Preparation method and application of near-infrared two-region linear fluorescence sensor

Abstract

The invention provides a preparation method and application of a near-infrared two-zone fluorescence sensor, chiral single-walled carbon nanotubes are separated by using an aqueous two-phase extraction method, and materials used in the separation process comprise deionized water, polyethylene glycol, glucan, lauryl sodium sulfate, deoxysodium cholate and sodium cholate hydrate; the chiral single-walled carbon nanotubes are dispersed and mixed with 4-nitrobenzene tetrafluoroboric acid diazonium salt to obtain the fluorescence sensor. The high-purity (6, 5)-SWCNT is efficiently, simply and conveniently separated through the aqueous two-phase extraction technology, the prepared SWCNT-NBD nano material has good optical performance including bright light emitting in a near-infrared second area and good penetrating power for biological tissue, a fluorescence detector (SWCNT-NBD) is low in detection line, wide in linear range and good in DA selectivity, preparation is convenient and fast, and the application prospect is wide. The cost is low.

Description

Technical Field

[0001] The invention relates to the technical field of nanosensors, and in particular to a preparation method and application of a near-infrared two-zone fluorescence sensor. Background Art

[0002] Dopamine (DA), as a catecholamine neurotransmitter, has an important impact on human health. Clinical studies have shown that patients with depression suffer from a lack of happiness due to disorders in the DA perception system, that is, the ability to feel happiness is destroyed. In addition, studies have shown that other human mental illnesses are also related to DA. In the central nervous system, DA content is also related to diseases such as Parkinson's disease and Alzheimer's disease. The concentration level of DA in living organisms is within 100nM. For example, the health of the human eye can be directly judged by the DA level. For example, Martin et al. used high-performance liquid chromatography electrochemical method to detect that the average DA concentration in the tears of healthy people was 58±33nM, while the DA concentration in the tears of people suffering from myopia would be reduced. Therefore, the development of DA fluorescent probes with good sensitivity, low detection limit and outstanding selectivity is of great significance for the discovery, prevention, diagnosis and treatment of diseases, and it is also a great challenge.

[0003] At present, the detection technologies for DA include enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography, electrochemical method, spectroscopy and colorimetry, etc. However, these detection technologies are not satisfactory in terms of sample preparation, detection stability, response time, anti-interference and detection cost. Cyclic voltammetry (CV) in electrochemical method has become a more commonly used DA detection method because of its simple detection, fast response and low cost. However, in untreated human body fluid samples, DA and are often together with molecules with similar structures. For example, catecholamine neurotransmitters such as epinephrine (EP) belong to the same class as DA. They have similar redox potentials, which will cause trouble for DA detection. In addition, due to the redox reaction on the electrode, the product will contaminate the electrode, shorten the electrode life, and the detection stability is low, the effect is poor, and the electrochemical method is also easily affected by the detection environment, such as pH, temperature, etc. Fluorescence sensors have excellent potential in biological imaging, but there are still problems with their complex preparation process, limited spatiotemporal resolution, selectivity and stability. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing a near-infrared two-zone fluorescence sensor, which solves the problems of high detection cost, poor selectivity, low sensitivity and inability to achieve linear sensing in the prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] A method for preparing a near-infrared two-zone linear fluorescence sensor, wherein chiral single-walled carbon nanotubes are separated by a two-phase aqueous extraction method, wherein the materials used in the separation process include: deionized water, polyethylene glycol, dextran, sodium dodecyl sulfate, sodium deoxycholate and sodium cholate hydrate; and the chiral single-walled carbon nanotubes are dispersed and mixed with 4-nitrobenzene tetrafluoroborate diazonium salt to obtain a fluorescence sensor.

