酸与偶氮苯化合物的摩尔比为7:3～9:1。
    步骤(B)中所述的H 2O/DMSO溶液中H 2O和DMSO的体积比为80：20；
    步骤(B)中所述的3-巯基丙酸的用量为按每升H 2O/DMSO溶液配比0.02～0.05mol 3-巯基丙酸计算。
  5. 根据权利要求1所述的基于偶氮苯-量子点的荧光探针的制备方法，其特征在于：
    步骤(A)中所述的搅拌的时间为10～45分钟；
    步骤(B)中所述的搅拌的时间为10～45分钟；
    步骤(C)中所述的反应的时间为0.5～2小时。
  6. 一种基于偶氮苯-量子点的荧光探针，其特征在于：通过权利要求1～5任一项所述的方法制备得到。
  7. 权利要求6所述的基于偶氮苯-量子点的荧光探针在制备开关型荧光传感器中的应用。
  8. 一种开关型荧光传感器，其特征在于，通过如下方法制备得到：
    (a)将权利要求6所述的基于偶氮苯-量子点的荧光探针溶于PBS缓冲液中，得到荧光探针溶液；
    (b)将生化分析物水溶液与荧光探针溶液混合后进行孵育，得到开关型荧光传感器；其中，生化分析物为连二亚硫酸离子、次氯酸离子或偶氮还原酶；
    步骤(a)中所述的PBS缓冲液的浓度为10～100mmol/L；
    步骤(b)中所述的连二亚硫酸离子的浓度为0.01～1μmol/L；
    步骤(b)中所述的次氯酸离子的浓度为0.05～0.5μmol/L；
    步骤(b)中所述的偶氮还原酶的浓度为0.02～2μg/mL；
    步骤(b)中所述的连二亚硫酸离子与所述荧光探针的摩尔比为10～1000：1；
    步骤(b)中所述的次氯酸离子与所述荧光探针的摩尔比为50～500：1；
    步骤(b)中所述的偶氮还原酶与所述荧光探针的摩尔比为10～200：1。
  9. 权利要求6所述的基于偶氮苯-量子点的荧光探针和/或权利要求8所述的开关型荧光传感器在化学化工，食品安全，环境检测以及非疾病诊断目的的生物医学领域中的应用。
  10. 一种利用权利要求8所述的开关型荧光传感器检测生化分析物的方法，其特征在于，包括如下步骤：
    (i)将至少3个浓度的生化分析物水溶液分别与权利要求6所述的基于偶氮苯-量子点的荧光探针溶液混合后孵育，然后测量FL光谱，得到荧光光谱强度；其中，生化分析物为连二亚硫酸离子、次氯酸离子或偶氮还原酶；
    (ii)根据步骤(i)得到的荧光光谱强度与生化分析物的浓度，制作工作曲线，得到线性方程；
    (iii)将待测样品与偶氮苯-量子点的荧光探针混合后孵育，然后测量FL光谱，获得荧光光谱强度，再根据步骤(ii)得到的线性方程，获得待测样品的含量；
    其中，步骤(i)和(iii)中所述的偶氮苯-量子点的荧光探针溶液与连二亚硫酸离子孵育的时间为5分钟以上；偶氮苯-量子点的荧光探针溶液与次氯酸离子孵育的时间为8分钟以上；偶氮苯-量子点的荧光探针溶液与偶氮还原酶孵育的时间为15分钟以上；
    步骤(i)中所述的连二亚硫酸离子的浓度为1～80nmol/L；
    步骤(i)中所述的次氯酸离子的浓度为4～150nmol/L；
    步骤(i)中所述的偶氮还原酶的浓度为0.02～1.2μg/mL。
PCT/CN2020/116110 2019-11-04 2020-09-18 基于偶氮苯-量子点的荧光探针及制备方法以及其在分子开关型荧光传感器中的应用 WO2021088529A1 (zh)

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