[0007] The present invention disperses chiral single-walled carbon nanotube powder in a 1% sodium deoxycholate solution at a ratio of 1 mg / mL to obtain a first mixed solution; the first mixed solution is subjected to tip ultrasound and centrifugation to obtain 80% of the upper clear liquid to obtain a second mixed solution; 2 mass% dextran, 12.5 mass% polyethylene glycol, 0.5 g / L sodium deoxycholate, and 18 g / L sodium dodecyl sulfate are mixed to obtain a first separation solution;

[0008] The second mixed solution, deionized water, a 20 wt / v% dextran solution, and a 40 wt / v% polyethylene glycol solution were respectively placed in a centrifuge tube in a volume ratio of 1:3:4:2 to obtain a third mixed solution;

[0009] The third mixed solution was centrifuged at 8000 rpm for 1 minute to separate the third mixed solution into an upper and lower phase, and the lower phase was taken as the fourth mixed solution;

[0010] The fourth mixed solution and an equal volume of the first separation solution were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the lower phase was taken as the fifth mixed solution;

[0011] The fifth mixed solution and an equal volume of the first separation solution were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the lower phase was taken as the sixth mixed solution;

[0012] The sixth mixed solution and an equal volume of the first separation solution were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the lower phase was taken as the seventh mixed solution;

[0013] The seventh mixed solution and the first separation solution of the same volume were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the upper phase was a high-purity (6,5)-chiral single-walled carbon nanotube dispersion;

[0014] The obtained (5,6) chiral single-walled carbon nanotubes are diluted with 0.1wt.% sodium dodecyl sulfate solution to an absorbance of 0.15 to obtain a first detection solution;

[0015] A 20 nM 4-nitrobenzene diazonium tetrafluoroborate solution was added to the first detection solution to obtain a fluorescent sensor.

[0016] An application of a fluorescence sensor, which is prepared by the method of the near-infrared two-zone linear fluorescence sensor of the present invention and is used for dopamine linear detection.

[0017] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0018] The present invention provides a preparation method and application of a near-infrared second-region fluorescence sensor. The present invention efficiently and conveniently separates high-purity (6,5)-SWCNT through a two-aqueous phase extraction technology. The prepared SWCNT-NBD nanomaterial has good optical properties, including bright luminescence in the near-infrared second region and good penetration into biological tissues. The fluorescence detector (SWCNT-NBD) has a low detection line, a wide linear range, good selectivity for DA, and is easy to prepare and low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0020] Figure 1 A detection principle diagram of a fluorescent sensor for dopamine detection provided by an embodiment of the present invention;

[0021] Figure 2 Steady-state fluorescence spectra, ultraviolet-visible-near-infrared absorption spectra, and Raman spectra of the first detection solution before and after adding dopamine provided in an embodiment of the present invention;

[0022] Figure 3 A linear detection effect diagram provided by an embodiment of the present invention;

[0023] Figure 4 A selective detection result diagram provided by an embodiment of the present invention;

[0024] Figure 5 This is a graph showing the actual detection results of DA concentration in a physiological saline environment provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0025] A preparation method of a near-infrared two-zone linear fluorescence sensor, wherein a chiral single-walled carbon nanotube (SWCNT) is separated by a two-phase aqueous extraction method, wherein the materials in the separation process include: deionized water, polyethylene glycol, dextran, sodium dodecyl sulfate, sodium deoxycholate and sodium cholate hydrate; and the chiral SWCNT is dispersed and mixed with 4-nitrobenzene tetrafluoroborate diazonium salt (NBD) to obtain a fluorescence sensor.

[0026] A method for preparing a near-infrared two-zone linear fluorescence sensor comprises dispersing SWCNT powder in a 1% sodium deoxycholate solution at a ratio of 1 mg / mL to obtain a first mixed solution; subjecting the first mixed solution to tip ultrasound and centrifugation, and taking 80% of the upper clear liquid to obtain a second mixed solution; mixing 2 mass% dextran, 12.5 mass% polyethylene glycol, 0.5 g / L sodium deoxycholate, and 18 g / L sodium dodecyl sulfate to obtain a first separation solution;

[0027] The second mixed solution, deionized water, a 20 wt / v% dextran solution, and a 40 wt / v% polyethylene glycol solution were respectively placed in a centrifuge tube in a volume ratio of 1:3:4:2 to obtain a third mixed solution;

[0028] The third mixed solution was centrifuged at 8000 rpm for 1 minute to separate the third mixed solution into an upper and lower phase, and the lower phase was taken as the fourth mixed solution;

[0029] The fourth mixed solution and an equal volume of the first separation solution were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the lower phase was taken as the fifth mixed solution;

[0030] The fifth mixed solution and an equal volume of the first separation solution were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the lower phase was taken as the sixth mixed solution;

[0031] The sixth mixed solution and an equal volume of the first separation solution were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the lower phase was taken as the seventh mixed solution;

[0032] The seventh mixed solution and the first separation solution of the same volume were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the upper phase was a high-purity (6,5)-SWCNT dispersion;

[0033] The obtained (6,5)-SWCNT was diluted with 0.1wt.% sodium dodecyl sulfate solution to an absorbance of 0.15 to obtain a first detection solution;

[0034] A 20 nM NBD solution was added to the first detection solution to obtain a fluorescent sensor.

[0035] Preferably, the first separation preparation solution of the present invention is stirred at a speed of 1200 rpm.

[0036] Preferably, the first mixed solution of the present invention is stirred for 20 minutes; the tip ultrasound of the first mixed solution of the present invention is performed under ice bath conditions; the tip diameter of the tip ultrasound of the first mixed solution of the present invention is 6 mm; the power of the tip ultrasound of the first mixed solution of the present invention is 1200 rpm; the tip ultrasound of the first mixed solution of the present invention is performed for 60 minutes; the rotation speed of the centrifugalization of the first mixed solution of the present invention is 12000 rpm; the first mixed solution of the present invention is centrifuged for 10 minutes.

[0037] Preferably, when the third mixed solution of the present invention is mixed, the speed of the vortex mixer is 2800 rpm; when the third mixed solution is mixed, the mixing time is 1 min.

[0038] Preferably, when mixing the mixed solutions of the present invention, the speed of the vortex mixer is 2800 rpm; when mixing the mixed solutions, the mixing time is 1 min.

[0039] Preferably, the absorbance of the (6,5)-SWCNT dispersion of the present invention is measured under UV-visible-near infrared absorption spectroscopy.

[0040] Preferably, the NBD solution of the present invention is added while the first test solution is being stirred.

[0041] Preferably, the stirring speed of the first detection solution of the present invention is 800 rpm.

[0042] An application of a fluorescence sensor, which is prepared by the method of the near-infrared two-zone linear fluorescence sensor of the present invention and is used for dopamine linear detection.

[0043] Use the following examples to verify the beneficial effects of the present invention:

[0044] Example 1: The preparation method of the fluorescent sensor for linear detection of dopamine in this example is carried out according to the following steps:

[0045] 1. Add 20 mg of single-walled carbon nanotube powder to 20 mL of 1% wt / v sodium deoxycholate solution to obtain a first mixed solution; add 1 g of dextran, 6.25 g of polyethylene glycol, 0.035 g of sodium deoxycholate, and 0.9 g of sodium dodecyl sulfate to 50 mL of deionized water to obtain a first separation solution;

[0046] 2. The first mixed solution is subjected to tip ultrasound in an ice bath to uniformly disperse the carbon nanotube powder, and then the completely dispersed carbon nanotube bundles and impurities are removed by centrifugation, and 80% of the upper clear liquid after centrifugation is taken to obtain a second mixed solution;

[0047] 3. Add 6 mL of deionized water, 2 mL of the second mixed solution, 8 mL of a 20% dextran solution, and 4 mL of a 40% polyethylene glycol solution into a 50 mL centrifuge tube, and mix well to obtain a third mixed solution;

[0048] 4. centrifuging the third mixed solution, and the lower phase separated by centrifugation is the fourth mixed solution;

[0049] 5. Take out the fourth mixed solution and add it to a 50 mL centrifuge tube, add an equal volume of the first separation solution, mix well to obtain a fifth mixed solution;

[0050] 6. Repeat step 5 three times to obtain the (6,5)-SWCNT dispersion in the upper phase after centrifugation.

[0051] 7. diluting the (6,5)-SWCNT dispersion with 0.1 wt / v% sodium dodecyl sulfate solution, and diluting the (6,5)-SWCNT dispersion to an absorbance of 0.15 under the characterization of UV-visible-near infrared absorption spectrometer;

[0052] 8. Take 2 mL of the above (6,5)-SWCNT dispersion with an optical density of 0.15 and mix it with 20 μL of NBD solution with a concentration of 1 nM to obtain a first dopamine probe solution;

[0053] 9. Weigh 0.9 mg of dopamine and add it to a 10 mL volumetric flask. Make up to volume with deionized water, and evenly oscillate with ultrasound to completely dissolve the dopamine. Dilute it 1000 times with deionized water to obtain a dopamine stock solution with a concentration of 1.96 μM.

[0054] 10. Weigh five different biomass small molecules (epinephrine, serotonin, glutathione, uric acid and gallic acid) and add them to 10 mL volumetric flasks respectively. Use deionized water and ultrasonic vibration to make different types of biomass small molecules completely dissolved to obtain different types of biomass small molecule stock solutions with a concentration of 1.96 μM.

[0055] The fluorescent probe for linear detection of dopamine prepared in this example was characterized by steady-state fluorescence spectroscopy, ultraviolet-visible near-infrared absorption spectroscopy and Raman spectroscopy. The test results are as follows: Figure 2 As shown. Figure 2It can be seen from the fluorescence spectrum that in addition to the intrinsic fluorescence emission of (6,5)-SWCNT at 980 nm, a red-shifted and more intense characteristic absorption peak was generated after the addition of dopamine, indicating that the fluorescent probe for linear detection of dopamine was successfully prepared;

[0056] This implementation also includes the detection performance test of the dopamine fluorescent sensor:

[0057] 1. Add 1, 10, 20, 40, 60, 60, 80, 100 μL of 1.96 μM dopamine stock solution and 100 mL of 1.96 μM biomass small molecule stock solution of different types to 2 mL of the first dopamine probe solution to obtain a mixed solution; magnetic stirring was performed at a speed of 800 rpm throughout the fluorescence detection process, and the target detection object was added and the reaction was waited for 10 minutes to stabilize the system before starting the fluorescence detection;

[0058] Second, with 565 nm as the excitation wavelength and the wavelength range of 800–1400 nm as the detection interval, the fluorescence emission spectra of the (6,5)-SWCNT dispersion, the first dopamine probe solution, and the dopamine stock solution and the different types of biomass small molecule stock solutions mixed with the first dopamine probe solution were detected. The detection results are as follows: Figure 2 , Figure 3 As shown, the linear detection results of dopamine are as follows Figure 4 shown.

[0059] like Figure 1 As shown, during detection, the dopamine fluorescent sensor of the present invention utilizes the reducing property of dopamine (DA) to reduce 4-nitrobenzene diazonium tetrafluoroborate (NBD) into aromatic free radicals, which are then covalently linked to the surface of carbon nanotubes, destroying the SP2 lattice structure of the carbon nanotubes, forming a quantum potential well, and generating bright luminescence in the near-infrared region II.

[0060] Figure 2 In a, the emission wavelength is plotted as the horizontal axis, the optical density is plotted as the left axis, and the fluorescence intensity is plotted as the right axis. It can be seen that the near-infrared fluorescence emission peak of the first dopamine probe solution under 565nm excitation light is at 980nm, and after dopamine is added, a red-shifted fluorescence peak with stronger luminescence intensity is generated at 1140nm. Figure 2 The two-dimensional fluorescence contour map in part b is drawn with the excitation wavelength as the vertical axis and the emission wavelength as the horizontal axis, which illustrates this point more intuitively. Figure 2 The Raman spectrum of c is plotted with Raman intensity as the ordinate and wave number as the abscissa. After DA is added, the D\G ratio increases significantly compared to the addition of DA, indicating that the SP of carbon nanotubes 2 The lattice is destroyed, forming SP 3Quantum defects have never been verified to emit fluorescence at 1140nm.

[0061] Combination Figure 3 In a, a three-dimensional waterfall chart is drawn with dopamine concentration, fluorescence intensity and emission wavelength as coordinate axes, which intuitively shows the relationship between different dopamine concentrations and the luminescence intensity of the fluorescent probe. It can be clearly seen that the intensity of the characteristic fluorescence peak at 1140nm becomes stronger with the increase of dopamine concentration. Figure 3 b, the average intensity of the characteristic fluorescence peak at 1140 nm before and after the addition of dopamine (II 0 ) / I 0 The vertical axis is the ordinate, and the dopamine concentration is the horizontal axis. Through analysis, it was found that there was a linear relationship between the brightness of the fluorescence peak and the dopamine concentration. The linear function between the fluorescence emission intensity (y) and the dopamine concentration (x) was fitted by data: y = 0.03x + 0.53, and the determination coefficient R 2 Up to 0.999.

[0062] Figure 4 In the figure, other biological small molecules are used as the horizontal axis, and the average intensity of the fluorescence characteristic peak at 1140nm before and after the addition of other biological small molecules (II 0 ) / I 0 By comparison, it can be seen that after adding other biological small molecule solutions, almost no red-shifted fluorescence peak appears, which shows good selectivity. Therefore, it can be seen that the first dopamine probe solution prepared in this example has the ability of linear detection and selective recognition of dopamine.

[0063] 3. When the probe solution A prepared in this embodiment is used for dopamine detection, in order to evaluate its actual detection ability, physiological saline is used to simulate the human body fluid environment for dopamine detection. The specific method is as follows: the (6,5)-SWCNT dispersion is diluted with physiological saline solution, and the (6,5)-SWCNT dispersion is diluted to an absorbance of 0.15 under the characterization of ultraviolet visible near-infrared absorption spectrometer to obtain a second dopamine probe solution;

[0064] Add 50 μL of 1.96 μM dopamine stock solution to 2 mL of the second dopamine probe solution to make the dopamine concentration reach 50 nM in the final test system. After fully shaking, perform fluorescence testing immediately. The actual test performance results are as follows: Figure 5 As shown in a, the fluorescence intensity is the ordinate and the dopamine concentration is the abscissa. Figure 5 As shown in b, Figure 5 Substitute the test results in Figure 3 The linear conclusion of b was verified (the average intensity of the characteristic fluorescence peak at 1140nm before and after the addition of dopamine (II0 ) / I 0 As the ordinate, dopamine concentration as the abscissa), after analysis, it was found that the final detection result was consistent with the derived fluorescence intensity and dopamine concentration function, which shows that even in the liquid phase environment of physiological saline, the dopamine fluorescent sensor can still accurately detect the dopamine concentration. This shows that the dopamine linear detection fluorescent sensor of this embodiment has the potential for practical application.

Claims

1. A method for preparing a near-infrared two-zone linear fluorescence sensor, characterized in that: Chiral single-walled carbon nanotubes are separated by aqueous two-phase extraction method. The materials in the separation process include: deionized water, polyethylene glycol, dextran, sodium dodecyl sulfate, sodium deoxycholate and sodium cholate hydrate; the chiral single-walled carbon nanotubes are dispersed and mixed with 4-nitrobenzene tetrafluoroborate diazonium salt to obtain a fluorescent sensor.

2. The method for preparing the near-infrared two-zone linear fluorescence sensor according to claim 1, characterized in that: Dispersing chiral single-walled carbon nanotube powder in a 1% sodium deoxycholate solution at a ratio of 1 mg / mL to obtain a first mixed solution; subjecting the first mixed solution to tip ultrasound and centrifugation, taking 80% of the upper clear liquid to obtain a second mixed solution; mixing 2 mass% dextran, 12.5 mass% polyethylene glycol, 0.5 g / L sodium deoxycholate, and 18 g / L sodium dodecyl sulfate to obtain a first separation solution; The second mixed solution, deionized water, a 20 wt / v% dextran solution, and a 40 wt / v% polyethylene glycol solution were respectively placed in a centrifuge tube in a volume ratio of 1:3:4:2 to obtain a third mixed solution; The third mixed solution was centrifuged at 8000 rpm for 1 minute to separate the third mixed solution into an upper and lower phase, and the lower phase was taken as the fourth mixed solution; The fourth mixed solution and an equal volume of the first separation solution were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the lower phase was taken as the fifth mixed solution; The fifth mixed solution and an equal volume of the first separation solution were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the lower phase was taken as the sixth mixed solution; The sixth mixed solution and an equal volume of the first separation solution were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the lower phase was taken as the seventh mixed solution; The seventh mixed solution and the first separation solution of the same volume were added to a centrifuge tube and mixed evenly, and centrifuged at 8000 rpm for 1 minute to separate into upper and lower phases, and the upper phase was a high-purity (6,5)-chiral single-walled carbon nanotube dispersion; The obtained (5,6) chiral single-walled carbon nanotubes are diluted with 0.1wt.% sodium dodecyl sulfate solution to an absorbance of 0.15 to obtain a first detection solution; A 20 nM 4-nitrobenzene diazonium tetrafluoroborate solution was added to the first detection solution to obtain a fluorescent sensor.

3. The method for preparing the near-infrared two-zone linear fluorescence sensor according to claim 2, characterized in that: The first separation preparation solution was stirred at a speed of 1200 rpm.

4. The method for preparing the near-infrared two-zone linear fluorescence sensor according to claim 2, characterized in that: The first mixed solution is stirred for 20 minutes; the tip ultrasound of the first mixed solution is performed under ice bath conditions; the tip diameter of the tip ultrasound of the first mixed solution is 6 mm; the power of the tip ultrasound of the first mixed solution is 1200 rpm; the tip ultrasound of the first mixed solution is performed for 60 minutes; the rotation speed of the centrifugalization of the first mixed solution is 12000 rpm; and the first mixed solution is centrifuged for 10 minutes.

5. The method for preparing the near-infrared two-zone linear fluorescence sensor according to claim 2, characterized in that: When mixing the third mixed solution, the speed of the vortex mixer is 2800 rpm; when mixing the third mixed solution, the mixing time is 1 min.

6. The method for preparing the near-infrared two-zone linear fluorescence sensor according to claim 2, characterized in that: When the above mixed solutions are mixed, the speed of the vortex mixer is 2800 rpm; when the above mixed solutions are mixed, the mixing time is 1 min.

7. The method for preparing the near-infrared two-zone linear fluorescence sensor according to claim 2, characterized in that: The absorbance of (6,5)-chiral single-walled carbon nanotube dispersion was studied under UV-visible-near infrared absorption spectroscopy.

8. The method for preparing the near-infrared two-zone linear fluorescence sensor according to claim 2, characterized in that: The 4-nitrobenzene diazonium tetrafluoroborate solution was added while the first test solution was being stirred.

9. The method for preparing the near-infrared two-zone linear fluorescence sensor according to claim 2, characterized in that: The stirring speed of the first detection solution is 800 rpm.

10. An application of a fluorescence sensor, prepared by the method of the near-infrared two-zone linear fluorescence sensor according to any one of claims 1 to 9, characterized in that Used for dopamine linear detection.

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